Exercise training improves insulin sensitivity in the obese Zucker rat

The effect of exercise on in vivo insulin sensitivity was examined in lean and obese Zucker rats. Rats (6 to 7 weeks of age) were swum two hours per day or kept sedentary for 8 weeks. Exercise decreased body weight gain as well as percent of fat in both genotypes. Sedentary obese rats had 62% higher gastrocnemius citrate synthase activity per gram of muscle than did lean rats. Exercise increased activity of this oxidative enzyme similarly in both genotypes. Compared to lean rats, obese rats had higher plasma-insulin levels and were less sensitive to insulin during an insulin tolerance test. Although training had no effect on plasma-insulin levels, exercise trained obese rats showed a greater drop in plasma glucose relative to sedentary controls following intravenous injection of three concentrations of insulin. It was concluded that moderate exercise training improved the insulin sensitivity of the obese Zucker rat.     

Increased renal angiotensin II AT1 receptor function in obese Zucker rat

Angiotensin II (Ang II) via the activation of AT1 receptors and subsequent stimulation of the tubular sodium transporters increases sodium and water reabsorption in the proximal tubule. An enhanced tubular action of Ang II is implicated in obesity related hypertension; however, the mechanism of such a phenomenon is unknown. Present study was designed to determine the AT1 receptor numbers and function in the proximal tubule of obese and lean Zucker rats. Obese Zucker rats were hypertensive and hyperinsulinemic. The plasma renin activity was similar in the lean and obese rats. Angiotensin II stimulated the Na,H-exchanger (NHE) activity in the proximal tubule, but the stimulatory response was markedly greater in obese than in lean rats. Similarly, Ang II caused greater inhibition in cAMP accumulation in the proximal tubule of obese compared to lean rats. The (125I]sar-Ang II binding revealed a 100% increase in the AT1 receptor number in the brush border membrane (BBM) of obese compared to lean rats. The Western blot analysis revealed a 36-51% increase in the Gi(alpha)1 and Gi(alpha)3 in the BBM of obese compared to lean rats. We conclude that increases in the AT1 receptor number and abundance of the Gi(alpha) on BBM may be responsible for the enhanced signaling and subsequent greater stimulation of NHE by Ang II in proximal tubules of obese rats. The greater stimulation of NHE by Ang II may contribute to the increased tubular sodium reabsorption and to the hypertension in obese Zucker rats.     

Renal function in the obese Zucker rat

The hyperphagic, genetically obese Zucker rat (fa/fa) exhibits both a greater kidney size and a progressive, premature glomerular sclerosis. In the present study, glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and renal tubular function were evaluated during study 1 in lean Zucker (FA/-), fa/fa, and lean Sprague-Dawley (S-D) rats. The GFR as measured by renal inulin clearance (ClIN) was not significantly different (P greater than 0.05) between S-D (1.36 +/- 0.18 ml/min) vs FA/- (1.36 +/- 0.33 ml/min) and FA/- vs fa/fa (1.25 +/- 0.42 ml/min). The ERPF as measured by renal p-aminohippurate (PAH) clearance (ClPAH) also was not significantly different between S-D (3.98 +/- 0.80 ml/min) vs Fa/- (3.71 +/- 0.81 ml/min) and Fa/- vs fa/fa (3.34 +/- 1.60 ml/min). There was a significant difference (P less than 0.05) in the renal tubular transport maximum (Tm) of PAH between S-D (2.23 +/- 0.40 mg/min) and Fa/- (1.64 +/- 0.63 mg/min) groups but not between Fa/- and fa/fa (1.29 +/- 0.61 mg/min) groups, indicating a strain effect in organic anionic renal transport. The Fa/- vs fa/fa comparisons were significant when GFR, ERPF and Tm were corrected for total body or kidney weight. In a second group of animals (study 2), GFR (as reflected by creatinine clearance [Clcr]) and histologic studies were performed in Fa/- and fa/fa rats. Clcr values were significantly higher in the fa/fa (2.10 +/- 0.44 ml/min) vs Fa/- (1.68 +/- 0.17 ml/min). Histologic studies in group 2 demonstrated no remarkable differences between Fa/- and fa/fa rats. These results suggest wide interanimal variation in obesity associated changes in renal function and possibly pathology in the fa/fa rat.     

Metabolic abnormalities of the hyperglycemic obese Zucker rat.

In a cross-sectional study, we evaluated the metabolic profiles of lean (Fa/?) and obese (fa/fa) Zucker male rats at 4 to 8 months of age. Although all of the obese rats (N = 108) demonstrated glucose intolerance, most of the obese rats exhibited only mild elevations of fasted and fed plasma glucose. Only 14 of the obese rats were severely hyperglycemic, which resulted in substantial elevations of glycohemoglobin (GHb) levels. The nerve and lens levels of glucose, sorbitol, and fructose were elevated, and the myo-inositol was depleted in all hyperglycemic obese rats, but not in the euglycemic obese rats. With increasing duration of hyperglycemia, the neural myo-inositol level approached normal, while the lenses became cataractous. All obese rats had increased urinary albumin excretion (UAE), which was dependent on age (r = .45, P less than .02) and independent of hyperglycemia, glucosuria, and polyuria. In conclusion, although the euglycemic obese rats exhibited some diabetic abnormalities, the hyperglycemic obese Zucker rat more closely resembled the altered metabolic profile associated with type II diabetes mellitus.     

Insulin sensitivity and response in eviscerated obese Zucker rats

In order to examine the contribution of the skeletal muscles to insulin resistance in obesity, fatty (fa/fa) or lean Zucker rats were functionally hepatectomized/nephrectomized by an evisceration procedure. Glucose loads, adjusted to produce plasma levels of about 500 mg/100 ml, were given IV postoperatively, and during the next four hours the uptake and utilization of glucose by peripheral tissues (muscles) was estimated from the rate of fall in the plasma glucose concentration. Glucose utilization in the fa/fa rats was similar to that in the lean, despite plasma insulin levels that were ten times higher. Furthermore, a marked reduction in uptake was revealed when plasma insulin in the fa/fa rats was normalized (by waiting one hour after evisceration) before the glucose load was administered. Glucose uptake was resistant to insulin over a range of submaximal plasma insulin concentrations, indicating a shift to the right of the dose-response relationship. The maximal response in the fatty rats was markedly depressed compared with that in the lean rats, and even large doses of insulin (up to 1000 mU, IV) failed to restore it. The results suggest that the skeletal muscles isolated by evisceration from fa/fa Zucker rats are less responsive as well as sensitive to insulin than the muscles of lean rats and that the muscles contribute significantly to the insulin resistance found in intact obese animals.     

Hypothalamic-pituitary-adrenal axis function in the Zucker obese rat.

The development of many endocrine, metabolic, and behavioral abnormalities characteristic of genetically obese Zucker rats is dependent upon the presence of glucocorticoids, the secretion of which is regulated by a neuroendocrine cascade initiated by hypothalamic release of CRF. Recent reports have inferred alterations in central CRF tone as a putative factor contributing to dysregulation of the pituitary-adrenal axis and of metabolic processes in this phenotype. In the current study the hypothalamic CRF system in Zucker lean (FA/?) and obese (fa/fa) phenotypes was functionally evaluated. Neither the stalk median eminence content of CRF or arginine vasopressin (AVP) nor hypothalamic levels of CRF or AVP mRNA differed in the lean and obese phenotypes. No phenotypic differences were observed in either basal or stimulated CRF release from hypothalamic tissue obtained from lean and obese rats. Furthermore, in intact rats the magnitude of pituitary-adrenal responses to various stressors was also similar between phenotypes. However, secretion of CRF and AVP into the hypophysial-portal circulation of obese rats was, respectively, 73% and 35% lower than that of the lean rats. Adrenalectomy was associated with a 3-fold elevation of hypophysial-portal CRF levels in obese rats compared to intact controls. Corticosterone infusion was more effective in suppressing portal CRF levels in adrenalectomized obese compared to adrenalectomized lean rats. Finally, neither CRF receptor number and affinity nor the magnitude of pituitary-adrenal responses to rat CRF challenge (1 micrograms, iv) differed between Zucker phenotypes. These observations lead us to infer that rats of the obese phenotype exhibit reduced hypothalamic CRF tone due to dysregulation of the HPA axis at a site proximal to the hypophysiotropic CRF system that mediates glucocorticoid feedback regulation.     

Endogenous opioids modulate ventilation in the obese Zucker rat

This study evaluated the modulatory role of endogenous opioids on ventilation in young and mature, lean and obese male Zucker rats. Naloxone, an opioid receptor antagonist, and saline (control) were administered subcutaneously to awake rats, and ventilation in air and in response to an hypoxic and an hypercapnic gas challenge measured. In response to naloxone young,obese but not lean rats exhibited a marked increase of ventilation in all three conditions. Older obese Zucker rats that were morbidly obese breathed at a frequency of over 200 breaths per minute and showed only a modest increase of ventilation in response to naloxone. Older lean rats increased ventilation with naloxone only when exposed to hypercapnia. Unlike the stimulatory effects hypoxia and hypercapnia had on ventilation in older, lean rats, the ventilatory responses of the obese, older rats to hypoxia and to hypercapnia were blunted. We conclude that the obese Zucker rat may be a good animal model to assess how chest wall loading and endogenous opioids interact in the development of ventilatory control abnormalities.     

Contribution of hyperinsulinemia to modulation of lipoprotein lipase activity in the obese Zucker rat

This study was designed to assess the contribution of hyperinsulinemia to the maintenance of high adipose and low muscle lipoprotein lipase (LPL) activity in the obese Zucker fa/fa rat. Insulinemia in obese Zucker rats was reduced for 4 days with a single injection of low-dose streptozotocin (STZ). Saline-injected intact obese (obese-INT) and STZ-injected obese (obese-STZ) rats were compared with a lean Fa/? reference group. LPL activity was assessed after a 12-hour fast, with or without a 1-hour refeeding period. Fasting serum insulin levels were 17-fold higher in obese-INT versus lean rats and were reduced to 60% of obese-INT levels in obese-STZ animals. In the postprandial state, serum insulin levels remained low in obese-STZ rats and were similar to the values in lean animals, whereas insulinemia increased in the obese-INT group to 18-fold the levels in lean rats. Serum glucose, nonesterified fatty acid (NEFA), and triglyceride levels, which were higher in obese-INT versus lean rats, were further increased in the obese-STZ group. Tissue weights of obese rats were unaffected by STZ treatment. Fasting LPL specific activity was higher in white adipose tissue ([WAT] +87%) and brown adipose tissue ([BAT] +167%) of obese-INT versus lean rats. Reducing the insulinemia in obese-STZ rats reduced fasting enzyme activity to the levels in lean animals in both WAT and BAT. Insulinemia and adipose LPL activity were positively correlated in the fasted state. Acute food intake increased WAT LPL activity in lean animals, but not in obese animals. Soleus LPL activity was lower in obese-INT compared with lean rats and was further decreased in obese-STZ animals. Heart LPL was decreased only in obese-STZ rats compared with the lean group. LPL in muscle tissue was not correlated with insulinemia, but an inverse relationship was found between serum NEFA levels and enzyme activity. It is concluded that in the obese Zucker rat, hyperinsulinemia is responsible for the maintenance of elevated basal LPL activity in adipose tissue independently of fat mass, whereas muscle enzyme activity appears to be more strongly and inversely related to the availability or tissue utilization of lipid substrates.     

Increased NAD(P)H fluorescence with decreased blood flow in the steatotic liver of the obese Zucker rat

The steatotic liver is characterized by deranged intrahepatic microvasculature that is believed to predispose it to ischemia-reperfusion injury. The aim of this study was to investigate the distorted hepatic hemodynamics and its impact on the redox status of the steatotic liver. Hepatic hemodynamic parameters, hepatic microcirculatory perfusion (HMP), and in vivo reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] autofluorescence, which reflects the mitochondrial redox status and tissue oxygen levels, were measured in obese (n = 7) and lean Zucker rats (n = 7). Portal venous and total hepatic blood flow per unit of liver weight were found to exhibit a 37.9% and 35.9% reduction, respectively, in the steatotic liver compared to the nonsteatotic liver of the lean group (P < 0.0001) as was HMP (obese, 96.1 +/- 18.1 PU; lean, 143.8 +/- 12.0 PU, P < 0.05) that showed a 33.2% decrease in the former. Hepatic arterial resistance, however, was 38.7% lower in the obese rat (83.1 +/- 9.1 mmHg. ml(-1). min) than in the lean rat (135.5 +/- 15.8 mmHg. ml(-1). min) (P < 0.05). NAD(P)H fluorescence intensity was significantly elevated in the steatotic liver (0.16 +/- 0.001 aU) compared with the lean one (0.14 +/- 0.007 aU) (P = 0.014). Our results suggest that, in response to a reduced portal venous blood flow, there is a significant decrease in hepatic arterial resistance that, nevertheless, cannot completely compensate for the drop in overall hepatic perfusion and oxygenation of the microvascular bed in the steatotic liver of the obese Zucker rat.     

Restoration of coronary collateral growth in the Zucker obese rat:

The metabolic syndrome (MS), a condition characterized by several risk factors for coronary artery disease, including obesity, is associated with endothelial dysfunction and oxidative stress. Because proper endothelial function is essential for signaling of certain growth factors (vascular endothelial growth factor, VEGF) we hypothesized that coronary collateral growth (CCG) is impaired in a model of the MS. To test this hypothesis, we stimulated coronary collateral growth in pre-diabetic Zucker obese fatty rats (OZR) and lean littermates (LZR) by using episodic, repetitive ischemia (RI: 40 s left anterior descending arterial occlusion, 24/d for 14 d). Myocardial blood flow (MBF, radioactive microspheres) was measured in the normal (NZ) and collateral-dependent (ischemic) zones (CZ); CCG was assessed as a ratio of CZ/NZ flow (unity represents complete restoration of CZ flow). In LZR, CZ/NZ ratio increased from 0.18 ± 0.03 to 0.81 ± 0.07 after RI (P < 0.05). In contrast, in OZR rats CZ/NZ did not increase after RI (0.15 ± 0.04 vs 0.18 ± 0.04). To rectify abrogated collateral growth in OZR, we employed VEGF gene therapy (VEGF-transduced, strained-matched, cultured vascular smooth muscle cells [cVSMCs], delivered intracoronary). VEGF therapy modestly but not significantly increased the CZ/NZ ratio after RI (0.16 ± 0.05 vs 0.33 ± 0.06). To facilitate VEGF signaling,we reduced oxidative stress by transducing cVSMCs with both ecSOD and VEGF. This increased the CZ/NZ flow ratio after RI to 0.52 ± 0.04 (p < 0.05 vs. OZR [(0.19 ± 0.04]) indicating partial restoration of collateral growth. Our results demonstrate that coronary collateral growth is impaired in a model of the metabolic syndrome and that growth factor gene therapy with VEGF is made far more effective when it is coupled to an intervention that reduces oxidative stress. Supported by NIH grants HL32788 (WMC), HL65203 (WMC), HL73755 (WMC), and COBRE RR018766 (FP, PR,WMC), and American Heart Association Scientist Development Grant (PR), AHA Postdoctoral Fellowship Award (NH, PR).     

Reduced tissue androgen receptors in the congenitally obese male Zucker rat

Androgen receptor (AR) concentrations were measured in the prostates and livers of the infertile congenitally obese male Zucker rat and their fertile non-obese litter-mates. In obese rats AR concentrations were significantly lower in both the liver (p less than 0.001) and the prostates (p less than 0.01) of the obese rats compared with the non-obese rats, despite similar plasma testosterone concentrations in both phenotypes. These findings suggest that a potential cause for the infertility observed in the obese Zucker rat is a reduced sensitivity of target tissues to circulating androgens.     

Serum testosterone and gonadotropins in the genetically obese male Zucker rat

Serum concentrations of testosterone, LH, and FSH were measured in obese and lean male Zucker rats 1--6 months of age. The serum concentrations of testosterone were lower in the 2-, 3-, and 4-month-old obese rats than in their lean controls. There was no correlation between fertility and serum testosterone concentration in the obese ras. Serum LH and FSH concentrations were normal, as was the response of the obese rats to LHRH stimulation. Testes morphology differed between the obese and the lean rats. Although the Leydig cells of the obese rat testes were hypertrophied, they contained numerous fat droplets and few signs of active hormone synthesis. These data suggest that the obese male Zucker rat has a defect in testicular testosterone production but has a normal pituitary response to hypothalamic stimulation.     

Hypertension-independent microvascular rarefaction in the obese Zucker rat model of the metabolic syndrome

OBJECTIVE: To test the hypothesis that reduced skeletal muscle microvessel density (MVD) in obese Zucker rats (OZR) is independent of chronic elevations in mean arterial pressure (MAP). METHODS: Microvessels in cross sections of gastrocnemius muscle from lean Zucker rats (LZR) and OZR were labeled with Griffonia simplicifolia I lectin, visualized with fluorescence microscopy and vessel number within sections was determined using imaging software. Rats were used at different ages to assess correlations between the temporal development of hypertension and microvascular rarefaction. Additionally, rats were chronically treated with captopril or hydralazine as antihypertensive therapies to examine the development of microvascular rarefaction in the absence of elevated blood pressure. RESULTS: MVD in muscle of OZR was reduced by approximately 17% versus LZR by 10-11 weeks of age, prior to any elevation in MAP. By 15-17 weeks, OZR exhibited a approximately 23% reduction in MVD and a approximately 25 mmHg increase in MAP. Treatment with hydralazine prevented elevated MAP in OZR, although this was not associated with an improved MVD. Captopril treatment also prevented elevated MAP in OZR, although a partial recovery of MVD toward normal levels was observed. This observation was associated with an improved insulin resistance. CONCLUSIONS: These results suggest that microvessel rarefaction in skeletal muscle of OZR manifesting the metabolic syndrome does not depend on an elevated mean arterial pressure and that other factors associated with the metabolic syndrome, possibly insulin resistance, may underlie the progressive reduction in MVD in these animals.     

The effect of dietary restriction on gastrin secretion in the obese Zucker rat

It has been hypothesized that G-cell hyperplasia secondary to increased food consumption in the obese Zucker rat was responsible for the hypergastrinemia observed in vivo and from the isolated perfused stomach preparation. This possibility was investigated in pair-feeding experiments wherein the food intake of obese animals was restricted to match that of lean littermates from 5 to 8 weeks of age. Dietary restriction reduced the antral G-cell population of the obese rat to a similar level as that seen in lean animals, supporting the view that hyperphagia is the trigger for G-cell hyperplasia. However, basal gastrin levels measured in vivo and in vitro from the stomach preparation of the pair-fed obese animals were not significantly lower than those of obese animals fed ad libitum. Thus, abnormal feeding behavior in the obese phenotype cannot be directly related to gastrin hypersecretion and G-cell hyperplasia is not the primary cause of hypergastrinemia.     

Glucocorticoid receptor numbers in the brain and liver of the obese Zucker rat.

Scatchard analysis of ligand binding activity has been used to investigate type I and type II glucocorticoid receptor subtypes in the hippocampus, hypothalamus and liver of lean and genetically obese fa/fa rats. Despite normal levels of corticosterone in the hypothalamus and hippocampus of fa/fa rats, maximum binding to both type I and type II receptors was increased in the hippocampus and hypothalamus. The KD value of type I receptors was normal in fa/fa rats (lean 0.13 nM, obese 0.15 nM) whereas the KD for corticosterone binding to type II receptors was increased (lean 0.48nM, obese 1.02nM). Adrenalectomy increased Bmax of type II receptors in lean rats in a time dependent manner but had no effect on binding to receptors of obese rats after the initial clearance of endogenous bound ligand. KD values were not altered by adrenalectomy in either genotype. There were no differences in binding of corticosterone to hepatic receptors of lean and obese rats seven days after adrenalectomy. The data suggests that glucocorticoid binding to type I and type II receptors is abnormally regulated in the brains of fa/fa rats.     

Pioglitazone-induced increase of insulin sensitivity in the muscles of the obese Zucker fa/fa rat cannot be explained by local adipocyte differentiation

Summary Thiazolidinediones are potent antidiabetic compounds, which act by enhancing peripheral insulin sensitivity. They are also activators of the peroxisome proliferator activated receptor γ in adipose tissue. Pioglitazone induces in vivo adipocyte differentiation in the obese Zucker fa/fa rat and hence the capacity of adipose tissue to utilize glucose. Nevertheless, muscles are the major site for insulin-mediated glucose disposal. The increase of muscle glucose utilization under thiazolidinedione treatment could be secondary to local adipose tissue differentiation. This possibility is supported by the fact that a thiazolidinedione-induced myoblast conversion into adipocytes has been described in vitro. To address this problem, we have studied the in vivo effect of a pioglitazone treatment on insulin-induced glucose utilization and the expression of genes exclusively expressed in mature adipocytes in three muscles differing by their fibre composition in Zucker (fa/fa) rats. Whereas pioglitazone treatment increased insulin-stimulated glucose utilization to the same extent in all muscle types, an adipocyte differentiation was only present in the oxidative muscle, the soleus. Soleus muscle was also the only one in which the presence of genes specific for adipose tissue could be detected before the pioglitazone treatment. There was no detectable expression of adipocyte specific genes in the extensor digitorum longus or in the epitrochlearis muscles before or after the drug treatment. We conclude that pioglitazone effects on muscle glucose metabolism cannot be due to a local adipocyte differentiation, and that the conversion of myoblasts into adipocytes under thiazolidinedione stimulation observed in vitro is, if it exists, a marginal phenomenon in vivo. [Diabetologia (1998) 41: 963–968] Received: 22 December 1997 and in final revised form: 9 March 1998     

Primary culture of adipoblasts from obese and lean Zucker rat adipose tissue

Adipocyte precursors derived from the epididymal fat pads of young adult lean (FaFa) and obese (fafa) Zucker rats were established in primary culture. The two types of culture were used to assess intrinsic cellular differences in proliferative capacity, lipoprotein lipase (LPL) activity and triglyceride (TG) accumulation related to genotype. Proliferative capacity was similar over seven days in vitro in lean- and obese-derived cultures. Heparin-releasable LPL activity was significantly greater in lean- than in obese-derived cultures grown in media supplemented with penicillin and streptomycin (pen-strep). However, when grown in media supplemented with cephalothin, heparin-releasable and total LPL activity increased significantly in obese-derived cultures and equalled LPL activity in lean-derived cultures. Substantial LPL activity was measurable in both types of culture at confluence, before exposure to media that promoted lipid-filling. TG accumulation was significantly greater in lean-than in obese-derived cultures in the presence of pen-strep but was similar in both culture types grown in cephalothin. These data support our hypothesis that the fa gene may affect mechanisms of protein turnover regulation, since pen-strep, but not cephalothin, has inhibitory effects on mammalian protein synthesis and degradation. The substantial LPL activity present in confluent, but unconverted cultures, suggests that some percentage of cells in the confluent monolayers are adipoblasts.