DIETARY REGULATION OF WHOLE BODY GLUCOSE DISPOSAL AND

CARBOHYDRATE METABOLISM IN HUMAN SKELETAL MUSCLE

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

TANYA PEHLEMAN

In partial fulfilment of requirements

for the degree of

Master of Science

August, 2000

OTanya Pehleman, 2000

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ABSTRACT

DIETARY REGULATION OF WHOLE BODY GLUCOSE DISPOSAL AND CARBOHYDRATE METABOLISM IN HUMAN SKELETAL MUSCLE

Tanya Lynn Pehlernan University of Guelph, 2000

Advisor: Dr. L.L. Spriet

This thesis investigated whole body giucose disposa1 and the adaptive

changes in skeletal muscle carbohydrate (CHO) rnetabolism after a 56-hr high

fat/low CHO diet (LCD; 5 % CHO, 73 % fat, 22 % protein). LCD increased the 90-

min area under the [glucose] and [insulin] curve (2 and 1 -25-fold) during an oral

glucose tolerance test (OGTT) (1 glkg). LCD increased resting pyruvate

dehydrogenase kinase (PDK) activity (0.1 91 0.05 vs. 0.083 + 0.02 min") and

decreased the activated f o n of pyruvate dehydrogenase (PDHa) at rest (0.38 +

0.08 vs. 0.79 + 0.1 0 mm01 acetyl-CoA/kglmin) and during OGTT (0.602 0.1 1 vs.

1 .O4 + 0.09) ( ~ 4 . 0 5 ) . LCD did not alter total GLUT-4 protein, total nexokinase

(HK) or glycogen synthase (GS) activity. It was concluded that glucose disposa1

was decreased after LCD and was related to restricted CHO oxidationi at the

level of PDH activation, secondary to increased PDK activity.

ACKNOWLEDGEMl3NTS

1 would like to thank my advisor Dr. Lawrence Spriet for his guidance and

support from start îo finish of this thesis. Lawrence's respect for his students and his

sincere involvement in their projects fosters the development of confidence to meet any

challenge with enthusiasm. 1 have considered it a pnvilege to work with him, and will be

forever grateful for everything 1 have learned in the process.

1 would also like to thank Dr, George Heigenhauser for his tirne and help over the

course of my graduate studies. In addition, 1 am grateful to Dr. Dave Dyck for always

taking the tirne to answer my questions when 1 knocked on his door dong the way!

A special thank-you to Dr. Sandra Peters who has been a wonderhl fnend and an

inspirationai role model, 1 have also been fortunate to work with Melissa Evans and

Ingrid Savasi. It is rare to work with a team of excellent students and friends.

Finaily, 1 would like to thank Steve for always believing in me, and rny family for

their encouragement.

TABLE OF CONTENTS

Acknowledgements ............................ .., ...................................... i

. . Table of Contents .....................~.....~............................................... LI

List of Tables .............................................................................. iv

List of Figures .................................................... .... ....................... v

List of Abbreviations .................................................................... vi

Introduction ................................................................................ 1

Literature Review ........................... ,... ..................................... Handling of a CHO load in normal subjects at rest ........................... Regulation of major sites involved in skeletal muscle glucose disposal at rest ................................................................................

Glucose transport ............................................................. .................................................... Glucose phosphorylation

Glucose storage ............................................................... Glucose oxidation ............................................................

Insulin Resistance .................................................................. Physiology ...................................... .. ............................. Mechanisms of insulin resistance .......................................... Tirnecourse of skeletal muscle adaptations to altered substrate availability that may downregulate insuiin-stimulated glucose

.................................... disposal .................................. .. ............................ Short-tem adaptations (min to 6-hrs)

Moderate-temz adaptations (>6-hrs tu 6-days) ................ Long-rem adaptations (> 6-days) .................... .. .........

Models used to study metabolic adaptations to altered substrate availability ........................................................................

Euglycemic hyperinsulinernic clamp + intralipid infusion ............ AnimaL Studies ...................................................... Humun Studies ......................................................

OGTT + intralipid infusion ................................................ High fat/iow carbohydrate diet ............................................

Animal Studies ...................................................... Human Studies .....................................................

Statement of the problem .............................................................. 40

.......................................... ................................... Hypotheses .. 41

Methods .................................... ... ...................................... 42 ...................................................... Pre-experimental protocol 42

........................................................... Experimental protocol 42 Bloodanalyses .......................... ... .................................. 45 Muscle analyses ...................... .... .................................... 45

...................................................................... PDK activity 45 ............................ Mitochondrial and total homogenate CS activity 47

...................................... .................... Enzyme activities ... 47 PDHa ...................................................................... 47

..................................................... GLycogen synthase 48 Hexokinase ............................................................... 49 .............................................................................. Pro tein 50

............................................................. Total GLUT-4 protein 50 ........................... ............................ Muscle metabolites .... 51

........................................................................ Calculations 51 ................................................................. Statistical analysis 52

....................................................................................... Results 53 ........................................................................ Diet analysis 53 ........................................................................ B lood results 53

Glucose and plasma insulin .................... ... ............... 53 Blood lactate, glycerol, b-hydroxybutyrate and plasma FFA ....... 54

............................................................. Muscle biopsy results 59 .......................................................... GLUT-4 protein 59 ........................................................ Enzyme activities 59

Hexokinase 59 . ....................................................... Glycogen synthase .............................................. 59

............................................................... PDK 60 PDHa .............................................................. 60

.................................................... Muscle îuels and metabolites 60 ......................................................... Muscle glycogen 60

...................................................... Muscle metabolites 60

.................................................................................. Discussion ............................................................... Insulin insensitivity

................................... Possible mediators of insulin insensitivity Sites of skeletal muscle adaptation to LCD ...................................

Glucose transport and phosphorylation .............................. ......................................................... Glucose storage

................................... Glucose oxidation .. ................ ..................................................... OGTT and acetyl-carnitine

Conclusion ........................................................................ .................................................................. Future directions

................................................................................. References Appendix .................... .... ........................................................

LIST OF TABLES

. ........................................ Table 1 Dietary analysis for CON and LCD 55

. Table 2 Muscle metabolites ........................................................... 64

Figure 1 . Potential sites of dietary adaptation leading to altered glucose ............................................................... disposal by skeletal muscle 6

Figure 2 . Regulation of glycogen synthase (GS) and glycogen phosphorylase ... 12

...................... Figure 3 . Acute regulation of pynivate dehydrogenase (PDH) 17

Figure 4 . Schematic of experimental protocol for both the CON and LCD trial .. 44

......... Figure 5 . Blood-[glucose] and plasma-[insulin] over Ume during OGlT 56

..... Figure 6 . Plasma FFA. and blood B.OH. glycerol and lactate during OGïT 57

............... Figure 7 . Western blot of total GLUT-4 protein for CON and LCD 61

Figure 8 . Basal (O-min) and insulin-stimulated (75-min) glycogen synthase .................................................................. fractional velocity (GSk) 62

...................................................... Figure 9 . PDK and PDHa activities 63

LIST OF ABBREVIATIONS

Acetyl-CoA AUC B-OH CHO CoA CON CS FFA G-6-P GFA GLYp GNG GS GS fv

GSK HK =NSP 1S&ly LCD LDH N2 NlDDM NMR OGTT PDH PDHa PDK PDP PFK PK PP1 PPARs RER TG VO2 VO2rna.x

acetyl-coenzyme A area under the curve beta-hydroxybutyrate carbohydrate coenzyme-A control citrate synthase fiee fatty acids glucose-6-phosphate glucose fatty acid cycle 2-hr area under the glucose curve during OGTT gluconeogenesis glycogen synthase glycogen synthase fiactional velocity glycogen synthase kinase hexo kinase 2-hr area under the insuiin curve during OGTT insulin sensitivity index high fat/low carbohydrate diet lactate dehydrogenase liquid nitrogen non-insulin-dependent diabetes mellitus nuclear magnetic resonance oral glucose tolerance test pyruvate dehydrogenase active pyruvate dehydrogenase pyruvate dehydrogenase kinase pyruvate dehydrogenase phosphatase phosphofiuctokinase pyruvate kinase protein phosphatase- 1 peroxisomal proliferator activating receptors respiratory exchange ratio triglyceride oxygen uptake maximum oxygen uptake

INTRODUCTION

Fat, in the form of free fatty acids (FFA), and carbohydrate (CHO) in the form of

glucose, are the two major fuel sources used by skeletal muscle to generate ATP under

most resting and exercise situations. The combination of fat and CHO used by the

muscle to meet an energy demand in resting and exercise situations is dependent on a

nurnber of factors including the training status of the individual, the oxygen uptake

required to meet the demand, and the concentration of substrates available to the muscle.

The net rate of skeletal muscle rnetabolism can increase by up to 100-fold during the

transition fiom rest to maximal exercise. However, in the resting situation, the energy

dernand and therefore the oxygen uptake by the muscie is low and relatively constant. A

function of resting muscle is to help clear a glucose load in the body by stonng glucose as

glycogen and by oxidizing some of the glucose load. The handling of a glucose load by

the resting skeletal muscle is the focus of the following work.

It is well known that increasing the acute availability of fat to the muscle

increases fat oxidation while decreasing the utilization of CHO. States of nutritional

alteration and disease where FFA levels are elevated andor CHO is restricted, are also

accompanied by a down-regulation in the handling of CHO by skeletal muscle. CHO

stores in the body are finite compared to fat stores, When CHO is not replaced in the

diet, liver glycogen depletes rapidly, within 24-hrs, and muscle glycogP ,n stores are

slowly depleted at rest. Tissues in the body that rely almost enürely on CHO for fuel

become cornprornised when CHO stores are depleted, making the ability of muscle to

preferentially oxidize fat and "spare" finite CHO stores advantageous in the face of

starvation or CHO restriction. In contrast, in a disease state such as non-insulin-

dependent diabetes mellitus -DM), CHO utilization by the muscle is downregulated

secondary to insulin resistance and contributes to the pathogenesis of the disease.

Recently in Our Iaboratory, a short-term high fat/low CHO diet was used to study

the regulation of pyruvate dehydrogenase (PDH) in skeletal muscle in response to a

decrease in CHO availability and an increase in fat availability for 3 and 6-days. In a

related pilot study, with two subjects, upregulation of pyruvate dehydrogenase kinase

(PDK) was found to be present as early as Zdays of the diet. In addition, disposai of an

oral glucose load was significantly decreased following only 2-days of LCD- This

suggested that the diet may have induced a state of decreased insuiin sensitivity, and

altered the handling of excess CHO by the resting muscle. Since our previous studies

indicated a rapid muscle adaptation at the Ievei of PDH regulation, we decided to M e r

examine the handling of glucose after a high fat/low CHO diet by investigating a number

of possible sites of CHO metabolism that may adapt and contribute to the altered

handling of glucose at rest. In the current work, a 56-hr high fatnow CHO diet (LCD)

was used to study the short-term adaptation of skeletal muscle to the disposal of a CHO

load following the alteration of substrate availability.

LITERATURE REVIEW

Handling of a CHO load in normal suhiects at rest

The postabsorptive state, defined as the period between an overnight fast and the

ingestion of a meal, is a common reference point for metabolic studies. After an

overnight fast, insulin concentrations are at basal levels, resulting in a low level of

glucose uptake by insulin-dependent tissues (20 to 25 %) (24). Non-insulin-dependent

tissues such as the brain and other tissues with an obligatory need for glucose, account for

75 to 80 % of the glucose utilizaton. Hepatic glucose output matches glucose utilization

in the postabsorptive state in order to maintain blood gIucose homeostasis. Hepatic

glucose output is a resuk of liver glycogenolysis (70 to 80 %) and glucogeogenesis

(GNG) (20 to 30 %). Liver glycogenolysis at this rate leads to the rapid depletion of

glycogen stores in this tissue during fasting. Liver glycogen is rapidly replenished with

the ingestion of CHO, and only 25 % of the repletion of liver glycogen following an oral

glucose load (lgkg) was due to splanchnic uptake of the glucose, suggesting that the

rnajority of Iiver glycogen repletion occurred via the GNG pathway (22).

When a glucose load is administered in the form of a meal or an oral glucose load,

the basal glucose disposa1 by the brain and other tissues remains constant and the increase

in peripheral glucose disposal ( ~ 3 0 0 %) is accounted for by insulin-dependent tissues. It

has been shown that after both oral (lg/kg) (22,42) and intravenous glucose

administration (18), 70 to 80 % of the glucose load is taken up by peripheral tissues,

predominantly s keletal muscle.

The metabolic fate of glucose within the muscle may not be identical after oral

and intravenous administration. Glucose storage is the major route of disposal of

intravenous glucose (18). During clamp studies the rate of glucose supply exceeds the

resting oxidative requirement and this may lead to overestimation of the role of glycogen

synthesis in the disposal of a normal glucose load (98). It is difficult to directly

quantitate changes in muscle glycogen concentration after both oral and intravenous

glucose administration due to the variability of biochemicd rneasurements in muscle

biopsies. One study used 13c-NM~ to demonstrate that fasted subjects (4 male, 4 fernale)

who ingested a 1900 kcal meal (60 8 CHO), stored 35 % of the CHO as glycogen in the

muscle (98). The increase in muscle glycogen content did not become significant until3-

hrs after the CHO meaI was ingested, despite the rapid timecourse of muscle gIucose

uptake measured after oral glucose (42).

In the basai state, the energy requirements of the muscle are supplied almost

entirely by iipid oxidation whereas the ingestion of oral glucose and subsequent

hyperinsulinemia stimulates CHO oxidation. Following an oral glucose load (lg/kg) at

rest, the overall energy requirements of muscle did not change as reflected by a constant

oxygen uptake (V02) over time (80-min) while the respiratory exchange ratio (RER)

increased significantly [Oldland, 2000 #135]. An increased RER following oral glucose

ingestion was associated with an increase in the calculated rate of CHO oxidation in this

study (basal; 44 & 22 to 75-min; 173 + 15 mg/min) and a similar increase following a 100

g oral glucose Ioad (85). The predominant fate of glucose taken up by the muscle

following oral CHO ingestion may be oxidation in contrast to glycogen synthesis.

Insulin, released in response to a glucose Ioad, is the main regulator that prevents

hyperglycaernia by promoting glucose uptake and storage in insulin-dependent tissues,

and by suppressing hepatic glucose release. In normal individuds, the ingestion of an

oral glucose load (lgkg) dramatically increases insulin-stimulated dnsposal of glucose

while hepatic glucose production is suppressed by approximately 50% (22).

When a CHO load is ingested at rest, blood glucose is iransported into skdetal

muscle by specific glucose transporters (GLUT-1 and GLUT4), and is subsequently

phosphorylated to G-6-P by hexokinase (HK). Inside the muscle, G-6-P is a substrate for

glucose storage as glycogen, and for glycolysis followed by oxidative glucose

metabolism. The two main fates of CHO inside the muscle are contralled by the activity

of glycogen synthase (GS) and pyruvate dehydrogenase (PDHa), respectively (Figure 1).

Since a i l of these processes are involved in the disposai of an oral glucose load, an

alteration at one or more sites may be involved in a state of insulin imsensitivity, where

glucose disposal by the skeletal muscle is reduced.

Fiare 1, Potential sites of dietary adaptation leading to aitered glucose disposal by skeletal mriscle. 1) glucose uptake, 2) glucose phosphorylation, 3) glucose storage, and 4) glucose oxidation

Muscle ceiI

' + Glucose - G-6-P -* -+ GLYCOGEN

1 PYRWATE

1 Mitochondria ,...*-

6 PDK i ri

Regulation of the ma-ior sites involved in skeletal muscle glucose disposa1 at rest

Glucose transport

Circulating blood glucose is taken up into cells by a family of integral carrier

proteins, known as glucose transporters (64). The two transporters found in skeletal

muscle are GLUT-1 and GLUT-4. GLUT-1 is found in low levels at the muscle

membrane, and regulates basal glucose uptake to support basic muscle cell fünctions.

The predominant glucose transporter in skeletal muscle is GLUT-4. GLüT-4 is localized

in intracellular vesicles under basal conditions and is translocated to the muscie ce11

membrane in response to insulin (64). The translocation of GLUT-4 to the muscle

membrane is mediated through an insulin-signaling pathway involving

phosphatidylinositol-3-kinase (34). The rapid redistribution and fusion of GLUT-4 with

the membrane results in a 30 to 40-fold increase in the ce11 surface content of the

transporter and pIays a dominant role in the disposal of glucose by the muscle under

conditions of insulin-stimulation. Most studies have shown that insulin increases glucose

transport through an increase in the Vmax and this appears to be mainly due to an

increased number of transporter proteins in the ce11 membrane.

Glucose phosphorylation

The hexokinases catdyze the ATP-dependent phosphorylation of hexose to

hexose-6-phosphate. In skeletal muscle, hexokinase (HK) catalyzes the transformation of

glucose to G-6-P:

Glucose + ATP G-6-P + ADP + Pi

There are four isofonns of hexokinase in mammalian tissues (HKI to IV) that differ in

tissue distribution, regulation and catalytic properties. HKII is the predominant isoform

expressed in skeletal muscle, but HKI is also present (28). HKI and II have a Km for

glucose in the subrniliimolar range and are allosterically inhibited by G-6-P.

The calculated Km of HKII for glucose in human skeletal muscle, which shares 94

% sequence hornology with the rat muscle isoform, is 0.13 mmol/l(56). G-6-P is a

potent inhibitor of HKII in skeletal muscle (28). The knowledge of the functional

organization and possible structure of HKII has rnainly been established from studies of

other HK isoenzymes. Studies of rat HKI suggest that ATP and glucose bind to the

catalytic C-temiinal of HK inducing a conformational change that prevents binding of G-

6-P at its allostenc site in the regulatory N-terminai (105). Binding of G-6-P induces a

conformational change that prevents the binding of ATP at the substrate site of the C-

terminal haif, but does not prevent the binding of glucose. Studies of human HKII have

shown that both the N- and the C- terminal halves of the enzyme have cataiytic activity

and are inhibited by G-6-P (4).

In vivo, hyperinsulinernia for a period of 4 to 6-hrs induces the expression of

HKII mRNA in human and rat skeletal muscle (56,77). In human skeletal muscle, only

one group (56) demonstrated that HKII activity was increased after 4-hrs of

hyperinsulinemia, while a representative study by KeIIey et al. (43) shows that this has

not been confirmed by other groups of researchers. Therefore, although insulin regulates

HKXI expression in skeletal muscle, the tirnecourse of the associated increase in HKII

protein and activity remains unconfïrmed. A single bout of exercise followed by 3-hrs of

rest also increased HKII mRNA in rat skeletal muscle (63) and increased HKTI mRNA

and activity in humans (50). The evidence for rapid induction of HKII expression and

possibly HKII activity in skeletal muscle suggest that it may be an important regulatory

site in situations where CHO utilization is altered.

Glucose storage

Gl ycogen s ynthase (GS) catalyzes the formation of a- 1,4-gl ycosidic linkages b y

transferrïng glycosyl units from uridine diphosphate glucose (UDP-glucose) to an

existing oligosaccharide chah:

UDP-glucose + glycogen. - UDP + glycogen,~

GS exists in two distinct isoforms, muscle-type GS and Iiver-type GS. Human muscle

GS has only 69% sequence homology to the liver isoforrn (61), whereas human and

rabbit muscle GS share 97% identity (1 10). It should be noted that other isoforms of GS

may exist. Two forms of glycogen exist in human skeletal muscle (1). MacrogIycogen is

recognized as the classic glycogen molecule, with a low protein and high CHO content.

Proglycogen exists as a stable intermediate form of glycogen and has a high protein

content. It has been proposed that the biosynthesis of proglycogen is mediated by a

separate proglycogen synthase, however so far this has not been well substantiated

biochemicaily (P.J. Roach, personai communication).

The initial stsp in glycogen biosynthesis involves the covalent attachrnent of

glucose to the priming protein glycogenin. Glycogenin has the ability to self-glucosylate

and catalyzes the sequential addition of glricosyl residues from UDP-glucose, in a

~ n ' + / ~ ~ * + - d e ~ e n d e n t reaction (76). The self-glucosylation results in the formation of a

chah of 8 to12 residues which is further elongated by GS when glycogenin and GS are

complexed together.

GS catalyzes the rate-limiting step in glycogen synthesis and is regulated by

reversible covalent phosphorylation and by Alostenc modification. The phosphorylated

forms of GS are inactive in skeletal muscle, while the dephosphorylated form of the

enzyme is active (60). The phosphorylation state of GS is determined by the activity of

regulatory protein kinases and a phosphatase enzyme (Figure 2). Traditionally, there are

at least three protein kinase enzymes that are known to phosphorylate and inactivate GS.

These are cyclic AMP-dependent protein kinase (also known as glycogen synthase

kinase-1 or GSK- l), phosphorylase kinase (dso known as GSK-2), and GSK-3 (60).

GSK-3 activity is specific for GS, while the other two participate in the coordinated

regulation of GS and glycogen phosphorylase, so that glycogen synthesis is inhibited in

situations where glycogenolysis predominates and vice versa (Figure 2). In vitro, a

number of additional protein kinases have been s h o ~ to phosphorylate GS (P.J. Roach,

personal communication), however the in vivo significance of the additional kinases has

not been determined. The dephosphorylation and activation of GS is achieved by a single

phosphatase enzyme, protein phosphatase- 1 (PP 1).

There are three important regulators that determine the relative rates of glycogen

synthesis and breakdown in the muscle (Figure 2). Ca* and CAMP (secondary to

elevated epinephrine) directiy and indirectly stimulate phosphorylase kinase to increase

glycogenolysis and decrease GS activity simultaneously (60). More importantly at rest,

insulin is thought to activate GS by increasing the activity of PP1 (19) or by decreasing

the activity of protein kinases, both of which would simultaneously decrease

glycogenolysis. Studies of rabbit muscle indicate that phosphate is released mainly fiom

three sites (3a, 3b and 3c) in response to insulin (67). The most important regulatory site,

3b, is specifically phosphorylated by GSK-3 (87). In addition, insulin was also shown to

promote dephosphorylation of site 2, which strongly inactivates GS and c m be

phosphorylated by a number of protein kinases in vitro (14) including phosphorylase

kinase. Therefore, insulin may activate GS by inhibiting a number of protein kinases or

by stimulating PPl, or both.

At physiological concentrations of G-6-P (0.1-0.5 mM), phosphorylated forms of

GS are much less active than the dephosphorylated form. G-6-P can allosterically

activate the phosphorylated form of GS (49), however G-6-P does not change drastically

in most aerobic situations in skeletal muscle (60). Therefore, the activity o f GS is rnainly

dependent on the overall phosphorylation state of the enzyme. Glycogen concentration

also plays a critical role in regulating GS activity through a negative feedback mechanism

(59).

Fiare 2. Regulation of glycogen synthase (GS) and glycogen phosphorylase. GSK- 3(glycogen synthuse kinasej), PK (CAMP-dependent protein kinase), PHOS-K (phosphoryl~se kinase), PPI (protein phosphatuse- 1).

Glycogen Phosphorylase (INACTIIE)

INSULIN PP I

(CAMP) : (Ca", INS ULZ-W

PK PHOS-K

Glycogen Phosphorylase - P (ACTIVE)

G-6-

~ 1 + 5 PHOS-K

GS-P GS - (INACTIVE) (ACTIVE)

Glycogen Phosphorylase - P (ACTIVE)

Glucose oxidation

Pymvate dehydrogenase (PDH) is a multienzyme complex that catalyzes the

conversion of pyruvate to acetyl-CoA:

Pyruvate + CoA + NADC ---+ Acetyl-CoA + NADH + COz

Since this is the first irreversible step in the oxidation of CHO, PDH controls the flux of

CHO-derived carbon into the TCA cycle. The complex contains multiple copies of three

enzymes; pyruvate dehdrogenase (El), dihydrolipoarnide transacetylase (E2), and

dihydrolipoamide dehydrogenase (E3). In addition to these catalytic components, PDH

contains a binding protein (protein X), which Links E3 to the complex, and two inbinsic

regulatory enzymes, a kinase and a phosphatase. Pyruvate dehydrogenase kinase (PDK)

phosphorylates the El subunit of PDH, resulting in inactivation of the complex, while

PDH phosphatase (PDP) dephosphorylates and activates PDH. The relative activities of

PDK and PDP determine the amount of complex in its active form, PDHa (8 1,94). In

contrast to GS, there is no direct allosteric regulation on the PDH complex itself, and the

metabolic effectors that regulate PDHa work through increasing and decreasing the

activities of PDK and/or PDP.

Acutely, PDK is regulated by intramitochondrial metabolic effectors (Figure 3).

Specificdy, a high NADHMAD+, acetyl-CoNfree CoA, and ATP/ADP ratio activate

PDIC, whereas pyruvate decreases PDK activity. At rest, PDK activity is high due to low

pyruvate, and high rnitochondrial effector ratios. PDH phosphatase (PDP) requires Mg+

and is activated by Ca+. During exercise, the increase in intraceliïlar Caf activates PDP

and the increase in pyruvate, as well as the decrease in ATP/ADP inactivate PDK.

Insulin has been shown to increase PDHa in rats (21) and in humans (57). In rats, the

acute effect of insulin to increase PDHa in alloxan diabetic rats was not mediated through

decreased PDK activity, suggesting that PDH is acutely activated by insulin through

direct PDP activation (21).

In the long-term, stable increases in PDK activity have been observed in

association with nutritional conditions (starvation, high fat feeding) or disease States

(alloxan diabetes). The mechanisms involved in the long-term regulation of PDK have

not been conclusively defined. However, possible rnechanisms may include a stable

upregulation of PDK specific activity or an increase in protein synthesis. Recent

evidence has indicated the existence of four isoenzymes in mammalian tissues (PDKl to

PDK4) that differ in tissue distribution, specific activity, and sensitivity to effectors (1 1,

29, 86). PDKl to 4 are present in human skeletal muscle with low Ievels of PDKl and 3

expression, while rat muscle expresses only the PDKl, 2 and 4 isoenzymes.

The kinetic properties and abundant expression of PDK2 in most tissues are

consistent with the idea that it is the isoenzyme mostly responsible for rnetabolic

regulation of PDH activity. Starvation and alloxan diabetes are common models used to

study the long-term regulation of PDK secondary to CHO restriction and elevated FFA.

A representative study by Feldhoff et al. (21) showed that PDK increased and PDHa was

decreased in 48-hr alloxan diabetic rats. Since P D M expression increases several-fold in

rat heart and skeletal muscle during starvation (72,95, 107), it is thought that this

isoenzyme may be important in the long-term adaptation of PDK. Adding to this idea is

the observation that PDK4 has a relatively high specific activity and is much less

sensitive to inhibition by dichloroacetate, the synthetic analogue of pyruvate (1 1).

Therefore, an increased expression of PDK4 would be consistent with an increase in total

PDK activity and a decrease in the ability of pyruvate to reactivate PDH, seen in rat

cardiac rnyocytes and skeletal muslce after 48-hrs of starvation (78,95). In addition,

Bowker-Kinley et al. (1 1) determined that PDK4 does not respond to NADH in

combination with acetyl-CoA. Therefore, if PDK4 is overexpressed in long-term

adaptation, the total kinase activation by these effectors rnay be blunted. This rnay be

important in situations where fat oxidation is elevated, leading to increâses in the

concentration of NADH and acetyl-CoA. In humans, a high fat diet caused a stable

increase in skeletal muscle PDK activity as quickly as 3-days, with a hrther increase at

6-days (71). A subsequent study (70) showed that PDK4 protein and mRNA increased

dunng the first 3-days of the high fat diet. The expression of PDK4 rnay be important in

the stable adaptation of PDK activity in human skeletal muscle, as suggested in rats.

There are a number of potentiai mediators that rnay play a role in the stable

increase in PDK activity. Elevated FFA and ketones contribute to the increase in PDK in

starvation and diabetes secondary to increased oxidation of these substrates which

increases acetyl-CoAKoA and NADWNAD'. There is also evidence ro suggest that

insulin rnay be directly involved in the stable adaptation of PDK. In 48-hr starved rats,

the rapid decrease in circulating insulin levels correlated with a stable increase in cardiac

PDK activity (65). The effects of insulin rnay be due to an indirect effect of circulating

FFA levels, however evidence from culture experiments shows that insulin rnay also have

a direct effect on PDK (65,97). In cultured cardiomyocytes, insulin opposed the effects

of n-octanoate and dibutyrl-CAMP to increase cardiac PDK activity (65), suggesting that

a decrease in insulin levels or a decrease in insulin sensitivity rnay be obIigatory for

stable increases in PDK (97). Insulin increased PDHa activity in diabetic rats within 1-

hr, while 72-hrs of low dose insulin (2 units/kg/day) or 48-hours of high dose insulin (40

unitskg/day), was required to decrease PDK activity to control values (21)- This

indicated that the long-term effects of insulin may directly invoIve PDK whereas the

acute effects of insuiin on PDHa may be PDK-independent.

In humans, Majer et al. (53) showed that the expression of the PDK2 and PDK4

isoforms in skeletal muscle fiom Pima hdians correlated with indicators of the severity

of insuLin resistance. PDK2 and PDK4 mRNAs wem positively correlated with fasting

plasma insulin, 2-hr plasma insulin in response to oral glucose and percentage body fat,

and were negatively correlated with insulin-mediated glucose uptake rates. Dunng a

100-min euglycemic hyperinsulinernic clamp, the Ievels of PDK2 and PDK4 rnRNA

decreased in response to insulin. These results demonstrated that insulin had a direct

effect on the expression of two PDK isoforms, and that an increase in PDK activity in

insulin-resistant subjects could be linked to a deficiency in insulin-stimulated

downregulation of PDK expression. In this case, decreased glucose oxidation would be a

consequence of insu!in resistance.

Fimire 3. Acute regdation of pyruvate dehydrogenase (PDH). Pymvate dehydrogenase kinase (PDK) phosphorylates and inactivates PDH. Pyruvate dehydrogenase phosphatase (PDP) dephosphorylates and activates the complex (PDHa). PDK and PDP are regulated by concentrations of intramitochondnd effectors.

CoA + NAD' NADH + COz

PYRUVATE PDHa

ACETYL-COA

Insulin Resistance

Physiology

The main actions of insulin following an oral glucose load include decreasing

blood glucose levels by promoting the utilization and storage of glucose in peripheral

tissues. Both an increase in the blood glucose Ievel at a given insulin concentration

andor an increase in the plasma insulin Ievel at a given glucose concentration indicate

that the tissues have become less sensitive to insulin (5). Specifically, insulin resistance

can be defined as a state in which insulin produces a subnormal biological response (41).

There are several methods used to measure insulin sensitivity clinically and

experimentally. The administration of an oral glucose load, followed by measurement of

blood glucose at timed intervals is called an oral glucose tolerance test (OGTT). Another

approach, the euglycemic hyperinsulinemic clamp, is often used experimentaily to study

insulin resistance. This method is performed under nonphysiological steady state

conditions, where a defined concentration of insulin is infused intravenorisly and variable

amounts of glucose are infùsed sirnultaneously to maintain a constant Ievel of blood

glucose. Since glucose and insulin are administered intravenously, physiological

hormonal responses and the kinetics of glucose absorption and insulin secretion are lost

(5).

Belfiore et al. (5) developed an insulin sensitivity index (ISIgi,) to quantify insulin

sensitivity using the glycemic and insuhnemic results of OGTT: ISIgly= 2/[(INSp X

GLYp)tl], where PNSp and GLYp are insulinernic and glycemic areas recorded during

O G n . Zt should be noted that discrepancy has been observed when comparing results

of OG?T and euglycemic hyperinsulinemic clamps. It is thought that the consistent

hyperinsulinemic suppression of FFA during the clamp technique may alter the short-

t e m effects of elevated starting FFA and remove an important causal component of

insulin resistance in certain populations.

Since the pnmary physiological indication of decreased insulin sensitivity is the

elevation of glucose andor insulin levels, Belfiore et al. (5) concluded that OGTT is a

simple procedure that can be used to determine whole-body insuLin sensitivity under

physio1ogical conditions.

Mechanisms of insuiin resistance

Altered insulin-stirnulated glucose disposal by skeletal muscle occurs as a result

of elevated fat availability andor CHO restriction, and may be due to adaptive changes at

a number of sites in CHO metabolism. These inciude: 1) glucose transport into skeletal

muscle (GLUT-1 and GLUT-4 transporters), 2) the subsequent phosphorylation of

glucose to G-6-P (HK), 3) the storage of glucose as glycogen (GS), and 4) the oxidation

of glucose (PDKIPDHa) (Figure 1). It is unlikely that experimentally induced insulin

resistance secondary to elevated fat availability over the short-term would alter these

mechanisms to the sarne degree as that of chronic insulin-resistant disease sthtes such as

NIDDM. Kowever, experimental data in normal subjects may provide some insight into

the development of these conditions. A number of models have been used to study the

interaction between insulin-stimulated CHO disposal and substrate availability. It is

important to separate models that induce short-term regulatory changes (minutes to 6-hrs)

vs. models that cause moderate (>6-hrs to 6-days) and long-term (> 6-days) adaptations

in insulin-stimulated substrate utilization. Different sites rnay be involved in

downregulating glucose disposal at different stages of skeletal muscle adaptation to

substrate availability in the development of insulin resistance.

Thecourse of skeletal muscle adaptations to altered substrate availability that may downregutate insulin-stimulated gIucose disposal

Short-term adaptations (min to 6-hrs)

The idea that dtering substrate availability changes the handIing of fat and CHO

by skeletal muscle was originally proposed by Randle et al. in 1963 (38). The reciprocal

interaction between fat and CHO rnetabolism was formally termed the "glucose fatty acid

cycle" (GFA), and was suggested as a possible mechanism for altered CHO metabolism

in diabetes, where FFA are elevated. The GFA was based on observations of the acute

effects of FFA (minutes) to decrease the uptake and oxidation of glucose in contracting

rat heart and resting diaphragm muscle. The central mechanism for the GFA was

proposed to be an increase in the mitochondrial acetyl-CoA and citrate content,

secondary to the increased delivery and oxidation of FFA. The increase in acetyl-CoA

was believed to inhibit the activity of PDH by increasing PDK activity, and the increase

in mitochondrial citrate was believed to increase transport of citrate into die cytoplasm

where it would inhibit the glycolytic enzyme phosphofructokinase (PFK). The secondary

accumulation of G-6-P was proposed as an indirect mechanism leading to the inhibition

of HK and ultimately glucose uptake. These mechanisms were supported in classical

studies by in vitro inhibition of PDH, PFK and HK by acetyl-CoA, and by high levels of

citrate and G-6-P, respectively (16,74,79,99). Re-examination of PFK regulation using

physiological enzyme concentrations reveded less potent effects of citrate than originally

proposed (108). It was subsequently shown that the most potent inhibition of PFK is

present at resting citrate concentrations (73), and it was speculated that additional

increases in citrate achieved by experimental elevation of FFA would not lead to further

increases in PFK inhibition.

In addition to the ciassic indirect role of HK in rnediating glucose disposal,

evidence for rapid adaptation at the level of HK is also based on changes in HKII rnRNA,

and possibly in HKII activity, during hyperinsulinernia (4 to 6-hrs) and 3-hrs following a

single bout of exercise, as discussed previously. The rapid alteration of HKII rnRNA

rnay be important in regulating glucose disposal since total HK protein is related to the

total activity.

In vivo, the short-term (hrs) inhibitory effects of FFA on insulin-stimulated

glucose disposal and oxidation have been repeatedly shown by indirect cdorimetry in

conjunction with euglycemic hyperinsulinernic clamps. These studies will be discussed

in a subsequent section. In sumrnary, fat infusion during euglycernic hyperinsulinernic

clamps in rats and humans rapidly decreases glucose oxidation within 2-hrs, and glucose

disposal within 3 to 4-hrs, although most of these studies do not measure glucose

transport, HK, PDHa and PDK or metabolite effectors directiy. A decrease in non-

oxidative glucose disposal ar;d GS activity occurs after 4 to 6-hrs. This would be

expected in a situation where glucose is continuously administered while glucose

oxidation is suppressed by artificidly elevated FFA, leading to extra glycogen

accumulation within the muscle.

OveralI, there is evidence to suggest that many sites of CHO metabolism may

adapt rapidly to elevated fat availability. In an acute situation, this may be due to altered

levels of metabolic regulators in skeletal muscle. More direct rneasurements in skeletd

muscle are required to determine the mechanisms underlying the reduction in insulin-

stimulated glucose disposal in short-term manipulations of fat availability.

Moderate-term adaptations (>6-hrs to 6-days)

Although studies that alter substrate availability over a moderate timecourse are

lirnited, the literature does suggest that the acute effects of increasing FFA availability

rnay be supplemented with longer-term mechanisms that downreguiate CHO utilization

over a period of hrs to days. The main models used to study this timecourse are

starvation, chemically induced diabetes (alloxan diabetes) and high fat/low CHO diets.

In contrast to FFA infusion studies where FFA leveis are elevated, in starvation, diabetes,

and CHO restriction, eievated FFA as well as low CHO and insulin levels (or decreased

insulin sensitivity) may be important in the adaptation of the skeletal muscle.

In general, early diet studies showed that 5-days of a high fatflow CHO diet

decreased glucose tolerance in human subjects (30). Recently, a study in Our laboratory

(7 1) used a high fat/low CHO diet mode1 in humans to directly examine the adaptation of

skeletal muscle to altered dietvy substrate availability at the level of PDK activity over a

sirnilar timecourse. The administration of a high fatnow CHO diet for 3 and 6-days

resulted in a stable increase in PDK activity (3 to 5-fold). This may be an important site

of downregülation of CHO utilization that could be involved in the altered insulin-

stirnulated glucose disposal following a high fatAow CHO diet. The stable adaptation of

PDK in humans was consistent with PDK findings in rats following 24 to 48-hrs of

starvation, a state where substrate availability is altered such that FFA and ketones are

elevated. Rodent studies have s h o w that starvation increased the activity of PDK 2 to 3-

fold in extracts of heart (65), skeletal muscle (90) or liver (93) rnitochondria. Alloxan

diabetes (48-hrs) aiso resufted in an increased PDK and decreased PDHa (21). These

studies provided evidence for a longer-tenn mechanism for the stable activation of PDK

that persisted through rigorous rnitochondrial extraction procedures, and incubation of the

rnitochondna with an uncoupler to convert PDH to PDHa. As discussed earlier, some

evidence suggests that decreased insulin levels associated with these conditions rnay

potentiate the stable increase in PDK resulting in decreased CHO metabolism.

In summary, the stable adaptation of PDK described above indicates that this is

one important site that rnay be involved in the shift in glucose handling by skeletal

muscle secondary to an elevated supply of FFA and ketones and/or to a decrease in CHO

availability. With respect to the other possible sites of adaptation in resting skeletal

muscle, adaptations in glucose transport, HK and GS secondary to altered substrate

availability have not been exarnined over this tirnecourse. Since some of these sites can

adapt to altered substrate availability over min to hrs, it would seem possible that

adaptations at these sites may also be extended into the short-term to days).

Long-term adaptations (>6-days)

Studies examinhg skeletal muscle adaptations to substrate avaiIability over the

Iong-term often involve human subjects with non-insulin-dependent diabetes mellitus

(NTDDM), since this condition is associated with chronic muscle insulin resistance.

Also, animai models use chronic high-fat feeding (typically 28-days) to induce skeletai

muscle insulin resistance and to study the possible underlying mechanisms.

Euglycemic hyperinsulinemic clamp s tudies in NIDDM subjects have s ho wn bo th

a decrease in skeletal muscle G-6-P (84) as well as an increase in intracellular free

glucose and G-6-P concentrations (55). These results suggest defects in glucose transport

or phosphorylation, and defects in a step distal to the G-6-P pool respectively, as possible

rate limiting steps in CHO metabolism in NIDDM. Vestergaard et al. (103) found

decreased HKII mRNA, protein and activity in NIDDM patients, however Kelley et al.

(43) did not observe a difference in total or HMT activity in the vastus IateraIis of

NIDDM subjects vs. non-diabetic controls. Glucose transport is a critical step in the

regulation of glucose metabolism and changes in the concentration of GLUT-4 and/or the

response of GLUT-4 to insulin may be involved in insulin resistance. Total GLUT-4

protein and gene expression is normal in NIDDM patients (69), however the translocation

of GLUT-4 to the plasma membrane in response to insulin may be impaired (1 11).

Long-term regulatory alterations leading to insulin resistance may involve chronic

adaptations in insulin-stirnulated GS activation, as seen in full-blown NIDDM. The

major defect in insulin stimulated muscle glucose metabolism in NIDDM is reported to

be decreased storage of glucose as glycogen, calculated based on indirect calorimetry

(100). Basal md insulin-stimulated GS mRNA expression were found to be reduced in

subjects with NIDDM (102, 104). It is interesting to note that decreased insulin

activation of GS Fractional velocity (GSn) has been observed in NIDDM patients during

euglycemic hyperinsulinernic clamps (40), but not during hyperglycemic

hyperinsulinemic clamps (55). Since defects in the GS gene itself have not been found,

aiterations in the pathway of insulin-stimulated GS activation may be important in insulin

resistance.

Long- term alteration of insulin sensitivity may involve an increase in PDK

mRNA and protein, or specific activity. Similar to starvation (48-hrs), high fat feeding

(28-days) in rats Ieads to a stable increase in PDK activity (65). Altered expression of

PDK isoforms has more recently been proposed as a mechanism invotved in the

adaptation of PDK activity and kinetics in response to disease or nutritional

manipulation, as discussed previously. In insulin-resistant Pima Indians with elevated

PDK rnRNA (53), a 100-min eugykemic hypennsulinemic clamp decreased PDK2 and

PDK4 mRNA to normal levels, indicating a direct effect of insulin on PDK expression.

This recent evidence suggests that altered expression of PDK isoforms and insufficient

insulin-mediated downregulation of PDK mEWA may be a cause of defects in insulin-

mediated glucose disposal in chronic insulin-resistant States. Further support for this

mechanism was observed in cardiac mitochondria of 28-day high fat fed rats (96) and in

alloxan diabetic rats (21), where acute insulin administration (1 to 6-hrs) failed to

suppress elevated PDK activity, in cornparison to control rats.

In sumrnary, evidence from subjects with NIDDM and from long-term dietary

manipulation of fat and CHO availability suggests that glucose transport/phosphorylation,

glucose storage and glucose oxidation may al1 be sites of skeletal muscle adaptation

involved in a state of insulin resistance.

Models used to study the metabolic adaptation to altered substrate availabilitv

The adaptation of skeletal muscle to altered substrate availability and the

subsequent changes in substrate handling and insulin sensitivity have been studied

extensively in animais including the hurnan. It has been shown repeatedly that elevating

the supply of FFA andor restricting CHO levels over different timecourses (min to days)

causes adaptations in skeletal muscle that inhibit glucose disposal, oxidation and storage.

Fat rnetabolism is also reciprocally downregulated when CHO supply is abundant (94).

However, the mechanisms underlying the adaptations of skeletai muscle in these

situations have not been conclusively defined. This review will focus on examining the

metabolic adaptations that occur in skeletal muscle from the perspective of alterations in

CHO metabolisrn in the presence of elevated fat availability and/or decreased CHO

availability. In addition, only studies that examine these interactions and mechanisrns in

normal subjects will be included, in contrast to those that examine adaptations secondary

to chronic disease States.

Two cornmon models used to experimentally decrease insulin sensitivity and alter

the metabolic handling of CHO are euglycemic hyperinsuhernic clamps with intralipid

infusion (vs. saline infusion), and a high fat and/or low CHO diet (vs. normal dietary

intake or a high CHO diet). A srnall number of studies have used the OGTT as a means

of studying aiterations in metabolism during the elevation of FFA. These models will be

reviewed in both animals and humans, where data is available.

Euglycemic hyperinsulinemic clamp + intralipid infusion

In humans and rodents, an increase in fat availability has been achieved by

infusing lipidheparin during euglycemic hypennsulinernic clamps to prevent the nomal

insulin-mediated suppression of FFA. In humans, continuous indirect calorimetry is used

to detennine rates of whole-body glucose oxidation during the clamp. The idmion of 3-

3~-glucose or 6,6-Dz-glucose coupled with measurements of specific activity in blood

sarnples is most often used to measure overall glucose turnover and is also used in

conjunction with calorimetry data to calculate nonoxidative glucose disposal. In rats,

insulin-stimulated whole body glucose utilization, glycolysis, and glycogen synthesis are

estimated based on tracer concentrations in the plasma and in the muscle glycogen pool.

Studies that attempt to directiy examine the mechanisms underlying altered substrate

handling using this mode1 usudly measure GS activity and G-6-P, and occasionaily

measure PDHa in skeletal muscle biopsies. However, in humans one study (83) used

13c-~MR spectroscopy to quanti@ the rate of muscle glycogen synthesis, and 3 L ~ - ~ ~ ~

spectroscopy to measure changes in G-6-P concentration in the muscle dunng clamp

experiments.

G-6-P is the end result of glucose transport and subsequent phosphoryiation by

HK in skeletal muscle, and is the initial substrate in the glycogen synthesis pathway. For

this reason, the concentration of G-6-P in the muscle is often measured dunng the clamp

protocols as an indicator of which of these mechanisms are altered to cause reduced

glucose disposd under experimental conditions of insulin resistance. It should be

recognized that G-6-P is also a source of pyruvate for PDHa. If the reduction in glucose

disposal is due to inhibition of insulin-stimuiated GS activity or decreased oxidative

glucose disposal, we would expect to see an accumulation of G-6-P in the muscle. If

glucose transport andor phosphorylation is irnpaired, we would expect to see a decrease

in the G-6-P pool.

Animal Studies

In rats, lipidhzparin infusion during the euglycemic hyperinsulinemic clamp

procedure does not consistently lead to the development of impaired insulin action at the

whole body Ievel during the first 2-hrs, however glucose disposal decreases with

iipidheparin infusion in the 3d hr, and thereafier. Consistent with human data, the effect

of the 2-hr clamp with elevated FFA on GS activity and glycogen rnass is variable.

When the lipidheparin infusion was extended to 5-hrs, Chalkiey et ai. (13) observed the

development of insulin resistance after 2 to 3-hrs and found that 3 to 5-hrs of FFA

elevation significantly impaired GS activity in rat skeletal muscle. Park et al. (66) also

found that glucose disposal during euglycernic hyperinsulinemia was only significantly

lower after 3 to 5-hrs of lipid elevation, and that GS activity significantly decreased at

this time. In the latter study, whole body glycoIysis was calculated to be lower

throughout the clamp with lipid/heparin infusion, consistent with a rapid downregulation

of glucose oxidation in the presence of elevated FFA. During the initial 3-hrs, this was

compensated by an increase in calculated whole body glycogen synthesis and an increase

in the accumulation of radioactivity in muscle glycogen. Also, in the initia1 hours of the

clamp, muscle G-6-P was elevated, suggesting that the FFA-induced inhibition of

glycolysis (PFK, PDH) resulted in the accumulation of G-6-P, which would then

stimulate GS activity. Another study (47) found that decreased glucose transport was

secondary to decreased glycolysis and the accumulation of G-6-P during a 3-hr intralipid

clamp.

In a more chronic elevation of FFA (24-hr inaalipid infusion) that examined

glucose transport as a possible limiting mechanism, whole body glucose disposal

decreased in lipid perfüsed rats (52). The amount of total GLUT4 protein was not

different, suggesting that penpheral insulin resistance after the elevation of plasma FFA

may have been due to a defect in translocation of the transporters to the plasma

membrane or transporter intrinsic activity.

Human Studies

The clamp model in normal human subjects has consistentiy resulted in impaired

insulin action at the whole body level, suggesting a state of decreased insulin sensitivity.

While oxidative glucose metabolism decreases rapidly under these conditions, a decrease

in non-oxidative glucose disposal only occurs after 4 to 6-hrs of intralipid infusion (8-10,

44). A representative study by Yri-Jarvinen et al. (109) is typical of some studies that

have not shown a decrease in insulin-stimulated GS activity during a 2-hr clamp. It is

accepted that this discrepancy is due to the time dependence of the fatty acid inhibition of

GS in the euglycemic hyperinsulinemic clamp situation (10).

As suggested earlier, muscle G-6-P levels rnay provide insight into the

mechanisms underlying altered skeletal muscle glucose disposal in this experimentaI

model. Roden et al. (83) measured a decrease in muscle G-6-P concentration starting at

1.5 hours of a euglycemic hyperinsulinemic clamp with elevated plasma FFA. After 3-

hrs, a decrease in glucose disposal was observed accompanied by a reduced rate of

glucose oxidation and glycogen synthesis, suggesting that initially, glucose

transport/phosphorylation was inhibited followed by a reduction in oxidative and non-

oxidative glucose disposal. Boden et al. (9) also found that decreased insulin-stimulated

glucose disposal only occurred after 3 to 4-hrs of fat infusion, despite decreased glucose

oxidation and an elevated acetyl-CoAfCoA ratio within the first hour. The decrease in

glucose disposal was associated with a decrease in GS activity, although no change in G-

6-P was rneasured. The elevated acetyl-CoNCoA was consistent with an inhibition of

PDHa in skeletal muscle secondary to FFA infusion, as measured by Kelley et al. (44). In

a subsequent study (8), the effects of FFA on decreasing glucose disposai were shown to

be dose dependent. After 4 to 6-hrs of fat infusion in normal subjects, high FFA

concentrations (750uM) were associated with impaired GS activity and an increase in G-

6-P concentration. This high FFA concentration is similar to that seen in NIDDM, where

non-oxidative glucose metabolism is thought to be the main defect responsible for

decreased glucose disposai. In contrast, Iower FFA concentrations (550uM) were

associated with a decrease in G-6-P and a speculated reduction in glucose

transport/phosphorylation. In normal subjects, at the lower FFA concentrations,

decreased CHO oxidation and glycogen synthesis contnbuted equaily to the overd1

decrease in glucose disposal.

Whereas most of the studies discussed above are. based on systemic measurements

of glucose metabolism, Kelley et al. (44) used the leg artenovenous balance technique to

more directly assess the effect of fat infusion on skeletai muscle glucose metabolism in

normal subjects. Maintenance of FFA for 4-hrs dunng hyperinsulinernia resulted in a 36

% decrease in the rate of glucose uptake. The calculated rate of glucose storage

accounted for 23 % of the glucose disposal during intralipid infusion (vs. 5 1 % in the

control clamp), and this was accompanied by a decreased GS activity at the end of the

clamp. The contribution of glucose to leg muscle oxidation was Iower when FFA were

rnaintained during the clamp (53% vs. 76 %), and PDHa activity was decreased

accordingly. The results cited above (44) were calculated based on data collected from

210 to 240-min and enzyme activities were measured only at 240-min, therefore it is

difficult to obtain a sense of the components contributing to muscle glucose disposal in

the early stages of the clamp, which may be physiologically relevant.

It should also be noted that although muscle glucose transporters are important in

insulin-mediated glucose homeostasis, studies investigating transport as a rate-Iimiting

step directly in humans are lirnited. Glucose transport is often combined with the

phosphorylation step and these are based on G-6-P levels as mentioned above. Both

gIucose transporters and HK should be examined directly in human skeletai muscle under

experimental conditions of insulin resistance.

Overall, euglycemic hyperinsulinemic clamp studies show that fat infusion

decreases CHO oxidation rapidly, probably by PDK-mediated inhibition of PDHa, and

that elevated FFA may also decrease glucose transport or phosphorylation. Fat infusion

decreases insulin-stimulated glycogen synthesis in a time and dose dependent manner. It

is suggested that the accumulation of glycogen within the initial hours of a euglycemic

hyperinsulinemic clamp may later inhibit GS activity. In addition, decreased levels of G-

6-P secondary to decreased glucose &ansport and phosphorylation may also lead to

decreased glycogen synthesis even when insulin-stimulation of GS is not impaired.

It is clear fiom studies using this mode1 that direct measurements of glucose

transport and glucose phosphorylation by HK are lacking and that the contribution of

these mechanisms to insulin resistance in this situation is not defined.

It is important to examine differences between these results and those measured

under more physiological conditions. The clamp studies examine the effect of elevated

FFA on the disposal of glucose without the physiological increases and fluctuations in

glucose and insulin concentrations that occur following the normal ingestion of CHO.

OGTT + intralipid infusion

The mechanisms involved in lipid-induced insulin resistance during euglycernic

hyperinsulinemia do not necessarily define the mechanisms that alter glucose metabolism

in the postprandial state of insulin-resistant subjects, where plasma glucose levels are

elevated. Although euglycemic clamps offer the advantage of precise control ofglucose

and insulin levels in experimental situations, the effects of elevated lipids may be more

relevant if evaiuated under the physiological conditions of OG?T

Euglycemic hyperinsulinemic clamp studies provide evidence for decreased

oxidation and storage of glucose during infusions of triglyceride (TG) emulsions,

however the same TG infusions in nomal subjects did not result in decreased glucose

storage during the acute hyperglycaemic conditions of an OGïT (85). More recently

(82), OGïT using double-labeled glucose with lipid infusion in normal subjects resulted

in an increase in non-oxidative glucose disposal.

It should be noted that glucose oxidation was decreased during OG'IT with

intralipid infusion (82, 85) and that this was accompanied by a moderate, although

significant glucose intolerance (85). An earlier study (26) also demonstrated that glucose

tolerance decreased despite higher serurn insulin levels in normal subjects during OGTT

(100 g glucose) with intralipid infusion. Indirect calorimetry in this study indicated that

glucose oxidation was lower during OGTT.

Although studies using OGTT are limited, they consistentiy show that glucose

oxidation is decreased in response to a glucose load when FFA are elevated during

OGTT, whereas non-oxidative disposal is not inhibited in this short-term insulin resistant

state. The non-oxidative results are in contrast to the results of clamp studies where a

constant infusion of glucose and insulin during the artificial elevation of FFA over a

period of hours leads to a decrease in the storage of glucose.

High fat/low CHO diet

Animal studies

In animals, high. fat feeding has been used as a mode1 to study the metaboiic

interactions and insulin resistance associated with elevated FFA and CHO restriction.

Chronic high fat feeding (2 to 4-wks) has been shown consistently to induce insulin

resistance in rats (45,46, 51,92), measured using the euglycernic hyperinsulinemic

clamp. The diets studied included those high in saturated fat (66.5 % fat as calories,

mainly shortening) (45,46) and (59% fat as calories, edible tallow and safflower oil)

(92), those high in polyunsaturated fat (59 % fat as calories, mainly saffiower oil) (51,

92), and those high in monounsaturated fat (59 % fat as calories, mainly olive oil).

Results of the various studies indicated that high fat feeding impaired insuIin-stimulated

glucose metabolism at the level of glucose transport, glycolysis, and glycogen synthesis.

The effects of high fat feeding in rats has been shown to be dependent on the type of fat

ingested (25,92). Diets high in saturated fat decreased muscle insulin sensitivity,

whereas diets substituted with n-3 polyunsaturated FFA reversed this effect (92). In one

study (25), the effects of a 28-day high fat diet (47 % fat by energy; 43 % lard and 4 %

corn oil) to increase PDK activity were completely suppressed with the dietary

substitution of 7 % n-3 polyunsaturated FFA (40 % lard and 7 % n-3) within 24-hrs.

In a recent study (43, rats were fed a high saturated fat (mainly shortening) diet

(high fat; 66.5 % fat, 12.5 % CHO, 21 % protein by calories) for 3-wks (vs. control; 12.5

% fat, 66.5 % CHO, 21 % protein by calories). Glucose metabolism was measured using

data from a 2-hr euglycernic hyperinsulinemic clamp, and directly by measuring enzyme

activities and protein levels. Foliowing the 3-wk high fat diet, plasma FFA

concentrations were elevated compared to the control diet. In contrast to the euglycemic

hyperinsulinemic clampAipid infusion studies, where FFA levels were artificially

elevated, FFA levels decreased sirnilarly in both groups during hypennsulinemia- The

high fat diet decreased insulin-stimulated glucose uptake. Results also showed that G-6-

P was increased, accornpanied by a decrease in GS activity and a reduced accumulation

of 3~-glycogen in skeletal muscle. There was no change in total GLUT-4 protein content

or in HKII activity. It was concluded that high fat feecling induced insulin resistance at

sites distal to the G-6-P pool, indicating that impaired glycolysis and glycogen synthesis

were mostly responsible for the decrease in glucose uptake. Also, the possibility of

decreased insulin-stimulated translocation of GLUT-4 cannot be mled out, despite the

unaltered levels of total GLUT-4 protein. It should be noted that in the study by Kim et

al. (43, glucose oxidation was not measured. We would expect that the oxidation of

glucose rnay also have been reduced since high fat feeding in rats increased PDK activity

(25) and subsequentiy decreased PDHa activity . Decreased oxidative disposal of

glucose rnay also have been contributing significantiy to the downregulation of glucose

disposal and the accumulation of G-6-P.

Other studies have also shown that high fat feeding is associated with insulin

resistance and a decreased insulin-stimulated glycolysis and glycogen synthesis (5 1, 9 1,

92). It is interesting to consider that rat studies have suggested that decreased insulin-

stirnulated GS activity rnay not be the primary mechanism responsible for insulin

resistance foliowing high fat feeding. In one study (46), insulin-stimulated GS activity

was increased during the initial days of high fat feeding and then decreased to control

values after 2-wks of the diet. Insulin-stimulated glycolysis decreased rapidly, dunng the

initial Zdays of high fat feeding. Impaired insulin-stimulated GS activity rnay not be

involved as a mechanism underlying insulin resistance until2 to 3-wks of high fat

feeding in rats (45,46). Therefore, glucose oxidation rnay be an important primary event

leading to decreased glucose disposal under the conditions of a high fat diet. Chronic

high fat feeding (28-days) is known to induce a stable increase in PDK activity in rat

skeletal muscle (25), suggesting that this rnay be an important mechanism involved in the

adaptation of skeletal muscle to a high fat diet.

High fat diets have been shown to decrease insulin-stimulated glucose transport

without changing the total protein content of the transporters (31, 32,45, 112). The

decreased glucose transport is presumably due to a decrease in the translocation of

GLUT-4 to the plasma membrane in response to insulin, as confirmed by Zierath et al.

(1 12) in high fat fed mice. More proIonged exposure to a high fat diet (>IO-wks) resulted

in a decrease in GLUT-4 mRNA in rat soleus muscle (48). Nthough it is not measured

as ofien as the glucose transport step, post-transport phosphorylation of the glucose

seems to be unaffected in rodents following high fat feeding (1 12), as indicated by

unaltered HKII activity &ter a high fat diet.

Overail, chronic high fat diet studies in animal models seem to indicate that the

elevation of FFA induces insulin resistance and that a number of mechanisms may be

involved. Altered transport of glucose may contribute to the insulin resistance due to

decreased insulin-stimulated translocation of GLUT-4, whereas the major defects in

glucose disposal are distal to G-6-P, including glucose oxidation and glycogen synthesis.

Human studies

Studies using high fat andlor low CHO diets to examine the interaction between

elevated FFA and insulin resistance are lirnited. In an early study, Gordon et al. observed

a rise in FFA in normaI subjects foLlowing dietary restriction of CHO (27). Hales et al.

(30) measured plasma glucose, FFA and insulin dunng OGTT (100 g glucose) in normal

subjects following a 5-day low CHO diet (less than 50 g CHO per day, fat and protein

unlimited). The dietary alteration led to a reduction in glucose tolerance as evidenced by

higher gIucose concentrations at 30 and 60-min during OGTT, and maintename of

elevated plasma insulin at 60 and 150-min during O G T . FFA were elevated following

the low CHO diet, and were suppressed during OGTï in the control and diet condition.

The suppression occurred rapidly in the control condition, but was delayed following the

low CHO diet- Another study (3) showed that increasing the fat content of the diet from

43 to 65 % and decreasing the CHO content fiorn 40 to 20 %, resulted in a marked

deterioration of glucose tolerance in normal subjects. In a different study (88), a 3-day

high fat/low CHO diet (65% fat by energy) with normal daily energy intake resulted in an

enhanced piasma insulin response during OGTT (1.5g glucosekg). Therefore, subjects

required a greater level of insulin after LCD to achieve a similar rate of forearm glucose

uptake and to maintain simiiar blood glucose concentrations.

Anderson et ai. (2) demonstrated that a 5-day low CHO diet (57 g CHO/day) did

not impair glucose tolerance in normal men if the fat content remained sirnilar to their

normal dietary fat intake (145 dday). However, when the fat content of their diet was

increased to 16 % higher than normal (168 g/day), the low CHO diet caused impaired

glucose toIerance. The glucose levels measured during OGTT were higher between 30

and 150-min after the 16 % increase in fat content of the diet. These results suggest that

the increased fat content of high fat diet models is important for the development of

glucose intolerance.

Decreased CHO oxidation may be involved in the downregulation of glucose

disposa1 seen after a high fat diet. The direct effects of a high fat diet on PDHa are not

well known. A significant reduction in resting RER has been observed following 3 and

5-days of a high fat diet (65 % and 72 % fat, respectively), suggesting that CHO

oxidation was downregulated by the dietary alteration (39, 88). Putman et al. examined

PDHa directly following exhzustive exercise and a 3-day high fat/low CHO diet (3 %

CHO, 51 % fat, 46 % protein) and found that resting skeletd muscle PDHa was

decreased vs. a high CHO diet (80). Cutler et al. (17) examined the effects of longer-

term (21-days) ingestion of a high fatnow CHO diet (8% CHO, 75% fat, 17% protein) on

glucose metabohm and insulin sensitivity. Indirect calonmeûy data indicated that

glucose oxidation was decreased during a euglycemic hyperinsulinemic clamp, and this

was accompanied by decreased PDHa activity measured in muscle biopsies.

Overall, diet studies in rodents and in humans indicate that elevating the dietary

intake of fat and/or restricting the intake of CHO is associated with a decrease in insulin-

stimulated glucose disposal. Results from Limited hurnan studies indicate that glucose

oxidation is downregulated whereas rodent studies suggest a nurnber of possible sites of

adaptation in CHO metabolism including transport, oxidation and glycogen synthesis. In

general, the mechanisms underlying the decreased insulin-stimulated glucose disposal

following a high fat and/or low CHO diet remain undefined, and the majority of the

studies in normal human subjects have not directly measured enzymes, proteins and

metabolites in the muscle. Therefore, although a decrease in insulin sensitivity following

a high fat/low CHO diet is not a novel finding, a comprehensive approach to examining

the possible sites of adaptation underlying the insulin resistance in humans is lacking in

the current literature.

More recently, in o u lab, enzymatic adaptation to a high fatnow CHO diet was

studied directly in muscle biopsies h m normal subjects. The diet (5 % CHO, 63 % fat,

33 % protein) resulted in a dramatic and rapid increase in PDK activity at 3-days (0.35 +

0.09 vs. 0.10 + 0.02 min-'), with a further increase at 6-days (0.49 + 0.06 min-'). Resting

PDHa activity was decreased accordingly (0.17 + 0.04 vs. 0.63 + 0.17 mm01 acetyl-

~ o ~ . m i n - ' - k ~ - ' ) after 6-days of the high fatllow CHO diet. In conjunction with elevated

blood FFA, glycerol and B-hydroxybutyrate levels following the diet, the decreased

PDHa strongly indicated that the diet shifted fuel metabolism by increasing the utilization

of fat and ketone bodies, and reducing CHO metabolism. A subsequent pilot study in Our

Iaboratory showed that PDK activity increased afier only 2-days of a high fatnow CHO

diet (10 % CHO, 63 % fat, 27 % protein) in normal subjects. In addition, blood glucose

increased to a higher peak and remained elevated throughout a 3-hr OGïT (lgkg)

following the high fat/low CHO diet when compared to pre-diet OGïT results. This

suggested that penpheral glucose uptake and utilization may be decreased as early as 2-

days after a high fat/low CHO diet, resulting in decreased glucose tolerance in normal

subjects.

STATEMENT OF THE PROBLEM

As discussed throughout this review, there are many possible sites of regulation

that may decrease the disposd of a CHO load when FFA are elevated and CHO is

restricted during LCD. Some of these sites include decreased glucose uptake (GLUT-

1,4), inhibition of glycolytic enzymes (PFK, HK), inhibition of glucose oxidation

(decreased PDHa, mediated by PDK), and decreased glucose storage (mediated by GS).

In addition, insulin receptor binding and insulin signaling events that precede these

glucose disposd mechanisms may also be irnpaired, however these were not discussed

within the scope of this review. Adaptations at any number of these sites may alter the

disposal of glucose by skeletal muscle.

A short-term (56-hr) high faülow CHO diet (LCD) mode1 was used to examine

adaptations in whole-body glucose disposal and insufin sensitivity following dietary

alteration in normal individuds. The first purpose of this study was to c o d m that the

short-term LCD altered whole body glucose disposai and insulin sensitivity in normal

subjects. The second purpose was to identify potential sites in the muscle where the

handling of CHO was altered. Specifically, the effect of LCD on total GLUT4 protein,

and on GS, total HK and PDWPDHa activity was examined.

HYPOTEESES

The overail hypothesis was that the short-term (56-hr) LCD wouid decrease

whole body glucose disposal and that this would be related to an increase in skeletal

muscle PDK activity and a decrease in PDHa.

Specific hypotheses

1. Whole body glucose disposal would be decreased following LCD.

2. Resting PDK activity wouId increase, and resting PDHa would decrease after

LCD. It was hypothesized that PDHa would be iower after the administration of

oral glucose in LCD vs. CON.

3. Total GLUT-4 protein would not be altered by LCD.

4. Total HK activity would not be different after LCD vs. CON.

5. Basai and insulin-stimulated GS activity would not be altered by the short-term

LCD.

METHODS

Six heaithy, male university students volunteered for this study, mean age and weight

(22.8 + 1.0 yr and 74.1 + 12.9 kg). Al1 of the subjects were aerobically active on a

regular basis (3 to 6 times per wk). The mean relative maximum 0 2 consumption

(VOzma) was 55.4 + 8.3 ml-rnin-L-kg-l (range 43.6 to 67.6 ml.min-'-kg-'). Subjects were

informed of the study protocol and associated nsks before giving their written informed

consent. The study was approved by the Ethics Cornmittees of McMaster University and

of the University of Guelph.

Pre-experimentalprotocol. Prior to the expenment, VOzm was determined using a

continuous incremental protocol on a cycle ergorneter (Excalibur, Quinton Instruments,

Seattle, WA) with a metabolic cart (SensorMedics Mode1 2900, Yorba Linda, CA).

Subjects also completed 3-day dietary records. Dietary records were analyzed using

Nutripro Diet Analysis Software (West Publishing, Salem, OR) to determine normal

dietary intake. This software was then used to design experimental diets. If necessary,

slight adjustments were made to the normal diet to achieve a standardized pre-diet (CON;

5 1 % CHO, 29 % fat, 20 % protein). A 56-hr LCD was designed for each individuai

subject (LCD; 5 % CHO, 73 % fat, 22 % protein). CON and LCD were eucaloric with

the normal diet of the subjects. The dietary composition (% CHO, % fat, % protein) is

reported as the mean of the six individual diets.

ExperimentaiprotocoL During the expenment, subjects reported to the laboratory on

two separate occasions (Figure 4). For 3-days prior to their first visit, subjects consumed

the standardized pre-diet, CON. Physical activity was restricted for 24-hrs before the

CON trial. On their first visit to the laboratory (DAY O), subjects arrived after an

overnight fast (10 to 12-hrs). A catheter was inserted into an antecubital vein of one

forearm, and a resting blood sample (-30-min) was obtained. Patency was maintained

with a sterile isotonic saline solution. One leg was prepared for muscle biopsies taken

from the vastus lateralis under local anesthetic, as previously described (7). A second

resting blood sample was taken followed by two resting muscle biopsies (O-min). A

portion of the frst biopsy was dissected free of blood and connective tissue, and

mitochondria were extracted on the fresh muscle for determination of PDK activity. An

oral glucose load (1 @kg) (Tmtol75, Custom Laboratories Inc., Baltimore, ML) was then

administered and blood samples were taken at 30-min intervals over a period of three

consecutive hours. Seventy-five min after the administration of the oral glucose, a third

muscle biopsy was taken from the vastus lateralis (75-min). An additional biopsy was

taken at this time if the amount of muscle was inadequate for the necessary analysis.

After completion of the 3-hr OG'M', subjects irnrnediately began their LCD. Detailed

dietary guidelines were provided for each subject in addition to al1 of the required food

for the 56-hr period. Physical activity was restncted to activities of daily living during

LCD, and alcohol was prohibited.

Following LCD, subjects fasted ovemight (10 to 12-hrs) and retumed to the

laboratory in the moming (DAY 3). The above protocol was repeated in the LCD

condition. Muscle biopsies were taken from the altemate leg at this time.

Fimire 4. Schematic of experimental protocol for both the CON and LCD trial.

Following the CON trial, subjects began LCD for 56-hrs followed by an overnight fast.

Subjects then returned to the laboratory for the LCD trial.

CONLCD caîheter lgkg glucose

-30 O 30 60 90 120 150 180 (minutes)

t

Blood t f t t f t

Fasted (IO-12h)

Muscle biopsy Oû fIU (75-min)

overnight 1 LEG PREP OGTT

Blood analyses. BIood samples (3 to 4 ml) were dram from the indwelling catheter.

One portion of wholz blood (200 pl) was added to 0.6 N perchloric acid (800 pl),

vortexed and centrifuged at 10,000 rpm for 1-min. The supernatant was removed for

analysis of glucose, phydroxybutyrate (B-OH), lactate and glycerol(6). A second

portion of whole blood was cenerifuged and 400 pl of plasma were removed into 5 M

NaCl (100 pl) and incubated for 30-min at 56OC to inhibit lipoprotein Lipase activity.

Plasma free fatty acids were measured in the plasma using a Wako NEFA C test kit

(Wako Chernicals, Richmond, VA). The remaining aliquot of plasma was analyzed for

insulin using a Coat-a-Count Insulin test kit (Diagnostics Products, Los Angeles, CA).

Muscle analyses. A portion of fresh muscle (-50 to 60 mg) was separated from the first

resting muscle biopsy and processed for the extraction of intact mitochondria to measure

PDK and citrate synthase (CS) activities. A second portion of the biopsy (-20 mg) was

frozen immediately in liquid nitrogen (&) for analysis of total homogenate CS. CS

activities were used to calculate mitochondnal recovery, and the quality of the

rnitochondrial extraction (discussed later). Extra muscle from the first biopsy was frozen

separately in N2 and was used for the measurement of muscle glycogen.

The second resting biopsy as well as the 75-min biopsies were frozen immediately

in N2 and stored for Iater analysis, including PDHa, HK, GS and muscle metabolites.

GLUT-4 protein was measured in the 75-min biopsy samples only.

PDK act ive . Intact mitochondria were extracted from the muscle homogenate using

differential centrifugation, as previously descnbed (38,54). The final rnitochonckial

suspension was incubated for 20-min at 30' C in a buffer containing 10 p M carbonyl

cyanide rn-chlorophenyl-hydrazone, 20 mM Tris-HCI, 120 mM KC1,2 rnM EGTA, and 5

mM potassium phosphate (monobasic), pH 7.4. This incubation decreases ATP

concentration to zero thereby causing complete conversion of PDH to the active form,

PDHa (20). Mitochondria were pelleted (7,000 g, 10-min) and stored in Nz for later

analysis of PDK activity.

PDK activity was measured following the protocol previously outlined in our lab

by Peters et al. (71). Briefly, the rnitochondnal pellet was resuspended in a phosphate

buffer, pH 7.0, and freeze-thawed twice to break al1 of the mitochondria. The suspension

was warmed to 30' C and two aliquots were removed into a sodium fluoride,

dichloroacetate buffer, pH 7.8 (dilution 1: 1) to lock PDHa activity through inhibition of

the phosphatase and kinase. These samples represent "zero-tirne" or "total P D H

Magnesium-ATP (final concentration 3 mM) was added to the remaining suspension and

timed samples were removed from the original suspension every 30-sec for 2-min, and

then every min up to 5-min. The samples were placed immediately into the sodium

fluonde, dichioroacetate buffer and stored on ice for analysis of PDHa by the

radioisotopic measurement of acetyl-CoA production as previously described (15,80).

PDK activity is reported as the apparent first-order rate constant of the inactivation of

PDH (min-') or as the slope of ln[%(PDHa activity with AïJ? addition)/(total PDH

without ATP addition)] vs. time (20, 101). The slope was deterrnined by regression

analysis.

Mirochondrial and total homogenate CS activity. CS activity was measured

spectophotometrically using an enzymatic method linked to a colored product, as

previously described (89). CS activities in the total muscle hornogenate (CShm), and in

mitochondrial suspensions were used to calculate the recovery and quality of the

rnitochondrial preparations (71). In the rnitochondrial suspension, extrarnitochondrial CS

(CS,,) was measured, and total CS (CS,) was measured after freeze-thawing to break the

mitochondria:

Fractional recovery = (CSts - CSed/CSh,

% Intact mitochondria = 100 X (CSts - CSem)/CSts

PDNa, A srnaII piece of frozen wet muscle (approxirnately 10 mg) was removed from

each of the biopsies under Nz for the determination of PDHa using tAe methods of

Constanin-Teodosiu et al. (15) as modified by Putman et al. (80). Briefly, muscle

samples were homogenized in a homogenizing buffer, pH 7.8 (30 p1:l mg), containing

NaF and dichloroacetate to inhibit PDH phosphatase and kinase respectively. PDHa

activity was deterrnined at 37OC by adding muscle hornogenate (30 pl) to a reagent

mixture, containing the necessary coenzymes (CoA, NADH and thiamine

pyrophosphate), as previously outlined (80). The reaction was initiated using pyruvate

foliowed by the removai of aliquots (200 pl) at precisely tirned intervals (1,2 and 3-min)

into 0.5 M PCA (40 pl) to stop the reaction. Samples were neutralized using 1.0 M

K2C03. The neutralized extracts were srored at -80°C for subsequent radioisotopic

analysis of acetyl-CoA (12). Linear regression of plots of acetyl-CoA vs. time was

performed to determine reaction rates. Total creatine content was measured in

neutralized PCA extracts of PDHa homogenates, using the method of Bergrneyer et al.

(6). PDHa activity was corrected to the highest creatine concentration in a set of biopsies

from a given subject. This allowed for compensation for the presence of blood and

connective tissue in the muscle biopsy samples. PDHa activity was calculated as mm01

acetyl-CoA per kg wet muscle per minute.

GZycogen synthase. A second piece of frozen wet muscle (6 to 10 mg) was removed from

the biopsies under N2 for the determination of GS activity. GS activity was calculated as

nmol of UDP-glucose incorporated into glycogen per minute per mg of protein, and was

used to calculate GS fractional velocity (GSfv). GSrv is defined as the activity of GS at 0.1

rnM G-6-P (active GS) divided by the activity at 10.0 rnM G-6-P (total GS). G& is a

sensitive indicator of in vivo GS activity (49) and the ratio increases in response to insulin

infusion in humans (56,57).

GS was measured fluorometrically as described by H e ~ k s s o n et al. (35) with

modifications. The muscIe samples were homogenized by hand in buffer (50 pl: I mg)

containing 50 mM Tris-HCI, 5 mM EDTA, 20 mM NaF, and 5 mM DT, pH 7.2-7.4.

Homogenates were centrifuged (7000 g / 8300 rpm, + 4 O C ) for 5-min and aliquots of the

cytosolic fraction (supernatant) were incubated for the determination of active and total

GS activity. The incubation media consisted of 50 mM Tris-HC1,2 mM EDTA, 10 rnM

NaF, 10 mM glycogen, 0.5 mM Dm, 0.02% BS A, and either 0.1 rnM or 10.0 mM G-6-

P, pH 7.2 to 7.4. Muscle homogenate (100 p1) was incubated with 450 pl each of 0.1

mM and 10.0 mM incubation media for 45411 at 37OC. The reaction was started with

the addition of 50 pl of UDP-glucose (final concentration 8 rnM), and was stopped by

heating at 90° C for 2.5-min. Samples were centrifuged and the supernatant was removed

for fluoromettic assay of UDP using the reactions catalyzed by pyruvate kinase (PK)

(PEP + UDP + Pynivate + UTP) and lactate dehyrogenase (LDH) (Pyruvate + NADH

i Lactate + NAD?. Bnefly, samples, blanks and standards (UDP, 25 pM to 150 pM)

were added to an assay reagent containing 20 mM Tris-HCl, 30 mM KC1,4 mM MgC12,

0.4 mM phosphoenolpyruvate, 20 p M NADH, 0.4 U/rnl LDH, pH 7.6. Pyruvate kinase

was added (3.0 U/ml) to stitrt the reaction. The samples were incubated for 15-min at

room temperature followed by fluorometric determination of NADH.

Note: In order to carry out fluorometric measurements after a 15-min incubation

period, a series of initial experirnental assays were run. These determined the endpoint of

the assay reaction and confirrned that any drift inherent to the assay over time was

consistent between blanks and standards as well as samples. In the initial assays, samples

were read fluorometrically followed by the addition of PK to start the reaction. Samples

were read at 1 and 10-min, and then every 5-min for a total of 40-min. Blanks, standards

and samples continued to drift between 1 and 10-min, however after 10-min the

fluorometric readings leveled out (ie. the reaction was completed) and were constant up

to 40-Mn with no indication of further dnfting. Therefore, in the final experimental

assays, an initial reading was taken followed by a 15-min incubation with PK at room

temperature and then a final reading.

Hexokinase. Maximat HK activity was measured at room temperature as origindly

described by Henriksson et al. (35) and modified by Phillips et al. (75), using a small

piece of wet muscle (3 to 5 mg). HK activity was determined in muscle homogenates by

measuring the fluorescence of NADPH (linked to [Gd-PI) against a range of G-6-P

standards (1.67 to 10.0 mM). HK activity was calculated as mol D-glucose incorporated

into G-6-P per hour per kg protein.

Protein. The protein content for GS and HK activity was rneasured using a BCA reagent

kit containing BS A standard (2 rng/rnl) and reagents (Pierce, Rockford, L).

Total GLUT-4protein. GLUT-4 protein content was deterrnined in collaboration with

Dr. Bonen at the University of Waterloo (Department of Kinesiology). Western blotting

procedures were used to determine the total protein, as previously described by this group

(58,75). A portion of frozen muscle (50 mg) fiom the 75-min biopsy samples was

homogenized and total membranes were isolated by centrifugation (250,000 g). Samples,

containing 30 pg protein were mixed with 62.5 pl of L a e d sarnple buffer containing

2.5 % dithiothreitol and brought to 125 pl with buffer containing 25 mM Tris (pH 8.3),

0.19 mM glycine, and 1 % sodium dodecyl sulfate (SDS). Sarnples were separated by

SDS-polyacrylarnide electrophoresis on a 12 % resolving gel and transferred to an

irnrnobilon polyvinylidene difluoride membrane (Millipore, Malton, ON) by

elecromembrane transfer (i 10 V, constant voltage, 90-min). GLUT-4 protein was

detected using a polyclonal immuno-A purified GLUT-4 antibody (1:750, East Acres

Biologicals) followed by incubation with a horseradish peroxidase-labeled second

antibody (15000, anti-rabbit imrnunoglobuIin G; Arnersharn). GLUT-4 protein was

visualized using an enhanced cherniluminescence detection system (Amersharn) and the

blots were quantified using a Macintosh LC cornputer with an Abaton scanner and

appropriate software (Scan Analysis, Biosoft, Cambridge, UK). GLUT-4 protein was

measured as the intensity of a fixed area and expressed in relative absorbante units.

Musck metabolites. The remainder of the frozen muscle was f'reeze-dried, powdered and

dissected of ail visible blood, connective tissue and fat. The muscle metabolites were

measured using standard enzymatic methods that link the concentration of the metabolite

to a product that absorbs light or fluoresces at a given wavelength, or to a labeled product

that can be measured radioisotopically. Glycogen content was measured in the resting (0-

min) samples (2 to 3 mg), as descnbed by Hanis et al. (33). The rest of the metabolites

were measured in resting and 75-min biopsies on neutralized PCA extracts. Creatine,

phosphocreatine (PCr), ATP, lactate and G-6-P were deterrnined by spectrophotometric

andysis (6,33), pyruvate was measured fluorometrically (68), and acetyl-CoA and

acetyl-camitine were determined by radioisotopic measurement (12). Al1 of the

metabolites were corrected to the highest total creatine concentration from a set of

biopsies to compensate for the presence of blood and connective tissue.

Calculations. A 90-min OGTï area under the curve (AUC) for blood-[glucose] and

plasma-[insulin] was calculated for each subject as follows; resting glucose and insulin

concentrations (O-min) were used as the baseline value and the total area of deviation

from the baseline was calculated between O and 90-min. Values above and below the

baseline were designated as positive and negative respectively.

An insulin sensitivity index for glycemia (ISI& was calculated to quantifi

whole body insulin sensitivity based on OGTï results, as descnbed by Belfiore et aL(5);

ISI(gly)= 2/[mSp x GLYp) + 11, where INSp and GLYp are 2-hr insulinernic and

glycemic areas during O G n . The change in ISI@ly) for each individual subject

following dietary aiteration was calculated by normalizing LCD results to a subject's

normal OGTT response (CON).

Resting or basal blood (glucose, P-hydroxybutyrate, glycerol and lactate) and

plasma (insulin and FFA) concentrations were calculated by averaging the -30 and O-min

concentrations.

Stutistical analyses. Paired student's t-tests were used to compare GLUT-4 protein

content, glycogen concentration, and PDK activity in muscle samples (CON vs. LCD).

AUC's for blood-[glucose] and piasma-[insulin] were cornpared using a paired student's

t-test. Basal concentrations of blood and plasma parameters were directly compared

between CON and LCD using a paired student's t-test. Blood parameters, enzyme

activities (PDHa, GS, HK), and muscle metabolite concentrations over time during

OG?T were analyzed using a two-way repeated rneasures ANOVA (time x diet) with a

Tukey post-hoc test for al1 painvise multiple comparisons. Al1 blood and muscle data are

presented as means t SE. Siopificance was accepted at pc0.05.

RESULTS

Diet analysis

Dietary compliance during CON and LCD was monitored and subjects completed

check-lists outlining specific quantities of foods as well as preparation instructions. If

subjects required slight modifications to the diet during the LCD, these were precisely

recorded after verbal approval, and then adjusted in a final dietary analysis. The subjects

consumed 51 + 0.5 % CHO, 29 + 0.5 % fat, 20 + 0.8 % protein in the CON diet (range

50.0 to 53.a %, 27.0 to 30.0 %, 17.8 to 23.0 %) and 5 + 0.2 % CHO, 73 + 0.6 % fat, 22 +

0.5 % protein (range 4.0 to 5.0 %, 71.2 to 75.0 %, 20.0 to 23.7 %) in the LCD diet. The

total energy intake was eucaloric with the subjects normal dietary intake. The total

caloric intake and breakdown of the diets is presented in Table 1.

Blood Resuits

Glucose and piasma insulin

Basal blood-[glucose] and plasma-[insulin] were lower following LCD vs. CON

(3.39 + 0.19 vs. 4.22 + 0.19 mM and 5.64 I 0.3 1 vs. 8.54 + 0.67 pIU/rnl) (p<0.05).

During OGTT, the blood-&lucosel decreased to the baseline level in CON and

decreased aimost to baseline in LCD at 90-min (Figure 5). The plasma-[icsulin]

decreased towards baseline values at 90-min in both conditions and then leveled out at

this concentration with slight decreases toward baseline in the final hour (Figure 5). A

90-min area under the curve (AUC) was calculated to compare the response to the oral

glucose load between conditions, since the majority of the glucose and insulin response

occurred within this time. The calculated 90-min AUC blood-[glucose] was 2-fold

higher and the AUC plasma-[insulin] was 1.25-fold higher following LCD vs. CON

(pcO.05). It should be noted that the 120 and 180-min AUC for blood-[glucose] were

significantly higher in LCD vs CON (pc0.05). However, the data was disproportionate

to the acfual diet effects on glucose disposal since blood glucose levels were well below

baseline after 90-min in CON, but not in LCD. The 120 and 180-min AUC for plasma-

[insulin] were higher after LCD vs. CON (p~0.05 and p=0.055 respectively).

In addition, an insulin sensitivity index for glycemia (ISI~gi,l) was calculated to

quantify whole body insulin sensitivity based on OG?T results (descnbed in Methods)

(4). There was a signif~cant decrease in insulin seiisitivity following LCD vs.CON

(ISIgly = 0.32 + 0.07 vs. 1) ( ~ ~ 0 . 0 5 ) .

Blood lactate, glycerol, p-hydroxybutyrate and plasma free fat@ acids

Basal blood [%hydroxybutyrate] (B-OH) and plasma F A ] were significantly

elevated after LCD. B-OH increased from O. 1 I + 0.02 to 0.29 t 0.05 rnmoVl and FFA

increased from 0.3 1 + 0.05 to 0.48 + 0.05 rnmol/l following LCD (p<0.05). FFA, B-OH

and glycerol were suppressed significantly vs. basal levels (O-min) during OGTï in both

conditions (pe0.05) (Figure 6). Blood lactate was lower from O to 120-min during OG'IT

following LCD (Figure 6) (NS). (See appendix for individual blood results).

Table 1. Dietary analysis for CON and LCD. CHO (carbohydrate), PR (protein), g (grams),Tot FAT (total fat), sat (saturated fat), mono (monounsaturated fat), poly (polyunsaturated fat). SE are presented in brackets-

CON LCD

kcal

Yo CHO

Yo FAT

?4o PR

g CHO

g PR

g Tot FAT

g sat

g mono

9 PO~Y

Figure 5. Blood-[giucose] and plasma-bulin] over time during OGTT. During OGTT, blood-[glucose] decreased more slowly and plasma-[insulin] reached a higher peak following LCD. "Significant ciifference within a given time point (LCD vs. CON), pcO.05.

Tlme (min)

+CON

-Ci- LCD

+CON

-iï- LCD

-30 O 30 60 90 120 150 180

T h e (mM)

Figure 6. Plasma FFA, and blood B-OH, glycerol and lactate during OGTT. " Significant difference within a given time point (LCD vs. CON) (pc0.05). Significant difference vs. basal concentrations (-30 and O-min) in both CON and LCD (pc0-05)

-

b b b b

Fiare 6. Plasma FFA, and blood B-OH, glycerol and lactate during OGTT. a Significant difference within a given time point (LCD vs. CON) (p<0.05). Significant difference vs. basai concentrations (-30 and O-min) in both CON and LCD @<O.OS)

+CON

4 LCD

-30 O 30 60 90 120 150 180

lime (min)

-o- CON

-a- LCD

-30 O 30 60 90 120 150 180

Tirn e (m in)

Muscle b io~sv results

GLUT-4 protein. Total GLUT-4 protein was unaltered by LCD (Figure 7). The

intensity of a fixed area for GLUT-4 was normalized to CON. GLUT-4 protein was 1.13

+ 0.06 (LCD) vs. 1 (CON). -

Enzyme activities

Hexokinase. Maximal HK activity was not altered by LCD (CON; 0.42 + 0.04 vs. LCD;

0.36 + 0.04 mol-kg%fL). There was also no change in maximal HK dunng OGTT in

either condition, CON; 0.43 + 0.05 and LCD; 0.40 + 0.05 motkg-'*hfl).

Glycogen synthase. At rest, GS activity (in the presence of 0.1 mM G-6-P) was sirnilar

in CON vs. LCD (1.66 2 0.36 vs. 1.43 + 0.18 nm~l*rnin'~m~-~). OGTï significantly

increased the active GS to 3.04 + 0.46 and to 2.69 + 0.53 nrnol.min-'mg-' in the CON and

LCD condition, respectively. Total GS activity (in the presence of 10.0rnM G-6-P) was

unchanged throughout the experiment. Therefore the GS fractional velocity (GSf,,)

increased significantly in response to insulin in CON and LCD (Figure 8).

It shouId be noted that both HK and GS activities are reported here based on

normalization to muscie protein ievels. However, HK and GS were aIso corrected to total

creatine concentration in the samples and compared with the protein-normalized data,

since it is standard practice to correct to total creatine in some laboratones. The creatine

correction did not alter the outcome of the HK or GS results. Therefore the data is

reported based on protein norrnalization.

PDK. The rnitochondrial recovery for this study was 18.9 + 4.4% of the total

mitochondria, and the quality of the extraction (percentage of intact mitochondria) was

87 t 4%. Resting PDK activity was significandy increased by LCD (0.19 0.05 vs. 0.08

+ 0.02 min-') (pc0.05) (Figure 9). -

PDHa, Resting PDHa activity was significantly lower after LCD vs. CON (0.38 + 0.08

vs. 0.79 + 0.10 mm01 acetyl-CoA/kg/min). During OGïT, PDHa increased significantly

in both conditions, but remained lower after LCD (0.60 f: 0.1 1 vs. 1-04 + 0.09 mm01

acetyl-CoA/kg/min) (Figure 9).

Muscle fuels and metabolites

Muscle glycogen

Mean muscle glycogen, measured in the resting biopsy sarnple was not different

between triais (CON; 346.1 4 22.3 and LCD; 320.0 + 14.7 mmolkg dm).

Muscle metabolites

Resting muscle ATP and G-1-P were lower following LCD vs. CON (Table 2).

Muscle acetyl-CoA and acetyl-carnitine decreased significantly dunng OGTT in both

conditions.

Fimire 7. Western blot of total GLUT-4 protein for CON and LCD. Representative western blot of GLUT-4 protein for CON and LCD in three subjects (1 to 3). The band representing GLUT-4 protein is indicated by the black arrows. Total GLUT-4 protein was not significantly different in LCD vs. CON.

CON 1 LCD I CON 2 LCD 2 CON 3 LCD 3

Fimire 8. Basal (O-min) and insulin-shulated (OGTT, 75-min) glycogen synthase fractional velocity (GSfv). GSfv represents active (0.1 mM)/total (10.0 mM) GS activity . *Significantly different from O-min (pc0.05).

CON O C O N 7 5 LCDO L C D 7 5

Fimre 9. PDK and PDHa activities. PDK (top panel) was measured in intact mitochondria at rest. PDHa (bottom panel) was measured at rest (O-min) and during OGTI' (75-min). The delta increase in PDHa fiom O to 75-min was not different between trials. 'Significanly dflerent from CON, bsignificantly different fiom O-min (p<0.05)

CON O-min LCD O-min

O 7 5 O 7 5 CON LCD

TabIe 2. Muscle metabolites. Metabolites are reported as mmolkg dm and *pmoVkg dm. SE is reported in brackets. aSignificantly different from CON, b~ign5cantly different from O-min, (p<O.OS).

CON O CON 75 LCD O LCD 75

ATP

PCr

G-6-P

G-1-P

AC-COA*

Ac-carn

Lactate

Pyruvate

Glucose

DISCUSSION

An important finding of this study was that a 56-hr LCD (5 + 0.2 % CHO, 73 2

0.6 % fat, 22 + 0.5 % protein) was an effective mode1 to reduce whole-body insulin

sensitivity in normal human subjects. In the muscle, the activity of resting PDK was

increased following LCD and this resulted in a decreased PDHa at rest and following the

administration of oral glucose. GS activity was unaltered by LCD. These resuIts suggest

that decreased skeletal muscle glucose disposal after a LCD is related to decreases in

oxidative CHO disposal and is not related to decreases in glycogen storage. Although the

total GLUT-4 protein did not change following LCD, the concentrations of glycolytic

intermediates in the muscle indicated that the uptake of the oral glucose load into the

muscle was decreased. GLUT-4 translocation may have been downregulated secondary

to LCD.

Insulin sensitivity

The AUC for blood-[glucose] and plasma-[insulin] during OGïT increased

folIowing LCD. Despite higher circulating plasma insulin, the subjects were unable to

dispose of an oral glucose load as efficiently after LCD, resulting in higher blood glucose

levels during OGTT. In addition, the calculated insulin sensitivity index (ISIgly) (5) was

lower after the diet.

The majority of an oral glucose load (71%) is taken up by skeletai muscle, as

evidenced by the direct measurement of k g glucose uptake using fernoral venous

cathetenzation (42). This predicts that the majority of the decrease in insulin sensitivity

following LCD would be accounted for by decreased insulin-stirnulated skeletal muscle

glucose disposal.

It is also important to bnefly consider the involvement of the liver in deterrnining

the blood glucose level during O G T . In normal subjects afier an overnight fast? hepatic

glucose uptake accounted for 2 5 8 of the disposal of an oral glucose load (42) and hepatic

glucose output was suppressed by 50% (22). Therefore, in addition to a decrease in

muscle glucose uptake, factors that may contribute to an elevated AUC for blood-

[glucose] following LCD, are a decrease in hepatic glucose uptake or an increase in

hepatic glucose output. The suppression of hepatic glucose output during OGïT was

impaired only in severe NIDDM subjects vs. mild NIDDM and control subjects, and was

associated with a deficient insulin response (23). In our normal subjects, it is unlikely

that the suppression of hepatic glucose output during OGïT would have been impaired

after LCD. Secondly, we would speculate that hepatic glucose uptake would have been

higher in the LCD condition vs. CON in Our study, since the liver glycogen stores would

have been depleted to a greater extent after fasting and LCD vs. an overnight fast in CON

(37). Overall, the net contribution of the b e r to blood glucose levels rnay have actually

been less in the present study after LCD and masked the severity of the decreased insulin

sensitivity. Therefore, the majority of the elevation in blood-[glucose] after LCD would

have been due to reduced insulin-stimulated disposal of oral glucose by the muscle.

Possible mediators of insulin insensitivitv

Elevated FFA induce a state of insulin insensitivity as evidenced by euglycemic

hypennsulinemic clamp studies that infuse intralipid during hyperinsulinemia (8,9,44,

53). In studies that use starvation or high fatllow CHO feeding as a model to study

metabolic adaptation, FFA and ketones are elevated and there is a shift towards reiiance

on these metabolic fiels. In addition, circulating insulin levels (or insulin sensitivity)

may be decreased in the latter models, and this could be an important signal involved in

altenng substrate utilization. In Our study, LCD resulted in elevated resting FFA and

ketone levels, and lower resting plasma insulin, as expected. A previous study (71)

demonstrated that circulating insulin levels in the fed state were also suppressed by LCD

(7.0 2 1.7 vs. 21 .O + 6.7 plU/d). Therefore, possible mediators of reduced insulin-

stimulated CHO utilization in the LCD condition include decreased circulating insulin,

and increased FFA and ketone levels.

In the present study we were unable to assess the actual mechanisms by which the

mediators of insulin insensitivity may exert their effects. The main purpose of the study

was to examine the potential sites of adaptation in skeletal muscle that may contribute to

decreased insulin-stimulated glucose disposal following LCD. The four major sites

investigated were glucose transport (GLUT-4), glucose phosphorylation (HK), glucose

storage (GS), and glucose oxidation (PDK and PDHa). These sites will be discussed in

the following section.

Sites of skeletal muscle adaptation to LCD

Glucose transport and phosphorylation

Total GLUT-4 protein levels were not altered by 56-hrs of LCD. However, it is

possible that insulin-stimulated GLUT-4 translocation rnay have been dowmegulated

following LCD and contnbuted to a reduced glucose uptake into skeletal muscle. In most

rodent studies, long-term high fat feeding (3 to 8-wks) has resulted in decreased insulin-

shu la ted glucose transport without changing the total protein content of GLUT-4 (3 1,

32,45, 1 12), suggesting a decrease in GLUT-4 translocation. Due to the arnount of

muscle required to measure GLUT-4 translocation in humans, we were unable to directly

investigate the effects of LCD on this mechanism. However, total GLUT-4 protein levels

had not been measured foilowing a short-term diet mode1 in human subjects pnor to this

study. Since GLUT-4 protein increases within 5-days of endurance training (79, it was

possible that the protein levels may have adapted over the timecourse of LCD (2.33-

days) .

Total HK activity was not different after LCD. Studies in rodents have shown

that HKII activity was unaltered after a long-term high fat diet (3 to 4-weeks) (45, 113).

Total HK activity had not been measured previously in humans following a short-terrn

LCD. This was an important site that rnay have altered glucose disposal over the short-

tenn, based on the capacity of HKII expression to adapt rapidly to insulin levels (56,77).

In addition to direct measurements of glucose transport and phosphorylation, G-6-

P is often used as an indicator of the activity at these sites. This and other glycolytic

intermediates, including G-1-P, lactate and pyruvate, are indicative of the relationship

between the uptake of glucose into the muscle (coupled with phosphorylation) and the

utilization of glucose for storage and oxidation. In the case where the uptake of glucose

into the muscle is in excess of the utilization, these intermediates would accumulate. The

decrease in PDHa suggested that the oxidative glucose disposal was reduced following

LCD. Since none of the glycolytic intermediates accumulated in the muscle during

OGTT in the LCD triai (vs. CON), this suggests that the transport of glucose into the

muscle was decreased accordingly. In this situation it seems that HK worked in concert

with GLUT-4 to phosphorylate the incoming glucose, since intracellular glucose did not

accumulate in the muscle during the OGTï in the LCD condition. In a situation where

glucose uptake is chronically decreased HK rnay eventually adapt at the level of protein

expression to alter its total activity, as seen in NIDDM (103).

Glucose storage

LCD did not change the basal activity of GS. During OGTT, GS activity was

increased sirnilarly at 75-min in both conditions to contribute to the disposal of the oral

glucose load by storing some of the glucose as glycogen in the muscle. The fractional

velocity of GS, GSfv, is used as an indicator of the arnount of GS in the active

(dephosphorylated) form, and was increased in response to insulin during OGTT (75-

min) in both trials, as expected. Our values for basal and insulin stirnulated GS activity

and GSfv were in accordance with the literature from human skeletal muscle (56). It is

important to consider that the results of this study with respect to GS activity during

OGTT are only for one point in time and may not reflect GS activity over the duration of

the 90-min glucose disposa1 penod.

Euglycemic hyperinsulinemic clamp studies have demonstrated that in an induced

state of insulin resistance secondary to elevated FFA, glucose disposal is decreased after

3 to Chrs accompanied by a decrease in insulin-stimulated GS activity (8, 10,44). In

these studies, blood glucose and high insulin levels are constantly maintained while FFA

levels are artificially elevated over a penod of hours. It would be expected that glycogen

levels should be increasing in the muscle due to the inhibition of glucose oxidation by

FFA and the stimulation of GS activity by insulin, and that this could start to inhibit GS

activity. In contrast, in the current study, under physiological conditions of

hyperglycemia and elevated insulin for a shorter duration, it is unlikely that glycogen

accumulated sufficiently in the muscle to inhibit GS activity. The starting muscle

glycogen concentration was equivalent in CON vs. LCD in the present study. Based on

calculations for an average 75 kg subject, if ail of the oral glucose load was stored as

glycogen in the muscle (assuming 40% of rnass as muscle), there was the potential for a

59.7 rnrnol glucosyl units/kg dm increase that could occur in the muscle. Assuming that

only -80 % of the glucose was absorbed and -70 % of the absorbed glucose was taken up

into the muscle, less than 35.8 rnmol/kg dm would have accumulated in the muscle at 75-

min of OGïT. In addition, a portion of the oral glucose load is oxidized within the

muscle. Based on these calculations, it was not surprising that 75-min after the

administration of oral glucose, insulin-stimulated GS activity was not inhibited following

LCD.

Insulin-stimulated GS activity is decreased in chronic States of insulin resistance

(MDDM). A recent study in rodents (45) demonstrated decreased GS activity and a

decreased accumulation of muscle glycogen during a 2-hr euglycemic hypennsulinemic

clamp following a 3-wk high fat diet. This group aIso found that a decrease in insulin-

stimulated GS activity did not occur after 2-wks of a high fat diet, suggesting that it is not

an initial adaptation in the development of insulin resistance (46). Measurements of GS

activity in human skeletal muscle following a short-term LCD are lacking. Our results

indicate that insulin-stimulated GS activity is not inhibited during OGTT (75-min)

following 56-hrs of LCD. However, 4 of the 6 subjects had decreased GS activity during

OGTï after LCD vs. CON (subject I to 4, see appendix). The mean decrease in insulin-

stimulated GS foIIowing LCD was not statistically signifîcant. Therefore, the ability of

insulin to stimulate GS activity may be dowmegulated in some subjects by 56-hrs of

LCD. Longer dietary alteration may lead to an adaptation in the ability of insulin to

stimulate GS, as seen in rodents (45). Also, more frequent biopsy sampling during

OGTT would provide insight into the insulin-stimulated disposal of a glucose load by GS

following dietary alteration.

Glucose oxidation

An important finding in this study was the rapid and stable increase in resting

PDK activity following a 56-hr LCD. The PDK-mediated inhibition of PDHa may be

one mechanism involved in the decreased disposal of an oral glucose load following

LCD. We have previously measured a dramatic increase in PDK activity after 3-days of

LCD (7 l), and a pilot study with two subjects indicated that the increase rnay be

O C C U ~ ~ ~ as early as Zdays into the diet. Our current results confirm that PDK increases

rapidly, within 56-hrs of LCD.

Accompanying the increase in resting PDK activity was a corresponding decrease

in resting PDHa. A decrease in PDHa was measured in our previous study after 6-days

of LCD (7 L), however PDHa activity was not measured after 3-days when PDK was

found to be increased. When the oral glucose load was adrninistered, PDHa activity

increased in both CON and LCD, but remained lower in LCD. Assurning that PDHa is

representative of PDHa flux and that the 75-min biopsy sample is representative of the

entire 90-min period, the decreased PDHa would have contributed to the overall decrease

in the disposal of glucose folIowing LCD.

The increase in resting PDK after LCD was a stable increase that persisted

through a rigorous mitochondrial extraction. At rest (O-min) there were no major changes

in the known acute regulators of PDK activity, indicating that the increase in resting PDK

was not a function of acute regulation. As discussed earlier, a stable increase in PDK

activity may involve the altered expression of PDK isoforms following LCD. Recently,

it was shown that increased PDK activity during LCD was accompanied by an increase in

PDK4 protein and mRNA in human skeletal muscle, while PDK2 protein was unchanged

by the diet (70).

During OGTT, PDHa increased in both conditions. Acetyl-CoA decreased

sirnilarly in both conditions while pyxuvate and ATP were unchanged. We would also

expect that [ C a r would have been the same in CON and LCD. The decrease in acetyl-

CoA may have been involved in the increase in PDHa during OGTT by decreasing PDK

activity acutely. However, the decrease in acetyl-CoA observed in the present study mziy

not have been physiologically significant. A previous study (62) in Our lab measured an

increase in PDHa without a change in acetyl-CoA dunng a standard OGTT . PDHa

activation dunng OGïT in the present study may have been independent of acute

mediation of PDK. Increased insulin levels during OGTï may have directly increased

PDH phosphatase activity, as suggested in rat studies (21).

The downregulation of PDHa by PDK was shown to be an important site involved

in the initial adaptation of skeletal muscle to LCD. It seems that this is the major

mechanism that changes in concert with glucose uptake to decrease the ability of the

muscle to dispose of an oral glucose load at this stage of adaptation. For this reason, we

will bnefly discuss the possible regulators involved in the stable adaptation of PDK to

LCD. These mainly include an increase in FFA and a decrease in circulating insulin (or a

decrease in insulin sensitivity) secondary to the diet.

With prolonged reliance on fat metabolism in starvation, diabetes and high fat

feeding, PDK activity has been shown to increase in a stable adaptive manner in order to

downregulate CHO oxidation. In rat heart and skeletal muscle, the temporal increase in

circulating FFA in these models correlates with an increase in PDK activity and

subsequent decrease in PDHa (36,69). In cell culture experiments, 24-hr culture with n-

octanoate , dibutyryl-CAMP, or a combination of these mediators resulted in an increase

in PDK sirnilar to that seen in starved rats (65,90). However, the stable upregulation of

PDK with high-fat feeding is mediated through a CAMP-independent mechanism (96).

The effect of F'FA to increase PDK in high-fat-fed rats has been shown to be dependent

on the type of fat ingested. Only 24-hrs of an n-3 poIyunsaturated diet reversed the

increase in PDK activity due to the standard 28-day high-saturated fat diet typically used

in the rat studies (25). The exact mechanism by which FFA alter the activity of PDK is

not known. Recent evidence suggests that peroxisornal proliferating activating receptors

(PPARs) may be a possible link between FFA and metabolic regulation. PPARs are

nuclear steroid-like receptors that act as trans-activators of many genes involved in lipid

metabolism. Exposure to a PPAR-a agonist (3-days) increased skeletal muscle PDK

activity and PDK4 mRNA and protein to levels observed in diabetes or 48-hr starved

animals, suggesting that PPARs may play a roie in downregulating CHO metabolism

(106).

In starvation and diabetes, the decrease in circulating insulin concentrations (or in

insulin sensitivity) correlates with the stable increase in PDK in rat skeletal muscle (65).

The effects of decreased insulin may be due to an indirect effect of elevating circulating

FFA due to a decrease in insulin-mediated suppression of lipolysis. However, in ceil

culture studies, insulin directly opposes the effects of FFA and CAMP to increase PDK

activity (65,97). It has been suggested that a decfine in insufin Ievels (or a decrease in

insulin sensitivity) may be obligatory for a stable increase in PDK. In alloxan diabetic

rats that have a stable increase in PDK, the long-term administration of insulin is required

to decrease PDK to control values (21). More recently, a study in Pima Indians showed

that the expression of PDK isoforms was correlated with the severity of a subject's

insulin resistance (53). These authors demonstrated that insulin had a direct effect on the

expression of PDK2 and 4, and suggest that in insulin-resistant individuals, there may be

insuffkient insulin-mediated downregulation of PDK. This would mean that decreased

insulin responsiveness may be a cause and not a consequence of increased fat utilization.

OGTT and acetyi-carnitine

Acetyl-carnitine decreased during OGTT in both trials. The reason for this

decrease is not known. We have previously observed a similar decrease in acetyl-

carnitine during a standard OGTT (62). One possibility is that there was insufficient

pyruvate to match the activation of PDHy even &ter LCD when PDHa was decreased. In

this case, the muscle may draw on the acetyl-camitine stores as a source of acetyl-CoA,

since the contribution of fat to the acetyl-CoA pool is minimal during OGTT due to the

insulin-mediated suppression of lipolysis. If pyruvate was insufficient, a decrease in

pyruvate would have been expected during OGTT, and this did not occur. However, G-

6-P does tend to decrease at 75-min in both conditions, suggesting that GS may compete

successfully for G-6-P, leaving less available pyruvate than could be handled by PDHa.

Conclusion

The initial adaptation of human skeletal muscle to the short-term (56-hr) dietary

elevation of fat and resmction of CHO involves a stable upregulation of PDK activity and

a subsequent decrease in PDHa. This data strongly suggests that the ability of the muscle

to dispose of an oral glucose oxidatively is decreased following LCD. If CHO flux

through PDH is in fact reduced by LCD, and insilin-stirnulated GS activity is unaltered

in response to an oral glucose load, the decrease in PDHa would necessitate a decrease in

glucose uptake. This is in accordance with the observed decreased in glucose disposa1

over 90-min of OGTï in the current study, and the lack of accumulation of glycolytic

intermediates at 75-min. Studies measuring GLUT-4 translocation directiy in humans are

limited due to the current techniques available.

Future directions

Since this was the first study that attempted to obtain a comprehensive picture of

skeletal muscle adaptation to a short-term LCD in humans by directly measuring muscle

proteins, enzyme activities and metabolites, it was important to maximize the information

that we could obtain from a lirnited quantity of muscle. We have now been able to

identify sites of adaptation thar require further study to determine the mechanisms

underlying decreased insulin-stimulated glucose disposal by the muscle following LCD.

Future studies should directly address the insuiin-stimulated translocation of

GLUT-4 following a short-term LCD. A longer-term (weeks) dietary stimulus should be

used to investigate adaptations in GLUT-4 protein levels, and in HK (activity, mRNA

and protein) and GS activity.

Measurements of CS and PDHa activity by repeated biopsy sarnpling over time

during insulin-stimulated glucose disposal should be performed in order to obtain more

conclusive evidence for the altered fate of glucose inside the muscle after LCD.

Further work is required to determine the mechanisms by which FFA and /or

insulin may be involved in inducing skeletai muscle insulin resistance. Measurements of

intermediates in the insulin-signaling pathway would be an important contribution to this

work. In addition, it will be important to study dBerent dietary fat compositions and the

possible differential effects on PPARs.

References

1. Adamo, K. B., and T. E. Graham. Cornparison of traditional measurements with

rnacroglycogen and proglycogen analysis of muscle glycogen. J Appl Physiol84: 908-13,

1998.

2. Anderson, J. W., and R. H. Herrnan. Effects of carbohydrate restriction on

glucose tolerance of normai men and reactive hypoglycemic patients. Am J Clin Nutr 28:

748-55, 1975.

3. Anderson, J. W., R. H. Heman, and D. Zakim. Effect of high glucose and high

sucrose diets on glucose tolerance of noma1 men. Am J Clin Nutr 26: 600-7, 1973.

4. Ardehali, H., Y. Yano, R. L. Printz, S. Koch, R. R. Whitesell, J. M. May, and D.

K. Granner. Functional organization of marnrnalian hexokinase II retention of catalytic

and regulatory functions in both the NH2- and COOH- terminal halves. J Bi01 Chem 271:

1849-52, 1996.

5. Belfiore, F., S. Iannello, and G. Volpicelli. Insulin sensitivity indices calculated

from basal and OGTT-induced insulin, glucose, and FFA levels. Mol Gen and Metab 63:

134-41, 1998.

6. Bergmeyer, H. U. Mehods of Enzyrnatic Analysis. New York: Academic, 1974.

7. Bergstrom, J. Percutaneous needle biopsy of skeletal muscle in physiological and

clinical research. Scand J Clin Lab Invest 35: 609-16, 1975.

8. Boden, G., X. Chen, J. Ruiz, J. V. White, and L. Rossetti. Mechanisms of free

fatty acid -induced inhibition of glucose uptake. J Clin Invest 93: 2438-46, 1994.

9. Boden, G., F. Jadali, J. White, Y. Liang, M. Mozzoli, X. Chen, E. Coleman, and

C. Smith. Effects of fat on insulin-stimulated carbohydrate metabolism in normal men. J

Clin Invest 88: 960-66, 1991.

10. Bonadonna, R. C., K. Zych, C. Boni, E. Ferrannini, and R. A. DeFronzo. T h e

dependence of the interaction between iipid and glucose in humans. Am J Physid 257:

E49-56, 1989.

I l . Bowker-Kinley, M. M., W. 1. Davis, P. Wu, R. A. Harris, and K. M. Popov.

Evidence for existence of tissue-specific regulation of the rnammalian pyruvate

dehydrogenase complex. Biochem J 329: 19 1-6, 1998.

12. Cederblad, G., J. 1- Carlin, D. Constantin-Teodosiu, P. Harper, and E. Hultman.

Radioisotopic assays of CoASH and carnitine and their acetylated forrns in human

skeletd muscle. Anal Biochem 185: 274-78, 1990.

13. Chaildey, S. M., M. Hettiarachchi, D. J. Chisholrn, and E. W. Kraegen. Five-hour

fatty acid elevation increases muscle lipids and impairs glycogen synthesis in the rat.

Merabolism 47: 1 121-26, 1998.

14. Cohen, P. Muscle glycogen synthase. In: The Enzymes, 3rd edirian, edited by P.

D. B. a. E. G. Krebs. Orlando. FL: Acadernic Press, 1986.

15. Constantin-Teodosiu, D., G. Cederblad, and E. Hultman. A sensitive radioisotopic

assay of pyruvate dehydrogenase complex in human muscle tissue. Anal Biochern 198:

347-5 1, 199 1.

16. Cooper, R. H., P. J. RandIe, and R. M. Denton. Stimulation of phosphorylation

and inactivation of pyruvate dehydrogenase by physiological inhibitors of the pyruvate

dehydrogenase reaction. Nature 257: 808-9, 1975.

17. Cutler, D. L., C . G. Gray, S. W. Park, M. G. Hickrnan, J. M. Beil, and O* G.

Kolterman. Low-carbohydrate diet alters intraceLlular glucose metabolism but not overall

glucose disposal in exercise-trained subjects. Metabolism 44: 1264-70, 1995.

18. DeFronzo, R. A., E. Jacot, E. Jequier, E. Maedar, J. Wahren, and J. P. Felber. The

effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry

and hepatic and femoral venous catheterization. Diabetes 30: 1000-7, 1981.

19. Dent, P., A. Lavoinne, S. Nakielny, F. B. Caudweli, P. Watt, and P. Cohen. The

molecular rnechanism by which insuLin stimulates glycogen synthesis in marnmalian

skeletal muscle. Nature 348: 302-8, 1990.

20. Fatania, H. R., T. C . Vary, and P. J. Randle. Modulation of pyruvate

dehydrogenase kinase activity in cultured hepatocytes by glucagon and n-octanoate.

Biochem J 234: 233-6, 1986.

21. Feldhoff, P. W., J. Arnold, B. Oesterling, and T. C. Vary. Insulin-induced

activation of pyruvate dehydrogenase complex in skeletal muscle of diabetic rats.

Metabolism 42: 6 15-23, 1993.

22. Ferrannini, E., O. Bjorkman, G. A. Reichard, A. Pilo, M. Olsson, J. Wahren, and

R. A. DeFronzo. The disposal of an oral glucose load in healthy subjects. A quantitative

sîudy. Diabetes 34: 580-8, 1985.

23. Fery, F., C. Melot, and E. O. Balasse. Glucose fluxes and oxidation after an oral

glucose load in patients with nm-insulin-dependent diabetes mellitus of variable severity.

Metabolism 42: 522-30, 1993.

24. Flatt, J.-P. Use and storage of carbohydrate and fat. Am J Clin Nrrtr 61: 952s-

959S, 1995.

25. Fryer, L. G., K. A. OrfaIi, M. J. Holness, E. D. Saggerson, and M. C. Sugden. The

long-term regulation of skeletal muscle pymvate dehydrogenase kinase by dietary lipid is

dependent on fatty acid composition. Eur J Biochem 229: 741-8, 1995.

26. Gomez, E, E. Jequier, V. Chabot, V. Buber, and 5. P. Felber. Carbohydrate and

lipid oxidation in normal human subjects: its infiuence on glucose tolerance and insulin

response to glucose. Metabolism 2 1 : 38 1-9 1, 1972.

27. Gordon, R. S., and A. Cherkes. Unesterified fatty acid in human blood plasma. J

Clin Invest 35: 206-12, 1955.

28. Grossbard, L., and R. T. Sctiirnke. Multiple hexokinases of rat tissues.

Purification and cornparison of soluble forms. J Bi01 Chem 241: 3546-60, 1966.

29. Gudi, R., M. M. Bowker-Kinley, N. Y. Kedishvili, Y. Zhao, and K. M. Popov.

Diversity of the pymvate dehydrogenase kinase gene family in humans. J Bi01 Chem 270:

28989-94, 1995.

30. Hales, C. N., and P. J. Randle. Effects of Iow-carbohydrate diet and diabetes

mellitus on plasma concentrations of glucose, non-esterified fatty acid, and insuiin dunng

oral glucose-tolerance tests. Lancet i: 790-4, 1963.

3 1. Han, D. H., P. A. Hansen, H. H. Host, and J. O. Holloszy. Insulin resistance of

muscle glucose transport in rats fed a high-fat diet: a reevaluation. Diabetes 46: 1761-67,

1997.

32. Hansen, P. A., D. H. Han, B. A. Marshall, L. A. Nolte, M. M. Chen, M. Mueckler,

and J. O. Holloszy. A high fat diet impairs stimulation of glucose transport in muscle:

functional evaluation of potential mechanisms. J Bi01 Chem 273: 26157-163, 1998.

33. Harris, R. C., E. Hultrnan, and L. O. Nordesjo. Glycogen, glycolytic intermediates

and high-energy phosphates deterrriined in biopsy samples of musculus quadriceps

femoris of man at rest: rnethods and variance of values. Scand J Clin LAzb Invest 33: 109-

20, 1974.

34. Hayaschi, T., J. F. P. Wojtaszewski, and L. J. Goodyear. Exercise regdation of

glucose transport in skeletal muscle. Am J Physiol273: E1309-51, 1997.

35. Henriksson, J., M. M. Y. Chi, C. S. Hintz, D. A. Young, K. K. Kaiser, S.

Salmons, and 0. H. Lowry. Chronic stimuIation of mammalian muscle: changes in

enzymes of six metabolic pathways. Am J Physiol25 1: C614-32, 1986.

36. Holness, M. J., and M. C. Sugden. Pyruvate dehydrogenase activities during the

fed-to-starved transition and on re-feeding after acute or prolonged starvation. Bioclzem J

258: 529-33, 1989.

37. Hultrnan, E. Studies on muscle metabolism of glycogen and active phosphate in

man with special reference to exercise and diet. Scand J Clin Lab Invest 94: 1-63, 1967.

38. Jackrnan, M. R., and W. T. Willis. Characteristics of mitochondria isolated from

type 1 and type IIb skeletai muscle. Am J Physiol270: C673-8, 1996.

39. Jansson, E., P. Hjemdahl, and L. Kaijser. Diet induced changes in sympatho-

adrenal activity during submaximal exercise in relation to substrate utilization in man.

Acta Pizysiol Scand 1 14: 171-8, 1982.

40. Johnson, A. B., M. Argyraki, J. C. Thow, 1. R. Jones, D. Broughton, M. Miller,

and R. Taylor. Impaired activation of skeletal muscle glycogen synthase in non-insulin-

dependent diabetes mellitus is unrelated to the degree of obesity. Metabolism 40: 252-60,

1991.

41. Kahn, C. R. Insulin resistance: a common feature of diabetes mellitus [editorial].

N Engl J Med 3 15: 252-4, 1986.

42. Katz, L. D., M. G. Glickman, S. Rapoport, E. Ferramini, and R. A. DeFronzo.

Splanchnic and peripheral disposal of oral glucose in man. Diaberes 32: 675-9, 1983.

43. KeUey, D. E., M. A. Mintun, S. C. Walkins, J. A. Simoneau, F. Jadali, A.

Fredenckson, J. Beattie, and R. Theriault. The effect of non-insuiin-dependent diabetes

mellitus and obesity on glucose transport and phosphorylation in skeletal muscle. J Clin

Invest 97: 2705-13, 1996.

44. Kelley, D. E., M. Mokan, J.-A. Simoneau, and L. J. Mandarino. Interaction

between glucose and free fatty acid metabolism in human skeletal muscle. J Clin Invest

92: 91-8, 1993.

45. Kim, C.-H., J. H. Youn, J.-Y. Park, S. K. Hong, K. S. Park, S. W. Park, K. 1. Suh,

and K.-U. Lee. Effects of high-fat diet and exercise training on intracellular glucose

metabolism in rats. Am J Physiol278: E977-84,2000.

46. Kim, J. K., J. K. Wi, and J. H. Youn. Metabolic impairment precedes insulin

resistance in skeletal muscle dunng high-fat feeding in rats. Diabetes 45: 651-8, 1996.

47. Kim, J. K., J. K. Wi, and J. H. Youn. Plasma free fatty acids decrease insulin-

stimulated skeletai muscle glucose uptake by suppressing glycolysis in conscious rats.

Diabetes 45: 446-53, 1996.

48. Kim, Y., T. Tamura, S. Iwashita, K. Tokuyama, and M. Suzuki. Effect of high-fat

diet on gene expression of GLUT4 and insulin receptor in soleus muscle. Biochem

Biophys Res Commun 202: 5 19-26, 1994.

49. Kochan, R, G., D. R. Lamb, S. A. Lutz, C. V. Perrill, E. M. Reimann, and K. K.

Schlender. GIycogen synthase activation in human skeletai muscle: effects of diet and

exercise. Am J Physiol236: E660-6, 1979.

50. Koval, J. A., R. A. DeFronzo, R. M. O'Doherty, R. Pnntz, H. Ardehdi, D. K.

Granner, and L. J. Mandarino. Regdation of hexokinase II activity and expression in

human muscle by moderate exercise. Am J Physiol274: E3û4-8, 1998.

51. Kraegen, E. W., P. W. Clark, A. B. Jenkins, and D. E. James. Development of

muscle insulin resistance after liver insulin resistance in high-fat-fed rats. Diabetes 40:

1397-1403, 1991.

52. Magnan, C., M. Gilbert, and B. B. Kahn. Chronic free fatty acid infusion in rats

results in insulin resistance but no alteration in insulin-responsive gIucose tmnsporter

levels in skeletal muscle. Lipids 3 1: 1 141-49, 1996.

53. Majer, M., K. M. Popov, R. A. Harris, C . Bogardus, and M. Prochazka. Insulin

downregulates pyruvate dehydrogenase kinase (PDK) mRNA: PotentiaI mechanism

contributing to increased lipid oxidation in insulin-resistant subjects. Mol Gen and

Metab 65: 18 1-6, 1998.

54. Makinen, M. W., and C. P. Lee. Biochernicd studies of skeletal muscle

mitochondria. 1. Microanalysis of cytochrome content, oxidative and phosphorylative

activities of rnammalian skeletd muscle rnitochondria. Arch Biochem Biophjrr 126: 75-

82, 1968.

55. Mandarino, L. J., A. Consoli, D. E. Kelley, J. P. Reilly, and N. Nu rjham. Fasting

hyperglycemia normdizes oxidative and nonoxidative pathways of insulin-stimulated

glucose metabolism in noninsulin-dependent diabetes meilitus. J Clin Endocrinol Metab

71: 1544-51, 1990.

56. Mandarino, L. J., R. L. Pnntz, K. A. Cusi, P. Kinchington, R. M- O'Doherty, H.

Osawa, C. Sewell, A. Consoli, D. K. Granner, and R. A. DeFronzo. Regulation of

hexokinase II and glycogen synthase mlRNA, protein, and activity in human muscle. Am J

Physid 269: E7O 1-8, 1995.

57. Mandarino, L. J., K. S. Wright, L. S; Verity, J. Nichols, J. Beil, O. G. Kolterman,

and H. Beck-Nielsoen. Effects of insulin infusion on human skeletal muscle pyruvate

dehydrogenase, phosphohctokinase, and glycogen synthase; Evidence for their role in

oxidative and nonoxidative glucose metabolism. J Clin Invest 80: 655-63, 1987.

58. Megeny, L. A., P. D. Neufer, G. L. Dohm, M. H. Tan, C. A. Blewett, G. C. B.

Elder, and A. Bonen. Effects of muscle activity and fiber composition on glucose

transport and GLUT-4. Am J Physiol264: E583-93, 1993.

59. Munger, R., E. Temier, D. Jallut, E. Haesler, and J.-P. Felber. Correlations of

glycogen synthase and phosphorylase activities with glycogen concentrations in human

muscle biopsies. Evidence for a double-feedback mechanism regulating glycogen

synthesis and breakdown. Metabolisnr 42: 36-43, 1993.

60. Newsholme, E. A., and A. R. Leech. Regulatory mechanisms in glycogen

synthesis. In: Biochernistry for the medical sciences. Great Britain: John Wiley Br Sons,

1985, p. 589-91.

6 1. Nuttal, F. Q., M. C. Gannon, G. Bai, and E. Y. Lee. Primary structure of human

liver glycogen synthase deduced by cDNA cloning. Arch Biochem Biophys 3 11: 443-9,

1994.

62. Odland, M., G. I. F. Heigenhauser, T. L. PehIernan, R. J. Schmidt, and L. L.

Spriet. Skeletal muscle pyruvate dehydrogenase activity and malonyl-CoA content

following glucose ingestion in man. Submitted to Am J Physiol:, 2000.

63. O'Doherty, R. M., D. Bracy, H. Osawa, D. Wasserman, and D. Granner. Rat

skeletal muscle hexokinase II mRNA and activity are increased by a single bout of actue

exercise. Am J Physiol266: E l 7 1-8, 1994.

64. Olson, A. L., and J. E. Pessin. Structure, function, and regulation of the

marnrnalian faciiitative glucose transporter gene farnily . Annu Rev Nutr 16: 235-56, 1996.

65. Orfali, K. A., L. G. D. Fryer, M. J. Holness, and M. C. Sugden. Long-term

regulation of pynivate dehydrogenase kinase by high-fat feeding: experiments in vivo

and in cultured cardiomyocytes. FEBS Lett 336: 501-5, 1993.

66. Park, J.-Y., C.-H. Kim, S. K. Hong, K. 1. Suh, and L U . Lee. Effects of FFA on

insulin-stimulated glucose fluxes and muscle glycogen synthase activity in rats. Am J

Physiol275: E338-44, 1998.

67. Parker, P. J., F. B. Caudwell, and P. Cohen. Glycogen synthase from rabbit

skeletal muscle; effect of insulin on the state of phosphorylation of the seven

phosphoreine residues in vivo. Eur J Biochem 130: 227-34, 1983.

68. Passoneau, J. A., and 0. H. Lowry. Enzyrnatic Analysis: A Pracfical Guide.

Totawa, NJ: Huniana Press, 1993. p. 219-22.

69. Pederson, O., J. F. Bak, P. H. Anderson, S. Lund, O. E. Moller, J. S. Flier, and B.

B. Kahn. Evidence against altered expression of GLUTl or GLUT4 in skeletal muscle of

patients with obesity or NIDDM. Diabetes 39: 865-70, 1990.

70. Peters, S. J. Changes in activity and isoform expression of human skeletal muscle

pymvate dehydrogenase kinase during the first 3 days of a high-fatflow-carbohydrate

diet. Submitted to Am J Physiol:, 2000.

71. Peters, S. J., T. A. S. Amand, R. A. Howlett, G. J. F. Heigenhauser, and L. L.

Spriet. Human skeletal muscle pyruvate dehydrogenase kinase activity increases after a

low-carbohydrate diet. Am J Physiol275: E980-86, 1998.

72. Peters, S. J., R. A. Harris, G. J. F. Heigenhauser, and L. L. Spriet. Muscle fiber

type cornparison of pynivate dehydrogenase kinase activity and isoform expression in fed

and fasted rats- Submitted to Am J Physiol:, 2000.

73. Peters, S. J., and t. L. Spriet. Skeletal muscle phosphofnictokinase activity

examined under physiological conditions in vitro. J Appl Physiol78: 1853-58, 1995.

74. Petit, F. H., J. W. Pelley, and L. J. Reed. Regulation of pyruvate dehydrogenase

kinase and phosphatase by acetyl-CoNCoA and NADEUNAD ratios. Biochem Biophys

Res Commun 65: 575-82, 1975.

75. Phillips, S. M., X. Han, H. J. Green, and A. Bonen. Increments in skeletai muscle

GLUT-1 and GLUT-4 after endurance training in humans. Am J Physiol270: E456-62,

1996.

76. Pitcher, J., C. Smythe, D. G. Campbell, and P. Cohen. Glycogenin is the priming

gIucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletd

muscle. Eur J Biochem 176: 391-5, 1988.

77. Postic, C. A., A. Leturque, F. Rencurel, R. Printz, C. Forest, D. Granner, and J.

Girard. The effect of hyperinsulinernia on GLUT4 and hexokinase II mRNA and protein

in rat skeletal muscle and adipose tissue. Diabetes 42: 922-9, 1993.

78. Priestman, D. A., K. A. Orfali, and M. C. Sugden. Pyruvate inhibition of pyruvate

dehydrogenase kinase, effects of progressive starvation and hyperthyroidism in vivo, and

of dibutyryl cyclic AMP and fatty acids in cultured cardiac rnyocytes. FEBS J 393: 174-

8, 1996.

79. Purich, D. L., H. J. Fromm, and F. B. Rudolph. The hexokinases: Kinetic,

physical and regulatory properties. Adv Enrymol39: 249-326, 1973.

80. Putman, C . T., L. L. Spriet, E. Hultrnan, M. 1. Lindinger, L. C. Lands, R. S.

McKelvie, G. Cederblad, N. L. Jones, and G. J. F. Heigenhauser. Pyruvate

dehydrogenase activity and acetyl group accumulation during exercise after different

diets. Am J Physiol265: E752-60, 1993.

81. Randle, P. J., D. A. Priestman, S. C. Mistry, and A. Halsall. Glucose fatty acid

interactions and the regulation of glucose disposal. J Cell Biochem 55s: 1-1 1, 1994.

82. Rigalleau, V., M. Beylot, C. Pachiaudi, C. Guillot, G. Deleris, and H. Gin.

Mechanisms of glucose intolerance d u h g triglyceride infusion. Am J Physiol275: E641-

8, 1998.

83. Roden, M., T. B. Pnce, G. Perseghin, K. F. Petersen, D. L. Rothman, G. W. Cline,

and G. 1. Shulman. Mechanism of free fatty acid-induced insulin resistance in humans. J

Clin Invest 97: 2859-65, 1996.

84. Rothrnan, D. L., R. G. Shulman, and G. 1. Shulman. 3 1 ~ nuclear magnetic

resonance measurements of muscle glucose-6-phosphate . Evidence for reduced insulin-

dependent muscle glucose transport or phosphorylation activity in non-insulin-dependent

diabetes mellitus. J Clin Invest 89: 1069-75, 1992.

85. Rousselle, J., A. Buckert, P. Pahud, E. Jequier, and J. P. Felber. Relationship

between glucose oxidation and glucose toIerance in man. Metabolism 3 1: 866-70, 1982.

86. Rawles, J., S. W. Scherer, T. Xi, M. Majer, D. C. Nickle, J. M. Rommens, K. M.

Popov, R. A. Harris, N. L. Riebow, J. Xia, L X . Tsui, C . Bogardus, and M. Prochazka.

Cloning and characterization of P D M on 7q21.3 encoding a fourth pyruvate

dehydrogenase kinase isoenzyme in human. J Bi01 Chem 27 1 : 22376-82, 1996.

87. Rylatt, D. B., A. Aitken, T. Bilharn, G. D. Condon, N. Embi, and P. Cohen.

Glycogen synthase from rabbit skeletai muscle. Amino acid sequence at the sites

phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence

containing the site phosphorylated by phosphorylase kinase. Eur J Biochem 107: 529-37,

1980.

88. Sidery, M. B., 1. W. Gaiien, and 1. A. Macdonald. The initial physiological

response to glucose ingestion in normal subjects are modified by a 3 d high-fat diet. Br J

Nutr 64: 705-13, 1990.

89. Srere, P. A. Citrate synthase. In: Methods of En~ymology, edited by I. M.

Lowenstein. New York: Academic, 1969, p. 3-5.

90. Stace, P. B., H. R. Fatania, A. Jackson, A. L. Kerbey, and P. I. Randle. Cyclic

AMP and free fatty acids in the longer-term regulation of pyrvate dehydrogenase kinase

in rat soleus muscle. Biochem Biophys Acta 1 135: 20 1-6, 1992.

91. Storlien, L. H., D. E. James, K. M. Burleigh, D. J. Chisholm, and E. W. Kraegen.

Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure,

and obesity in rats. Am J Physiol251: E576-83, 1986.

92. Storlien, L. H., A. B. Jenkins, D. J. Chisholm, W. S . Pascoe, S. Khouri, and E. W.

Kraegen. Muence of dietary fat composition on developrnent of insufin resistance in

rats: relationship to muscle triglyceride and w-3 faty acids in musde phospholipid.

Diabetes 40: 280-9, 1991.

93. Sugden, M. C., L. G. D. Fryer, K. A. Orfali, D. A. Priestman, E. Donald, and M.

J. Holness. Studies of the long-term regulation of hepatic pyruvate dehydrogenase kinase.

Biochem J 329: 89-94, 1998.

94. Sugden, M. C., and M. J. Holness. Interactive regulation of the pyruvate

dehydrogenase complex and the carnitine palmitoyltransferase system. FASEB J 8: 54-

61, 1994.

95. Sugden, M. C., A. Kraus, R. A. Harris, and M. J. Holness. Fibre-type specific

modification of the activity and regulation of skeletal muscle pyruvate dehydrogenase

kinase (PDK) by prolonged starvation and refeeding is associated with targeted

regulation of PDK isoenzyme 4 expression. Biochem J 15: 65 1-7,2000.

96. Sugden, M. C., K. A. Orfali, L. G. D. Fryer, M. J. Holness, and D. A. Priestman.

MoIecular mechanisrns underlying the long-term impact of dietary fat to increase cardiac

pynivate dehydrogenase kinase: regulation by insulin, cyclic AMP and pyruvate. Mol

Ce11 Cardiol29: 1867-75, 1997.

97. Sugden, M. C., K. A. Orfali, and M. J. Holness. The pyruvate dehydrogenase

complex: nutrient control and the pathogenesis of insulin resistance. J Nutr 125: 1746s-

1752S, 1995.

98. Taylor, R., T. B. Pnce, L. D. Katz, R. G. Shulman, and G. 1. Shulman. Direct

measurement of change in muscle glycogen concentration after a mixed med in normal

subjects. Am J Physiol265: E224-9, 1993.

99. Tomheirn, K., and J. M. Lowenstein. Control of phosphofnictokinase from rat

skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate. J Bi01 Chem

251: 7322-28, 1976.

100. Vaag, A., F. Alford, F. L. Henriksen, M. Christopher, and H. Beck-Nielsen.

Multiple defects of both hepatic and peripherd intracellulac glucose processing contribute

to the hyperglycaemia of NIDDM. Diabetalogia 38: 326-36, 1995.

101. Vary, T. C . Increased p p v a t e dehydrogenase kinase activity in response to

sepsis. Am J Physiol260: E669-74, 199 1.

102. Vestergaard, H., C. Bjorbaek, P. H. Anderson, J. F. Bak, and 0. Pedersen.

Impaired expression of of glycogen synthase mRNA in skeletal muscle of NIDDM

patients. Diabetes 40: 174045, 199 1.

103. Vestergaard, H., C. Bjorbaek, T. Hansen, F. S. Larsen, D. K. Granner, and 0.

Pedersen. Impaired activity and gene expression of hexokinase 11 in muscle from

NIDDM patents. J Clin Invesr 96: 2639-45, 1995.

104. Vestergaard, H., S. Lund, F. S. Larsen, O. J. Bjerrum, and 0. Pedersen. Glycogen

synthase and phosphofnrctokinase protein and mRNA levels in skeletal muscle from

insulin resistant patients with non-insulin-dependent diabetes mellitus. J Clin Invest 91:

2342-50, 1993.

105. Wilson, J. E. Hexokinases. Rev Physiol Biochem Pharmacol 126: 65-198, 1994.

106. Wu, P., K. Inskeep, M. M. Bowker-Kinley, K. M. Popov, and R. A. Harris.

Mechanism responsible for inactivation of skeletai muscle pyruvate dehydrogenase

complex in starvation and diabetes. Diabetes 48: 1593-9, 1999.

107. Wu, P., J. Sato, Y. Zhao, S. Jaskiewicz, K. M. Popov, and R. A. Harris. Starvation

and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat

heart. Biochem J 329: 197-201, 1998.

108. Wu, T.-F. L., and E. J. Davis. Regulation of glycolytic flüx in an energetically

controlled ceil-free system: The effects of adenine nucleotide ratios, inorganic

phosphate, pH, and citrate. Arch Biochem Biophys 209: 85-99, 198 1.

109. Yri-Jarvinen, H., 1. mihakainen, and V. A. Koivisto. Effect of free fatty acids on

glucose uptake and nonoxidative glycolysis across human forearm tissues in the basai

state and during insulin stimulation. J Clin Endocn'nol Metab 72: 1268-77, 199 1.

110. Zhang, W. M., M. F. Browner, R. J. Fietterick, A. A. DePaoli-Roach, and P. J.

Roach. Primary structure of rabbit skeletal muscle glycogen synthase deduced from

cDNA clones. FASEB J 3: 2532-6, 1989.

11 1. Zierath, J. R., L. He, A. Guma, E. O. Wahlstrom, A. Klip, and H. Wallberg-

Henriksson. Insulin action on glucose transport and plasma membrane GLUT4 content in

skeletal muscle fiorn patients with NIDDM. Diabetalogia 39: 180-89, 1996.

112. Zierath, J. R., K. L. Houseknecht, L. Gnudi, and B. B. K&n. High-fat feeding

impairs insulin-stimulated GLUT4 recruitment via an early insulin-signaling defect.

Diabetes 46: 2 15-23, 1997.

APPENDIX

BLOOD RESULTS over time, during OGTT (O to 180-min)

1. Blood glucose, mmoVl PRE

1 2 3 4 5 6

MEAN Sem

LCD 1 2 3 4 5 6

MEAN Sem

2. Plasma Insulin, ulU/ml PRE

1 2 3 4 5 6

MEAN Sem

LCD 1 2 3 4 5 6

MEAN Sem

3. FFA, mmoUi PRE

1 2 3 4 5 6

MEAN Sem

LCD 1 2 3 4 5 6

MEAN Sem

PRE 1 2 3 4 5 6

MEAN sem

LCD 1 2 3 4 5 6

MEAN sern

PRE 1 2 3 4 5 6

MEAN Sem

LCD 1 2 3 4 5 6

MEAN Sem

5. Glycerol, mmoüi

1

6. Lactate, mmoüi

PRE 1 2 3 4 5 6

MEAN sem

LCD 1 2 3 4 5 6

MEAN Sem

MUSCLE RESULTS

1. GLUT-1 protein, intensity of fixed area

PRE LCD SET PRE =1

2. GLUT4 protein, intensity of fixed area

PRE LCD SET PRE =1

1 31056 48626 2 92226 118093 3 120502 84395 4 100198 119209 5 127339 115582 6 73497 82383

MEAN 90803 94715 sern 15718 12593

3a). HK activity, moükg proteinlhr

CON O 1 0.271 2 0.556 3 0.439 4 0.361 5 0.384 6 0.493

MEAN 0.417 Sem 0.041

CON 75 0.384 0.387 0.633 0 -274 0.397 0.520 0.432 0.051

LCD O 0.296 0.31 7 0.484 0.274 0.345 0.462 0.363 0.036

(O-rest, 75-during OGTT)

LCD 75 0.443 0.450 0.453 0.1 39 0 -476 0.407 0.395 0.052

PRE

1 1 1 1 1 1

1 O

PRE

1 1 1 1 1 1

1 O

LCD

0.67 0.90 2.20 0.88 0.99 1.51

1 .l9 0.25

LCD

1 -57 1 -28 0.70 1.19 0.91 1.12

1 . l3 0.06

3b). HK activity, mmoVg wet musclehin (corrected to total muscle Cr)

CON0 CON75 LCDO 1 4.550 4.51 0 4.1 10 2 3.860 2.860 2.690 3 3.760 4.1 20 3.090 4 2.480 2.390 1.990 5 4.710 5.1 70 3.71 O 6 2.460 3.1 40 4.01 0

MEAN 3.637 3.698 3.267 sem 0.399 0.437 0.339

4ai). GS activity, nmoUmin/mg cytosolic pr

CON (Omin)

0.1 mM 1 3.02 2 1.32 3 1.06 4 2.05 5 1.66 6 0.82

MEAN 1.66 Sem 0.36

CON (75min)

0.1 mM 2.39 3.12 4.5 4.03 2.22 1.98 3.04 0.46

4aii). GS fractional velocity (O.l/l OmM)

5. PDK activity, min -' CON0 LCDO

1 0.127 0.379 2 0.034 O -223 3 0.116 0.071 4 0.072 0.128 5 0.05 0.1 13 6 0.097 0.231

LCD 75 4.340 3.770 3.820 1.1 60 3-61 O 2.950 3.275 0.461

LCD (Omin)

0.1 mM 1-19 1 -84 1.98 0.96 1.27 1.32 1.43 0.1 8

LCD (75min)

0.1 mM 1-14 2.28 3.76 2.1 6 2.4 4.37 2.69 0.53

MEAN 0.083 0.1 91 sern 0.015 0.046

6. PDHa activity, mm01 acetyl-CoA'kg-'-min" (corrected to total muscle Cr)

CON0 CON75 LCDO 1 0.413 0.568 0.270 2 1.168 1 -220 0.1 97 3 0.663 0.743 0.723 4 0.752 0.828 0.462 5 0.847 1 .O22 0.448 6 0.908 1.298 0.1 92

MEAN 0.792 0.947 0.382 sem 0.103 0.1 16 0.084

7. Muscle gtycogen, mmoükg dw

CON LCD 1 315.29 303.61 2 377.23 366.56 3 428.84 314.33 4 303.76 265.59 5 367.53 351.68 6 284.20 318.44

8. Muscle metabolites

ATP

1

2

3

4

5

6

MEAN

Sem

PCr

1

2

3

4

5

6

MEAN

sem

CON O

29.092

25.973

25.468

27.31 7

27.994

23.978

26.637

0.830

CON O

91.309

95.1 13

89.098

87.252

88.102

85.866

89.457

1 -485

CON 75

25.467

25.601

29.487

27.599

26.1 25

24.1 1

26.398

0.844

CON 75

84.559

91.207

83.086

82.654

80.379

78.069

83.326

2.005

LCD O

23.27

23.003

21.733

24.1 08

26.1 35

23.621

23.645

0.652

LCD O

86.938

83.277

89.936

64.263

85.787

85.58

82.630

4.1 39

LCD 75 1.112 0.312 0.658 0.988 0.766 0.282

0.686 0.1 39

LCD 75 mmoikg dm

25.258

23.812

29.793

27.374

25.939

21 -737

25.652

1.250

LCD 75 mmoUkg dm

92.302

84.989

79.824

78.149

85.336

76.485

82.848

2.622

glucose

1

2

3

4

5

6

MEAN

sern

CON O

1.284

0.382

1 .O61

0.3

0.569

0.674

0.71 2

0.1 73

CON O

0.1 81

0.248

0.697

0.1 4

0.485

0.667

0.403

0.1 11

CON O

3.437

1 343

2.658

2.72

1.499

3.298

2.493

0.396

CON O

5.94

9 -38

10.79

13.89

6.32

5.88

8.700

1.456

CON 75

0.505

0.17

0.306

0.236

0.998

0-1 96

0.402

0.1 41

CON 75

0 .27

0.038

0-1 25

O

0.084

0.26

0.1 30

0.051

CON 75

19.1 98

1.481

1.593

1.897

2.1 97

17-41 6

7.297

3.824

CON 75

4.86

4.1 4

4.27

4.69

4.54

6.81

4.885

0.438

LCD O

1.4

0.265

0.486

0.721

1.153

0.299

0.721

0.209

LCD O

0.021

0-1 4

0.C66

0.1 13

0.049

0.288

0.113

0.043

LCD O

7.861

4.1 59

1.12

3.469

2.638

1.569

3.469

1 .O88

LCD O

9.1 6

7.65

7.32

8.642

10.09

8.99

8.642

0.457

LCD 75 mmoikg dm

0.596

0.71 5

0.378

0.244

0.738

0.63

0.550

0.088

LCD 75 mrnoflkg dm

0.02

0.048

0.095

0.1 4

0.108

0.092

0.084

0.01 9

LCD 75 mmoükg dm

4.627

1.595

9.764

6.891

1.989

5.355

5.037

1.374

LCD 75 umoükg dm

3.75

6.08

5.87

6.388

8.76

7.48

6.388

0.752

Lactate

1

2

3

4

5

6

MEAN

sem

Pyruvate

1

2

3

4

5

6

MEAN

sem

CON O

2.91

2.969

2.281

6.81

1 -75

5.607

3.721

0.901

CON O

6.481

2.203

2.01 5

3.647

2.637

6.223

3.868

0.898

CON O

0.1 42

0.061

0.043

0.067

0.1 26

0.379

0.1 36

0.056

CON 75

0.748

0.439

1.308

0.378

0.1 59

4.81 6

1.308

0.789

CON 75

8.597

3.679

5.43

2.594

2.1 35

10,145

5.430

1.473

CON 75

0.204

0.1 06

0.064

0.092

0.1 36

0.31 8

0.1 53

0.042

LCD O

4.592

2.809

2.049

2.482

3.607

6.931

3.745

0.806

LCD O

6.453

5.1 25

7.83

13.946

7.448

6.1 76

7.830

1.407

LCD O

0.065

0.086

0.C29

0.238

0.087

0.1 55

0.110

0.034

LCD 75 mmoükg dm

0.978

2.336

0.545

0.1 11

2.584 3-41 7

1.662

0.583

LCD 75 mmoükg dm

6.333

3.501

3.888

13.839

4.563

5.873

6.333

1 .il7

LCD 75 mmoükg dm

0.057

0.064

0.1 1 1

0.131

0.063

0.224

0.1 O8

0.029