postprandial insulin resistance in zucker diabetic fatty rats is associated with...

ORIGINAL ARTICLE

Postprandial Insulin Resistance in Zucker Diabetic Fatty Rats is Associatedwith Parasympathetic-Nitric Oxide Axis DeficienciesR. A. Afonso1*, A. B. Fernandes1*, C. Santos�, D. Ligeiro�, R. T. Ribeiro*, I. S. Lima*, R. S. Patarrao*, P. A. Videira*, J. Caldeira� and

M. P. Macedo*§

*CEDOC, Faculdade de Ciencias Medicas, Universidade Nova de Lisboa, Campo Martires da Patria, Lisboa, Portugal.

�REQUIMTE, Department of Chemistry, FCT-UNL, Caparica, Portugal.

�Centro de Histocompatibilidade do Sul, Hospital Pulido Valente, Alameda das Linhas de Torres, Lisboa, Portugal.

§Portuguese Diabetes Association - Education and Research Centre (APDP-ERC) Rua Salitre, Lisboa, Portugal.

Obesity, insulin resistance and type 2 diabetes are closely related

(1). The Zucker diabetic fatty (ZDF) rat is an obesity animal model

that it is prone to developing insulin resistance and type 2 diabetes,

which is reported to occur by 12 weeks of age (2,3), although the

literature is scarce at younger ages, such as 9 weeks old. From an

early stage, these animals present a pre-diabetic condition, charac-

terised by high adiposity and hyperinsulinaemia along with marked

hyperglycaemia that has been suggested to occur as a consequence

of b-cell decompensation (4). ZDF rats also present impaired fasting

glucose and glucose tolerance (3). However, because the glucose

used to perform oral glucose testing does not constitute an actual

meal, it can be considered that most of those studies were

performed either in the fasting state or under a poorly controlled

prandial state (5), leaving a gap concerning insulin resistance in the

postprandial state. Even though it is now consensual that, in the

course towards overt diabetes, the first and most deleterious

Journal ofNeuroendocrinology

Correspondence to:

M. P. Macedo, Department of

Physiology, CEDOC, Faculdade

Ciencias Medicas, Universidade Nova

de Lisboa, Campo Martires da Patria,

130, 1169-056 Lisbon, Portugal

(e-mail: [email protected]).

1These authors contributed equally to

this study.

The Zucker diabetic fatty (ZDF) rat is an obesity and type 2 diabetes model. Progression to dia-

betes is well characterised in ZDF rats, but only in the fasted state. We evaluated the mecha-

nisms underlying postprandial insulin resistance in young ZDF rats. We tested the hypothesis

that the overall postprandial action of insulin is affected in ZDF rats as a result of impairment

of the hepatic parasympathetic-nitric oxide (PSN-NO) axis and ⁄ or glutathione (GSH), resulting

in decreased indirect (PSN-NO axis) and direct actions of insulin. Nine-week-old male ZDF rats

and lean Zucker rats (LZR, controls) were used. The action of insulin was assessed in the fed

state before and after parasympathetic antagonism atropine. Basal hepatic NO and GSH were

measured, as well as NO synthase (NOS) and c-glutamyl-cysteine synthethase (GCS) activity

and expression. ZDF rats presented postprandial hyperglycaemia (ZDF, 201.4 � 12.9 mg ⁄ dl; LZR,

107.7 � 4.3 mg ⁄ dl), but not insulinopaenia (ZDF, 5.9 � 0.8 ng ⁄ ml; LZR, 1.5 � 0.3 ng ⁄ ml). Total

postprandial insulin resistance was observed (ZDF, 78.6 � 7.5 mg glucose ⁄ kg; LZR, 289.2 �24.7 mg glucose ⁄ kg), with a decrease in both the direct action of insulin (ZDF, 54.8 � 7.0 mg

glucose ⁄ kg; LZR, 173.3 � 20.5 mg glucose ⁄ kg) and the PSN-NO axis (ZDF, 24.5 � 3.9 mg glu-

cose ⁄ kg; LZR, 115.9 � 19.4 mg glucose ⁄ kg). Hepatic NO (ZDF, 117.2 � 11.4 lmol ⁄ g tissue;

LZR, 164.6 � 4.9 lmol ⁄ g tissue) and GSH (ZDF, 4.9 � 0.3 lmol ⁄ g; LZR, 5.9 � 0.2 lmol ⁄ g)

were also compromised as a result of decreased NOS and GCS activity, respectively. These

results suggest a compromise of the mechanism responsible for potentiating insulin action

after a meal in ZDF rats. We show that defective PSN-NO axis and GSH synthesis, together

with an impaired direct action of insulin, appears to contribute to postprandial insulin resis-

tance in this model.

Key words: Zucker diabetic fatty, insulin action, parasympathetic nerves, nitric oxide, glutathi-

one, postprandial state.

doi: 10.1111/j.1365-2826.2012.02341.x

Journal of Neuroendocrinology, 2012, 24, 1346–1355

ª 2012 The Authors.

Journal of Neuroendocrinology ª 2012 British Society for Neuroendocrinology

modifications in carbohydrate metabolism occur in the postprandial

state (6,7), the literature is scarce relating the pathophysiological

mechanisms involved in the uprising of postprandial insulin resis-

tance and their impact on diabetes.

It is known that autonomic dysfunction is commonly associated

with diabetes. Postprandially, insulin-dependent glucose uptake by

peripheral tissues relies both on the action of insulin per se (direct

action of insulin on target tissues) and on an hepatic-dependent

mechanism (8), which involves a parasympathetic-nitric oxide (PSN-

NO) axis (9–11). Although PSN-NO axis activation is required in the

liver, the increment in insulin-dependent glucose disposal after a

meal occurs mainly in extrahepatic tissues, namely the skeletal

muscle, as previously observed using arterial–venous gradients

across different organs, both by Xie and Lautt (9) and by Moore

et al. (12). More recently, our group used a double tracer technique

to quantify insulin-dependent glucose uptake by different tissues in

animals with or without hepatic PSN-NO blockade, and we were

able to confirm that skeletal muscle is the major tissue affected by

hepatic PSN-NO axis manipulation (13).

In addition to activation of the PSN-NO axis, hepatic glutathione

(GSH) is also required for insulin to achieve full hypoglycaemic

action after a meal (10). Thus, in a healthy subject, either human or

animal, insulin sensitivity increases significantly after a meal,

through a mechanism that requires the rise in hepatic GSH, on one

hand, and hepatic PSN-NO axis activation, on the other (10,14).

Although hepatic GSH levels increase as a consequence of nutrient

absorption, hepatic NO synthesis is increased after a meal through

hepatic parasympathetic (cholinergic) stimulation that leads to

activation of hepatic NO synthase (NOS), resulting in higher

NO production (11,15). Hepatic parasympathetic ablation and ⁄ or

pharmacological inhibition of GSH synthesis results in postprandial

but not fasting insulin resistance (10,16,17).

Intracellular GSH content is ensured mostly by de novo synthesis,

a two-step process catalyzed by c-glutamyl-cysteine synthase (GCS)

and GSH synthase. Both GCS activity and cysteine bioavailability

are limiting to GSH synthesis. GCS is composed of a catalytic

(cGCL) and a regulatory (called modifier, mGCL) subunits (18).

Nitric oxide is produced from L-arginine by a family of NOS

enzymes: endothelial (eNOS); neuronal (nNOS) and inducible (iNOS).

eNOS and nNOS are constitutively expressed in various cell types.

The NOS inducible isoform, iNOS, produces NO only when induced

by an inflammatory process.

We hypothesed that either the hepatic PSN-NO pathway or GSH,

or both, are impaired in young ZDF rats; such an impairment would

contribute to significant insulin resistance and hyperglycaemia in

these animals. We further hypothesised that PSN-NO axis dysfunc-

tion in ZDF rats results in decreased NO synthesis as a result of

ineffective NOS activity and ⁄ or expression.

Materials and methods

Animals and surgical procedures

Applicable institutional and governmental regulations concerning ethical use

of animals were followed, according to the NIH Principles of Laboratory

Animal Care (NIH Publication 85-23, revised 1985) and the European Union

Laboratory Animal Care Guidelines (86 ⁄ 609 ⁄ CEE).

We used 9-week-old male ZDF rats (n = 15) and lean Zucker rats (LZR;

n = 16) obtained from Charles River Laboratories (Barcelona, Spain). Animals

were maintained under a 12 : 12 h light ⁄ dark cycle with free access to food

and water.

On the day before the experiment, rats were submitted to 18-h fasting

period, followed by 1 h of free access to food (day of the experiment:

08.00–09.00 h) to ensure that the rats were fed at the beginning of the

experiment. Anaesthesia was induced by sodium pentobarbital (65 mg ⁄ kg,

i.p.). The trachea, left carotid artery and jugular vein were cannulated, and

an arterial–venous shunt was placed for arterial blood sampling and i.v.

drug infusion, as described previously (19). The arterial–venous shunt was

connected to a pressure transducer (ML750; ADInstruments, Colorado

Springs, Co., USA); blood pressure was monitored using LabView Software

(National Instruments, Austin, TX, USA). Anaesthesia was maintained by pen-

tobarbital continuous infusion (10 mg ⁄ h ⁄ kg, i.v.). Body temperature was

kept at 37.0 � 0.5 �C. After surgery, a 30-min period was allowed for sta-

bilisation.

Insulin sensitivity assessment

Peripheral insulin sensitivity was evaluated by the euglycaemic rapid insulin

sensitivity test (RIST); the glucose infused during the test [RIST index, milli-

gram glucose per kilogram body weight (BW)] was the parameter used to

evaluate insulin sensitivity, after administration of 50 mU ⁄ kg insulin, as

described previously (20). This is a modified euglycaemic clamp that allows

quantification of insulin-dependent glucose uptake specifically by peripheral

tissues in both fasted and fed states, at the same time as inhibiting hepatic

glucose production (9,21). The RISTs dynamic profiles were obtained from

the glucose infusion rates required to maintain euglycaemia at 0.1-min

intervals.

Glycaemia and insulinaemia determination

Arterial glycaemia was determined by the glucose oxidase method, using a

glucose analyser (1500 Sport Analyzer; Yellow Springs Instruments, Yellow

Springs, OH, USA). Insulinaemia was measured by radioimmunoassay (RI-13K

kit; Linco Research, St Charles, MO, USA), as described previously (22).

Measurement of GCS expression in the liver

Liver samples were homogenised and total RNA was isolated using GenElute

Mammalian Total RNA Kit (Sigma, St Louis, MO, USA). Total RNA was

reverse-transcribed into cDNA, using the random-primers-based High Capac-

ity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). A real-time

reverse transcriptase-polymerase chain reaction (RT-PCR) was performed

using Taqman probes methodology (23). Detected reference sequences and

location on the gene were: GCS catalytic subunit (Gclc), Rn00563101_m1;

GCS modifier subunit (Gclm), Rn00568900_m1 (Applied Biosystems). mRNA

expression was normalised using the geometric mean of the endogenous

control b-actin expression (4352931E; Applied Biosystems). Results are

expressed as percentage of endogenous b-actin and control rats (controls

are expressed as 100%).

Evaluation of hepatic GCS activity

GCS activity was measured as described by White et al. (24). The assay

consisted in 2,3-naphthalenedicarboxaldehyde (NDA) derivatisation.

NDA-GSH and NDA-c-glutamylcysteine were measured by fluorescence

intensity (472 excitation ⁄ 528 emission, Gemini Spectrum fluorescence plate

Insulin resistance and parasympathetic-NO deficiency in ZDF 1347

Journal of Neuroendocrinology, 2012, 24, 1346–1355 ª 2012 The Authors.


reader; Molecular Devices, Sunnyvale, CA, USA). Protein content was deter-

mined by the Bradford method.

Hepatic glutathione quantification

Hepatic glutathione content was determined by high-performance liquid

chromatography (HPLC), using N-ethylmaleimide (NEM) derivatisation and

2,4-dinitrofluorobenzene (FDNB) cromophore for detection. This technique

was adapted from a previous study (25) for application in liver samples,

allowing quantification of both reduced (GSH) and oxidised (GSSG) glutathi-

one with high reproducibility and sensitivity (< 2 lmol ⁄ dm3). Briefly, after

homogenisation with NEM, samples were deproteinised (tricholoroacetic

acid, 0 �C for 10 min; 30 000 g at 4 �C for 20 min). After alkalinisation

(Tris–HCl, pH 10), samples were incubated with FDNB (3 h in dark, room

temperature), acidified (HCl 37%) and loaded onto a LiChroCART Purospher

Star-NH2 column (250 · 4 mm; Merck, Darmstadt, Germany) for HPLC sepa-

ration (LaChrom L-7000; Hitachi, Tokyo, Japan). Elution (1.2 ml ⁄ min) was

performed using four solvents: 0–8 min, 14% water (solvent A); 3% acetic

acid (3 mol ⁄ dm3; solvent B); 3% sodium acetate (3 mol ⁄ dm3; solvent C);

and 80% methanol (solvent D); 8–13 min, linear gradient, 3–10% of sol-

vents B and C (0% of solvent A); 13–45 min, 0% A, 10% B, 10% C, 80% D.

Detection was made at 355 nm wavelength, using a LaChrom L-7400 UV

detector, Hitachi (chromatogram analysis performed using the LaChrom

D-2500 ⁄ D7500 Data File Conversion Utility; Hitachi). The area under the

curve was calculated with TABLECURVE 2D, version 5.01 (Systat Software Inc.,

Chicago, IL, USA). GSH and GSSG eluted at 15 and 23 min, respectively.

Hepatic NOS expression quantification

This assay was similar to the GCS expression assay. For each primer ⁄ probe

set, detected reference sequences and location on the gene were: neuronal

(nNOS ⁄ NOS1), Rn00583793_m1; inducible (iNOS ⁄ NOS2), Rn00561646_m1;

endothelial (eNOS ⁄ NOS3), Rn02132634_s1 (Applied Biosystems).

Hepatic NOS activity evaluation

Total NOS activity was assayed by conversion of radiolabelled arginine to ci-

truline ([3H]citruline), as described previously (26), using a NOS Activity

Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA). Protein content

was determined by the Bradford method.

NO assessment

Liver and plasma NO levels were assessed by chemiluminescence-based

measurement of nitrate (NO�3 ) and nitrite (NO�2 ) concentrations, as

described previously (27). This method consists of the vanadium III-induced

reduction of NO�2 and NO�3 to NO, at high temperature (90 �C), using a Sie-

vers 280 NO Analyzer (Sievers Instruments, Boulder, CO, USA).

Experimental protocols

After postprandial glycaemic baseline determination (90–120 min after feed-

ing), an initial insulin sensitivity assessment was made (control RIST). The

PSN-NO-independent action of insulin was achieved using atropine sulphate

(3 mg ⁄ kg, i.v.) and quantified by a post-atropine RIST.In a separate set of fed rats, without any other procedures, arterial blood

samples were collected for insulinaemia, and liver samples were collected

for glutathione, NO, GCS and NOS (activity and expression) quantification. In

the protocols in which atropine administration was performed, blood sam-

ples were also collected before and after atropine, for insulinaemia quantifi-

cation.

Statistical analysis

Data are presented as the mean � SEM. Differences significance was calcu-

lated through two-tailed Student’s t-tests (GraphPad Software Inc., San

Diego, CA, USA). P < 0.05 was considered statistically significant.

Results

BW, blood pressure, glycaemia and insulinaemia

ZDF rats were moderately obese compared to controls (LZR).

Accordingly, ZDF rats presented higher BW (291.3 � 2.7 g, n = 15)

than LZR (258.5 � 5.3 g, n = 16; P < 0.001). Such a moderate

increment in BW could be associated with the young age of the

rats.

Postprandial mean arterial blood pressure, determined before

control RIST, was higher in ZDF rats (142.8 � 8.9 mmHg, n = 6;

LZR, 105.5 � 6.9 mmHg, n = 6; P < 0.01). Blood pressure was not

significantly affected by atropine (ZDF, 134.3 � 10.1 mmHg; LZR,

100.8 � 5.1 mmHg; P < 0.05 ZDF versus LZR).

At this age (9 weeks), ZDF rats already showed a marked post-

prandial hyperglycaemia (201.4 � 12.9 mg ⁄ dl, n = 6) compared to

LZR (107.7 � 4.3 mg ⁄ dl, n = 6; P < 0.001). Glyceamia did not

change significantly throughout the entire protocol, either after

control or post-atropine insulin sensitivity assessments.

Interestingly, despite hyperglycaemia, postprandial insulinaemia

was higher in ZDF rats (5.9 � 0.8 ng ⁄ ml) compared to LZR (1.5 �0.3 ng ⁄ ml; P < 0.001), suggesting that, although the ZDF pancreas

was still functioning, its efficacy was declining. Atropine adminis-

tration did not change insulinaemia in either group.

Insulin sensitivity

Postprandial action of insulin (control RIST) was significantly

impaired in ZDF rats compared to LZR as a result of an impairment

of both insulin per se (post-atropine RIST) and a PSN-NO-depen-

dent component, with the latter obtained by subtraction of post-

atropine RIST from the control RIST index.

ZDF rats presented a severe decrease in overall postprandial

action of insulin (control RIST: ZDF, 78.6 � 7.5 mg glucose ⁄ kg BW,

n = 6; LZR, 289.2 � 24.7 mg glucose ⁄ kg BW, n = 6; P < 0.001;

Fig. 1B), which was caused by an impairment of both PSN-NO-inde-

pendent (ZDF, 54.8 � 7.0 mg glucose ⁄ kg BW; LZR, 173.3 �20.5 mg glucose ⁄ kg BW, P < 0.001; Fig. 1D) and the PSN-NO-

dependent action of insulin (ZDF, 24.5 � 3.9 mg glucose ⁄ kg BW;

LZR, 115.9 � 19.4 mg glucose ⁄ kg BW; P < 0.01; Fig. 1F).

PSN-NO contribution to the overall postprandial action of insulin,

given by atropine-induced inhibition of insulin sensitivity, was

40.3 � 6.6% in LZR and 32.0 � 5.9% in ZDF rats.

Considering the profiles of the action of insulin during the RISTs,

peak magnitude and duration of insulin hypoglycaemic curves were

analysed. ZDF rats present a decreased peak magnitude (ZDF, 5.4 �0.4 mg glucose ⁄ kg ⁄ min; LZR, 14.8 � 1.4 mg glucose ⁄ kg ⁄ min;

P < 0.001) and duration of total postprandial action of insulin (con-

trol RIST curves: ZDF, 22.4 � 2.1 min; LZR, 38.1 � 2.1 min;

P < 0.001) (Fig. 1A). PSN-NO-independent insulin dynamic profile,

1348 R. A. Afonso et al.

ª 2012 The Authors. Journal of Neuroendocrinology, 2012, 24, 1346–1355


obtained by the post-atropine RIST (Fig. 1C), was also affected in ZDF

rats (peak magnitude: 4.0 � 0.7 mg glucose ⁄ kg ⁄ min; duration of

action: 19.7 � 0.2 min) compared to LZR (peak magnitude: 10.0 �1.1 mg glucose ⁄ kg ⁄ min; duration: 34.2 � 2.6 min; P < 0.01). The

PSN-NO-dependent profile, given by subtraction of post-atropine from

control curves, was also altered in ZDF rats (ZDF: peak magnitude,

2.3 � 0.8 mg glucose ⁄ kg ⁄ min; duration, 13.7 � 1.0 min; LZR: peak

magnitude, 7.0 � 1.3 mg glucose ⁄ kg ⁄ min, P < 0.05; duration: 33.4 �2.7 min, P < 0.001) (Fig. 1E).

Hepatic glutathione synthesis and content

Considering hepatic GCS expression, and taking LZR as a reference

(100%, n = 10), both catalytic (cGCS) and modifier (mCGS) subunits

were expressed more in ZDF rats (cGCS, 155.9 � 12.3%; mGCS,

175.4 � 25.3%, n = 9; P < 0.05) compared to LZR (Fig. 2A).

On the other hand, hepatic GCS activity was impaired in ZDF rats

(14.2 � 1.2 lM ⁄ min ⁄ mg protein) compared to control rats (LZR,

21.2 � 1.0 lM ⁄ min ⁄ mg protein; P < 0.001) (Fig. 2B). Increased GCS

expression can be interpreted as a compensatory mechanism to

overcome the lower GCS activity in the liver of ZDF rats.

Postprandial hepatic glutathione levels were quantified in both reduced

(GSH) and oxidised (GSSG) forms (Fig. 3). Hepatic GSH content was reduced

in ZDF rats (4.9 � 0.3 lmol ⁄ g) compared to LZR (5.9 � 0.2 lmol ⁄ g;

P < 0.05). By contrast, GSSG was higher in ZDF rats (ZDF, 349.1 �78.0 nmol ⁄ g; LZR, 136.8 � 24.8 nmol ⁄ g, P < 0.01). GSH ⁄ GSSG ratio was

impaired in ZDF rats (ZDF, 23.3 � 3.7; LZR, 53.4 � 11.9; P < 0.05).

Hepatic NO synthesis and content

Concerning hepatic NOS expression, hepatic NOS mRNA levels in

ZDF rats showed a decrease in eNOS, which expressed only 18.3 �

Fig. 1. Postprandial hypoglycaemic action of insulin in Zucker diabetic fatty rats (ZDF, n = 6; bold line ⁄ hatched bars) and lean Zucker rats (LZR, n = 6; regu-

lar line ⁄ white bars). On the left, mean dynamic profiles of the action of insulin are impaired in ZDF: (A) overall postprandial action of insulin; (C) parasympa-

thetic-nitric oxide (PSN-NO)-independent action of insulin post-atropine; (E) PSN-NO-dependent action of insulin. On the right, ZDF rats present impairment of

both PSN-NO-dependent (F) and PSN-NO-independent (D) components of the action of insulin, resulting in total postprandial insulin resistance (B). Results are

the mean � SEM. **P < 0.01; ***P < 0.001 (versus LZR). RIST, rapid insulin sensitivity test.




2.6% of that observed in LZR (P < 0.001), whereas expression of

both nNOS and iNOS was increased to 303.8 � 40.5% and

45540 � 10346%, respectively (P < 0.001) (Fig. 4A).

Hepatic NOS activity was significantly lower in ZDF rats (1.2 �0.1 pmol ⁄ min ⁄ mg protein) compared to LZR (1.7 � 0.2 pmol ⁄ -

min ⁄ mg protein; P < 0.05) (Fig. 4B).

Plasma NO levels (Fig. 5A) were higher in ZDF rats compared to con-

trol rats (ZDF, 17.9 � 1.6 lM; LZR, 12.4 � 1.5 lM; P < 0.001), whereas

hepatic NO (Fig. 5B), which is essential for the PSN-NO-dependent

action of insulin, was impaired in ZDF rats (117.2 � 11.4 lmol ⁄ g tis-

sue) compared to LZR (164.6 � 4.9 lmol ⁄ g tissue; P < 0.001).

Discussion

The ZDF rat is an animal model of obesity known for its predisposi-

tion to develop overt diabetes, which has been described to occur

around the 12th week of age. Our data suggest for the first time

that the first manifestations of carbohydrate metabolism deficien-

cies in ZDF rats occur in the postprandial state at a very young age

(9 weeks old), as demonstrated by the pronounced postprandial

insulin resistance and hyperglycaemia in these animals.

Postprandial peripheral insulin resistance in ZDF rats appears to

be caused by a defect in both PSN-NO axis-dependent and -inde-

pendent components of the action of insulin. Hepatic GSH levels,

which are required for adequate action of insulin after a meal, were

also affected.

Methodological considerations

All the experiments described in the present study were performed

under sodium pentobarbital anaesthesia, which minimises the acute

effects of animal stress and does not affect glucose metabolism

(17,28,29).

The method used to assess insulin sensitivity was the transient

euglycaemic clamp RIST, which allows quantification of the action

of insulin in both fasted and fed states, being reproducible within

the same experiment (up to four consecutive RISTs) at the same

time as retaining high sensitivity (20). The RIST allows postprandial

quantification of peripheral insulin-dependent glucose disposal,

without induction of vagal impairment, as observed using methods

that require long insulin infusions resulting in hyperinsulinaemic

states (30). The insulin bolus used in the RIST ensures maximal

Fig. 2. Hepatic c-glutamylcysteine synthetase (GCS) expression (A) and

activity (B), in lean Zucker rats (LZR, n = 10, white bars) and Zucker diabetic

fatty rats (ZDF, n = 9, hatched bars). Hepatic expression of both GCS subun-

its (catalytic, cGCS; was increased in ZDF rats compared to LZR (A), possibly

as an adaptation to the lower GCS activity observed in the liver of ZDF rats

(B). mGCS, modifier GCS subunit. Data are expressed as the mean � SEM.

*P < 0.05; ***P < 0.001.

Fig. 3. Postprandial hepatic glutathione in lean Zucker rats (LZR, n = 6,

white bars) and Zucker diabetic fatty rats (ZDF, n = 7, hatched bars).

Reduced glutathione (GSH) is impaired (A), whereas oxidised glutathione

(GSSG) is increased (B) in ZDF rats compared to LZR animals. Accordingly,

the GSH ⁄ GSSG ratio is decreased in ZDF rats (C). Data are the mean � SEM.

*P < 0.05, ** P < 0.01.




suppression of hepatic glucose production (9), allowing the deter-

mination of peripheral glucose uptake. The RIST has been validated

in several animal models (17,19,31,32) and in humans (33), with

results comparable to those obtained using the insulin tolerance

test (34).

Atropine was used specifically to assess the PSN-NO-dependent

action of insulin, by subtracting the post-atropine RIST index from

the control RIST index. We observed that this PSN manipulation

does not alter circulating insulin levels and previous studies show

that hepatic glucose output is not affected either (9). The 3 mg ⁄ kg

i.v. dose of atropine was chosen because it allows higher hepatic

PSN-NO inhibition with minimal cardiovascular effects, as previ-

ously determined in several animal models (9,19,29,35,36). Because

surgical ablation of the hepatic anterior plexus and atropine admin-

istration induce PSN-NO axis impairment of similar magnitude

(9,36), atropine i.v. administration was chosen for being a less inva-

sive approach.

Postprandial hyperglycaemia and insulinaemia in ZDF rats

Our results show that fed 9-week-old ZDF rats are hyperglycaemic,

but not insulinopenic, even though they only show a moderate

increase in BW (approximately 11%). Postprandial glycaemia

(90–120 min after feeding) was approximately doubled in ZDF rats

compared to controls (LZR), and insulinaemia was also increased,

revealing that, at this age, b-cell exhaustion is not the main cause

of postprandial hyperglycaemia in ZDF rats.

These data are in accordance with recent reports suggesting that

ZDF rats present high insulinaemia at 6 weeks of age, although

b-cell morphological abnormalities are observed only at 14 weeks,

accompanied by postprandial hyperglycaemia (5), which was already

described for older ages (e.g. 19 weeks) (37).

Furthermore, ZDF rats do not appear to present any differences

in terms of hepatic glucose efflux (2), suggesting that the liver is

not the main contributor for the observed hyperglycaemia either.

On the other hand, hyperglycaemia is accompanied by glucose

intolerance (37), which, along with hyperinsulinaemia, already sug-

gested an inadequate capacity of insulin to promote peripheral glu-

cose uptake after a meal (i.e. postprandial peripheral insulin

resistance).

In the present study, we demonstrated that, at 9 weeks of age,

ZDF rats already present severe postprandial glucose homeostasis

alterations that involve hyperglycaemia and insulin resistance.

Postprandial action of insulin in ZDF rats

Although previous studies describe a sustained insulin resistance,

accompanied by hyperglycaemia and hyperinsulinaemia in ZDF rats,

those studies were performed in older animals, under noncontrolled

prandial or fasting conditions and using a methodology that

Fig. 4. Hepatic mRNA nitric oxide synthase (NOS) expression and NOS activity in lean Zucker rats (LZR, n = 10, white bars) and Zucker diabetic fatty rats

(ZDF, n = 9, hatched bars). (A) Expression of endothelial (eNOS) was decreased, whereas expression of neuronal (nNOS) and inducible (iNOS) was increased in

the liver of ZDF animals (expressed as a percentage of elevation ⁄ reduction in relation to the control value established as 100%, for LZR). (B) Hepatic NOS

activity was lower in ZDF rats than LZR, which was accompanied by an impairment in eNOS expression, suggesting that this isoform is the major contributor

for the parasympathetic-NO axis. Data are the mean � SEM. *P < 0.05; ***P < 0.001.




neglects the contribution of the PSN-NO with respect to postpran-

dial insulin sensitivity (2,30,38,39).

The importance of analysing glucose homeostasis in the post-

prandial state has been highlighted in several studies suggesting

that the loss of postprandial glycaemic control precedes the deteri-

oration of fasting glycaemia with worsening diabetes (40), leading

to new guidelines aiming specifically at postprandial glucose man-

agement (6).

We were able to quantify postprandial insulin sensitivity in ZDF

rats at an early stage (9 weeks old) and we observed that ZDF

present an impairment of approximately 73% compared to control

animals. Further analysis of the two components that constitute

the postprandial action of insulin revealed a severe defect in the

PSN-NO-dependent axis (79% impairment), which we previously

demonstrated to be physiologically absent in the fasted state (8).

PSN-NO axis activation occurs in the liver, although the effect of

PSN-NO on the action of insulin is seen specifically in peripheral

tissues (9,16). Indeed, the PSN-NO-dependent action of insulin

stimulates glucose uptake in the skeletal muscle (9,13), the main

fate of circulating glucose (41). Thus, inhibition of hepatic PSN-NO

axis, either by atropine or hepatic denervation, ultimately leads to

peripheral, but not hepatic, insulin resistance (9). Different ZDF rat

tissues present different magnitudes of insulin resistance (2), of

which the most affected is the skeletal muscle, with no significant

impact in adipose tissue or cardiac muscle (2,42).

As previously reported, both hepatic GSH and NO are necessary

for the proper action of insulin in the fed state (10,15,16). The first

appears to be obtained from nutrient absorption (e.g. cyste-

ine ⁄ methionine) and the latter results from meal-induced activation

of the hepatic PSN-NO axis (15,16,43). This double requirement in

GSH plus NO explains why the restoration of GSH levels and reduc-

tion of oxidative stress, without any NO-targeted approach, does

not reverse high blood pressure, nor hyperglycaemia ⁄ insulin resis-

tance, in ZDF rats, as observed previously (44).

The results obtained in the present study suggest that ZDF rats,

representing diet-induced obesity models, present a compromise in

the PSN-NO axis, which appears to be essential for physiological

regulation of postprandial insulin. Moreover, at a very young age,

ZDF rats have a diabetic predisposition, which already presents sig-

nificant postprandial insulin resistance without morbid obesity.

Thus, these animals might be considered as a relevant diabetogenic

model.

Oxidative stress and inflammation in ZDF rats

Both inflammation and oxidative stress are associated with the

development of insulin resistance and diabetes in ZDF rats(44). In

this context, reduced glutathione (GSH) and iNOS-derived NO are

extremely relevant because the first acts as a reactive species scav-

enger, thereby avoiding oxidative damage, whereas the latter is

itself an inflammatory, and potentially oxidative, mediator.

For the first time, we determined hepatic glutathione (GSH and

GSSG) concentrations in ZDF rats and observed that these animals

present an approximately 20% decrease in hepatic GSH, whereas

the oxidised form (GSSG) was increased, leading to an impairment

of the GSH ⁄ GSSG ratio. Taken together, these data suggest that the

lower GSH ⁄ GSSG ratio in ZDF rats results from decreased GSH syn-

thesis, caused by a lower GCS activity, which contributes to the oxi-

dative stress. The oxidative state can also induce a decrease in the

GSH ⁄ GSSG ratio by increasing GSSG formation, although it appears

that the impairment in GSH synthesis is the major contributor.

These observations establish an association between hepatic GSH

impairment and oxidative stress in ZDF rats, as previously described

in different tissues (44,45).

Additionally, our NO data also indicate a pro-inflammatory and

pro-oxidant state already in young ZDF rats because both plasma

NO and iNOS expression were significantly increased. Other studies

have reported augmented cytokines levels in ZDF rats (46) that

appear to be accompanied by an upregulation of heart iNOS

expression, which is related to inflammation and impaired cardiac

function (47). Although we did not measure plasma cytokine in ZDF

rats, our data suggest a pro-inflammatory condition derived from

the increase in hepatic iNOS expression and plasma NO. This iNOS-

related pro-inflammatory condition appears to contribute to the

impairment of hepatic function and decreased PSN-NO activation

(Fig. 6).

Fig. 5. Nitric oxide (NO) levels, assessed by nitrate (NO�3 ) and nitrite (NO�2 )

concentrations, in plasma (A) and liver (B) samples of lean Zucker rats (LZR,

n = 6, white bars) and Zucker diabetic fatty rats (ZDF, n = 7, hatched bars).

Plasma NO is higher in ZDF rats compared to LZR (A), whereas the hepatic

NO concentration is impaired in ZDF rats (B). Data are the mean � SEM.

*P < 0.05; **P < 0.01.




Taken together, both the GSH and NO data indicate a pro-

inflammatory and pro-oxidative condition in the young-adult

(9-week-old) ZDF rats that could be associated with insulin resis-

tance (Fig. 6).

Postprandial insulin resistance and the PSN-NO axis in ZDFrats

In addition to their relevance for the inflammatory ⁄ oxidative stress

status, hepatic GSH and NO are both required for adequate periph-

eral insulin sensitivity after a meal (10).

Low hepatic GSH can be directly linked to insulin resistance

(10,16,48). However, the GSH decrease in ZDF rats was much less

than the insulin sensitivity impairment. This suggests that, although

GSH impairment may contribute to oxidative stress and inflamma-

tion in ZDF rats, which are associated with insulin resistance, by

itself, it does not explain the full magnitude of postprandial insulin

resistance in these animals.

In addition to GSH, the PSN-NO axis is required for the full

action of insulin, in particular after a meal (8,17). In the present

work, we observed that PSN-NO axis is also impaired in ZDF rats.

We have previously studied different obesity models and shown

that autonomic dysfunction can be associated with obesity

(19,29). Furthermore, although autonomic dysfunction can also be

associated with insulin resistance and diabetes, the studies pub-

lished so far concerning autonomic function in ZDF rats are

scarce and quite inconclusive. In the present study, we tested

parasympathetic function in ZDF rats indirectly, by testing the

effect of cholinergic antagonism on the action of insulin. The

magnitude of atropine-induced inhibition of the postprandial

action of insulin was much lower in ZDF rats compared to con-

trols, suggesting a parasympathetic impairment in ZDF rats. The

genetic mutation of the leptin receptor present in this model is

consistent with our observation. Indeed, Li et al. (49) recently

showed that i.c.v. leptin administration improves glucose disposal

through a process partially dependent on hepatic parasympathetic

nerves. Such parasympathetic impairment in ZDF rats does not

exclude a defect downstream from parasympathetic activation.

One of the common mediators of parasympathetic function is NO,

and low NO levels are known to be associated with sympathova-

gal imbalance (50). Thus, to determine whether the PSN-NO axis

defect on the postprandial action of insulin was a result of

Fig. 6. Proposed mechanism for postprandial insulin resistance in the Zucker diabetic fatty (ZDF) rat, an animal model of obesity and type 2 diabetes. After a

meal, two feeding signals are crucial for an increase in peripheral glucose uptake: increased glutathione (GSH) levels, by de novo synthesis, through glutamyl-

cysteine synthase (GCS) as one of the regulatory enzymes, and activation of the hepatic parasympathetic system-nitric oxide (NO) axis, through activation of

nitric oxide synthase (NOS). A dysfunction in any of these signals will lead to insulin resistance through inactivation of hepatic GSH and ⁄ or NO bioavailability.

The loss of postprandial response to insulin could be on the genesis of type 2 diabetes, with increased inflammation and oxidative stress possibly also related

to the bioavailability of GSH and NO, in ZDF animals. Ach, acetylchloline; cNOS, constitutive NOS; iNOS, inducible NOS.




inadequate parasympathetic activation, decreased hepatic NO

production, or both, we evaluated hepatic NOS activity and

expression, as well as hepatic NO levels.

The results obtained in the present study suggest that the

impairment of PSN-NO activity is caused by decreased hepatic con-

stitutive NOS activity, namely eNOS, the major contributor for

hepatic whole NOS activity, leading to defective hepatic NO levels

produced by that constitutive isoform, which is determinant for

postprandial insulin resistance in ZDF rats (Fig. 6). The impairment

of hepatic NOS activity was accompanied by a decrease in hepatic

eNOS expression (Fig. 4). Abnormalities in endothelial NO synthesis

have been previously identified in diet-induced obese rats (51), as

well as in ZDF rats, but only in extrahepatic tissues (52,53). Our

observations suggest that also hepatic eNOS is impaired in these

animals, which results in a defective PSN-NO axis, therefore estab-

lishing the link between NO ⁄ NOS impairment and postprandial

insulin resistance in ZDF rats.

Altered NO production resultant from autonomic dysfunction is

also suggested as one of the most relevant causes for hypertension

in ZDF rats (54,55), which also agrees with our data. In the long

term, parasympathetic impairment in ZDF rats, with consequent

decrease in NO levels, may contribute to the observed hypertension

in these animals. In addition to blood pressure, in the present

study, we show that the fate of the hepatic eNOS-derived NO is

also related to the control of insulin sensitivity, which is impaired

in ZDF rats.

In conclusion, our experiments provide the pathophysiological

link between glutathione impairment, autonomic dysfunction and

altered NO production in ZDF rats, which largely explains the post-

prandial insulin resistance in these animals, as depicted in Fig. 6.

These data suggest that, at 9 weeks of age, fed ZDF rats are already

moderately obese, hyperglycaemic, non-insulinopenic and insulin

resistant. We show for the first time that postprandial insulin resis-

tance in ZDF rats appears to result not only from an impaired func-

tion of the action of insulin per se, but also from impaired hepatic

glutathione and the PSN-NO axis, through a mechanism that also

elicits oxidative damage at this young age. Future work in skeletal

muscle intracellular pathways should aim to clarify the sequence of

events occurring from obesity to overt diabetes.

Acknowledgements

The present study was supported by FCT grants (POCI ⁄ SAU-

OBS ⁄ 56716 ⁄ 2004; PIC ⁄ IC ⁄ 82956 ⁄ 2007). A. Fernandes was supported by a

FCT PhD fellowship (SFRH ⁄ BD ⁄ 29693 ⁄ 2006). None of the authors have any

conflict of interests to declare.

Received 19 January 2012,

revised 10 May 2012,

accepted 16 May 2012

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