Resistance to Leptin Action is Resistance to Leptin Action is the Major Determinant of the Major Determinant of

Hepatic Triglycerides Hepatic Triglycerides Accumulation in vivo.Accumulation in vivo.

BySigal Fishman, MD

Insulin resistance/Syndrome X• Obesity/abdominal obesity• Diabetes (Type 2)• Hyperlipidemia (low HDL)• Hypertension (increased AGT)• Thrombosis (increased PAI-1)• Inflammation (Cytokines)• NAFLD• Leptin resistance (High leptin; develops with insulin resistance)

• Resistance to both, insulin and leptin action Resistance to both, insulin and leptin action may be implicated in accumulation of may be implicated in accumulation of hepatic TG hepatic TG

• Which one is the major determinant??Which one is the major determinant??

Leptin role:

Browning JD JCI 2004

Insulin action on glucose metabolism

Peripheral Hepatic

glycogenolysis Gluco-neogenesis

Glycogensynthesis

Glycolysis

Clamp studies• Hyperinsulinemic clamp assesses peripheral

insulin action and hepatic• Insulin infused at a fixed rate (3mU/kg/min)

• In insulin sensitive states, glucose is driven into the cells, higher rate of glucose infusion required to maintain euglycemia

• In insulin resistant states, the amount of glucose required to maintain euglycemia is much lower.

• Glucose fluxes in to the peripheral tissues assessed by the use of tracers.

Clamp studies

• Blood glucose is a balance between Tissue glucose uptake (RD) and Hepatic glucose production (HGP)+ Glucose infusion rate (GIR).

• When hepatic glucose production goes down, glucose infusion rate should be increased to maintain euglycemia.

Liver

Muscle

25% glucose

Insulin action++

__

Diet

a. Glycolysis

b. Glycogen syntase

a. Glycogenolysis

b. Gluconeogenesis

Insulin glucose isotopes infusion

Blood

Sample

VF- IV catheter

Day of the clampRecovery

Day -3

Day 0

Tritiated glucose infusion

Insulin 3mu/kg/min+glu

Somatostatin

H2O3

OldYoung

r=-0.57

p=0.01

Relationship between hepatic TG and hepatic insulin action

0

1

2

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0 5 10

^ HGP (mg/kg/min)

Hep

atic

TG

Con

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(mg/

gr)

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0 5 10

^ HGP (mg/kg/min)

Hep

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Leptin reduces hepatic TG and improves hepatic insulin

sensitivity in young lean rats

1

0

0.5

1

1.5

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2.5

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TG (mg/g liver)

YoungLeptinPair-fed

Effect of chronic leptin delivery on the hepatic TG

P<0.01 vs. all

*

0

1

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7

HGP (mg/kg/min)

YoungLeptinPair-fed

Effect of chronic leptin delivery on the insulin-mediated suppression of HGP

P<0.001 vs. all

Role for SCD-1 in mediating leptin action:

Monounsaturated Fatty acid

Saturated Fatty Acyl CoA

Oxidation

TGVLDL Storage

Acetyl-CoA Malonyl-CoACPT-1

SCD-1

ACC

Cohen P. J Nutr 2004

Effect of chronic leptin delivery on hepatic SCD-1 expression

p<0.001 vs. youngSCD-1

young

leptin

pair-fed

0

0.5

1

1.5

Effect of chronic leptin delivery on hepatic ACC-1 expression

p<0.001 vs. youngACC-1

00.2

0.40.60.8

1

1.21.4

youngleptinpair-fed

Effect of chronic leptin delivery on hepatic ACC2 expression

P<0.05 vs. young

ACC2

01234567

youngleptinPair-fed

0

0.5

1

1.5

2

2.5

Malonyl Co-A ()

YoungLeptinPair-fed

Barzilai et al. JCI. 100:3105, 1997

Effect of chronic leptin delivery on hepatic Malonyl Co-A levels

P<0.01 vs. all

*

Jiang G. JCI 2005

Leptin:

• Improves hepatic insulin sensitivity.• Decreases hepatic TG stores

What happens in leptin resistant states?

Leptin’s effect on insulin suppression of glucose production in aging

-100

-80

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-20

0

Young Old

% S

uppr

essi

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Leptin vs. pair-fed

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TG levels after leptin administration in aging rats

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TG (mg/g liver)

Old LeptinOld Saline

Young– pair-fedYoung– leptin

Old– pair-fedOld – leptin

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9

0 2 4 6 8 10 12HGP (mg/kg/min)

Hep

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TG

Con

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(mg/

gr)

*#

**##

1A

1B

Improvement in hepatic insulin Improvement in hepatic insulin action by visceral fat removal action by visceral fat removal is associated with reduction in is associated with reduction in

hepatic TG content in old hepatic TG content in old obese rats…but also with obese rats…but also with

improvement in leptin improvement in leptin sensitivitysensitivity

2

Epidydimal fat removalEpidydimal fat removal

Visceral fat

• Associated with insulin resistance, abnormal glucose tolerance and diabetes across all ages.

• Labile fat depot.

• VF is associated with increased risk of hypertension, thrombosis and dyslipidemias.

• VF, in adolescents, correlates with insulin resistance.

• Increased visceral fat seen in aging.

Are the fat depots biologically distinct?• Sprague-Dawley rats were sacrificed after 12 hours fast.• RNA was isolated from perinephric (visceral fat) and

subcutaneous adipose tissues.• Experiments were performed using rat genomic microarrays

(RGU34A), a platform containing 9000 genes (Affymetrix, Santa Clara, CA).

• Results of gene array expression involving genes implicated in insulin resistance (PPAR-, leptin) or it’s syndrome (angiotensinogen and plasminogen activating inhibitor-1 {PAI-1}), were confirmed and quantified by real time PCR.

• Some of the genes that are involved in glucose metabolism but were not part of the gene array platform, such as Resistin and Acrp 30, were studied by real time PCR.

Out of approximately 8,000 full-length sequences and approximately 1,000 EST clusters

1660 were expressed

297 were up/down- regulated in each chip

Atzmon et. Al Horm Metab Res. 2002; 34:622

Beta 3-adrenergic receptor 5 -Phosphoenolpyruvate carboxykinase (GTP) PEPCK 4.3 -PPAR-gamma 4.1 -Hormone sensitive lipase 3.5 -Insulin-like growth factor I 3.2 -Fatty acid transporter 3.2 -Thioesterase II - 156Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase - 21.1Low molecular weight fatty acid binding protein - 10.1GLUT1 = glucose transporter 1 - 9.8Lipopolysaccharide binding protein - 9.7Lysosomal acid lipase = intracellular hydrolase - 4.6Fatty acid synthase - 3.9Type II cAMP-dependent protein kinase regulatory subunit 7.4 -Adipocyte hormone-sensitive cyclic AMP phosphodiesterase 5.7 -Potential-sensitive polyspecific organic cation transporter 5.6 -Retinol-binding protein (RBP) gene, exon 5 5.2 -Steroidogenic acute regulatory protein 5 -Growth hormone receptor 5 -Chaperonin 60 (Hsp60) and chaperonin 10 (CPN10) genes, nuclear genes encoding mitochondrial proteins

4.8 -

Aquaporin 7 4.3 -Angiotensinogen 4.5 -Glutathione-dependent dehydroascorbate reductase 4 -MHC class II antigen RT1.B-1 beta-chain 3.6 -Tricarboxylate carrier 3.6 -Thyroid stimulating hormone receptor 3.6 -Phosphodiesterase I 3.5 -Water channel aquaporin 3 (AQP3) - 20.8Carbonic anhydrase II - 11.8Wistar-Kyoto (Heidelberg) angiotensin converting enzyme - 7.4GST - 6.1Na-K-Cl cotransporter (Nkcc1) - 5.7Alpha-2-u globulin - 5.3Glutathione S-transferase Yc1 subunit - 4.7Wistar transforming growth factor beta-3 - 4.7Liver glutathione S-transferase Yc subunit - 4.4Polymeric immunoglobulin receptor - 4Alkaline phosphatase - 3.9

Cellular metabolism and other

Glucose homeostasis Insulin action and lipid metabolism

Atzmon et. Al Horm Metab Res. 2002 34:622

Visceral and SC fat are biologically distinct

Visceral fat and subcutaneous fat are biologically distinct.

Some of the significant changes are in the expression of fat-derived peptides that may have a role in insulin resistance (PPAR, leptin, resistin, and adiponectin) or its syndrome (PAI-1 and AT) and in factors affecting body fat distribution (leptin, adrenergic receptors, PPAR, IGF-1, GH).

Factors affecting expression of FDP

• Is there a role for nutrients in the expression of FDP?

• How do nutrients affect the two fat depots?

• Is the effect of nutrients on the two depots different?

• How do adipocytes “sense” excess nutrients?

FFA

Hexosamine Biosynthetic Pathway

Glucose

Glucose-6-P

Glc-1-P

UDP- Glc

Glycogen

F -6-P

Triose -P

Glycolysis

GlcN-6-P

UDPGlcNAc

GFAT1-3%

FFA

Glucosamine

Glycosylationsp1

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PAI-1

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VF

Nutrients, nutrient sensing, and induction of fat-derived peptides

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Resistin

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Angiotensinogen

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ADA, 2003

What are the biological reasons for the risks determined by increased

VF?1) VF expresses higher harmful fat-derived peptides.2) Nutrients induce the expression of fat-derived

peptides more in VF than other fat depots.

The metabolic syndrome

Apo BApo B InsulinInsulin GlucoseGlucose TGTG

Skeletal Muscle

IRIR LPLLPL

NEFAsNEFAs

Liver

HLHL

NEFAsNEFAs

Increased Visceral Fat

. . Thin fibrous cap

. Unstable plaque

. Impaired fibrinolysis

. Increased collagen

. Endothelial dysfunction

GlucoseFFA

Nutrient sensing (HBP?)

TNF-TNF-LeptinLeptin

PAI-1PAI-1IL-6IL-6

adiponectinadiponectin TNF-TNF-

AdiponectinAdiponectinResistinResistin

leptinleptin TNF-TNF-

Insulin’s Suppression of Hepatic Glucose production After VF removal

0-

2-

4-

6-

8-

10-

12-

HGP (mg/kg/min)

*

P<0.01

Old AL Old VF-

Reduction in Hepatic TG After VF removal

0

TG (mg/g liver)

*

P<0.01 vs. VF-

Old AL Old VF- Old SC-

2-

4-

6-

8-

Plasma FFA levels in this model (old VF-) do not relate to changes in hepatic TG

FFA (mmol/l) old VF- old AL Basal: 1.06 ± 0.13 0.83 ± 0.6 clamp: 0.77 ± 0.11 0.65 ± 0.1

Removal of visceral fat in rats results in coordinated changes in leptin level and leptin gene expression of SC fat.

VF-

Leptin

ß-actin

VF+ VF-

Leptin (expression)-VF-0

20

40

80

100

60 M SC*

E P M SC E P M SC E P M SC

0

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Leptin (ng/ml)

*

Old AL VF-

1

Decrease levels of hormone may be an index for improvement in its action!

VF removal might improved leptin sensitivity:

• Hormone level decreased ,combined with reduction in SC gene expression

• No change in food intake!

• In this model we can not dissociate again, insulin action from leptin role

Improvement in hepatic insulin Improvement in hepatic insulin Does not improve hepatic TG Does not improve hepatic TG

content in leptin resistant ZDF content in leptin resistant ZDF ratsrats

33

Removal of visceral fat improves glucose tolerance in Zucker diabetic rats .

ZDVF+ ZDVF-

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Glucose (mM)

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EGP (mg/kg/min)

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Insulin (µU/ml)

GIR 0 2.1 (mg/kg/min)

ZDVF+ ZDVF-

TG levels after VF extraction in ‘leptin resistance’ Zucker rats.

02468

101214161820

TG (mg/g liver)

VF+VF-

Hepatic TG stores improves by leptin independent of insulin sensitivity

Old – sham operationOld – VF-

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0 2 4 6 8 10 12HGP (mg/kg/min)

Hep

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Con

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(m

g/gr

)

ZDF– sham operationZDF – VF-

*

#

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2A

2B

Summery• Leptin and insulin resistance occur together with

obesity and overfeeding.• Leptin decreases hepatic TG stores by decreasing

lipogenesis and increasing ß-oxidation.(leptin studies in young)

• With leptin resistance hepatic TG stores are not decreased (in old obese animals).

• Reversal of insulin resistance is associated with decreasing hepatic TG stores. (Visceral fat removal)

• It is the leptin action and not the insulin action that modulates hepatic TG stores (in Zucker NASHI rats).

Nir BarzilaiRadhika HumuzumdarGil Atzmon Xiao-Hui MaXiao-man YangHong qiang Liang

Thank you!!!