Physiological difference between obese (fa/fa) Zucker rats and lean Zucker rats concerning adiponectin

Obese (fa/fa) Zucker rat is a spontaneous genetic obesity model and, by comparison with lean Zucker rat, exhibits hyperphagia, hyperinsulinemia, and hyperlipidemia. The aim of this study was to examine the physiological difference concerning adiponectin between obese (fa/fa) Zucker rats and control lean Zucker rats. We therefore measured plasma adiponectin level and analyzed adiponectin and adiponectin receptor 1 mRNA expression in retroperitoneal white adipose tissue (RT WAT), brown adipose tissue (BAT), liver, and soleus muscle. We also examined the tissue mRNA expression of peroxisome proliferator-activated receptor alpha (PPAR alpha), PPAR delta, and PPAR gamma, which regulate adiponectin expression sensitivity to a PPAR gamma agonist shown by brown adipocytes from obese (fa/fa) Zucker rats and lean Zucker rats, by measuring adiponectin release from these cells. Plasma adiponectin levels of obese (fa/fa) Zucker rats were significantly higher than those of lean Zucker rats. Adiponectin mRNA expression levels in RT WAT were lower in obese (fa/fa) Zucker rats than in lean Zucker rats, but those in BAT were higher. Adiponectin receptor 1 expression levels in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats were lower than in lean Zucker rats. The expression level of PPAR alpha, PPAR delta, and PPAR gamma in BAT was lower in obese (fa/fa) Zucker rats than in lean Zucker rats. Moreover, the PPAR gamma agonist increased adiponectin release only from the brown adipocytes isolated from lean Zucker rats. It is the conclusive difference between obese (fa/fa) Zucker rats and lean Zucker rats that plasma adiponectin levels of obese (fa/fa) Zucker rats are significantly higher than those of lean Zucker rats. Moreover, we clarified that mRNA expression level of adiponectin receptor 1 in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats is low despite high plasma adiponectin level, and low expression of PPARs in BAT leads to less sensibility of adiponectin release from brown adipocytes to a PPAR gamma agonist in obese (fa/fa) Zucker rats.     

The onset of liver glycogen synthesis in fasted-refed lean and genetically obese (fa/fa) rats

Summary Lean and genetically obese (fa/fa) rats were fed ad libitum, or fasted for 17 h and then meal-fed for varying time intervals. During refeeding, glucose-6-phosphatase activity of lean rats declined to the low value that was present in livers of fasted obese rats and which remained unchanged in the obese group during the meal. Refeeding also resulted in increases in hepatic concentrations of glucose-6-phosphate and fractose-6-phosphate, fructose 1,6-bisphosphate, fractose-2,6-bisphosphate, -glycerophosphate, pyruvate and lactate in lean and obese rats, absolute values being higher in the fasted obese than in the fasted lean group. Obese animals had higher postprandial portal blood insulin, glucose and lactate concentrations than lean animals. In spite of this, the rate of hepatic glycogen deposition was the same in both groups and was accompanied by similar glycogen synthase a levels. Following refeeding, phosphorylase was transiently inactivated in livers of lean but not of obese animals, while glycogen synthase was inactivated in both groups. The data suggest that (1) in lean animals refeeding was associated with a stimulation of liver glycolysis, presumably by insulin; (2) in fasted obese rats hepatic glycolysis was already in a stimulated state and was only slightly enhanced further after the meal, in keeping with their unaltered hyperinsulinaemia; (3) there was an increased turnover of liver glycogen or a resistance to insulin stimulation of glycogen synthesis in fa/fa rats during refeeding.     

Acute intravenous corticotropin-releasing factor administration: effects on insulin secretion in lean and genetically obese fa/fa rats.

The effect of an i.v. administration of different doses (250, 500, and 1000 pmol) of ovine CRF (oCRF) on plasma glucose and insulin levels in lean and genetically obese fa/fa rats was investigated. In both phenotypes, i.v. CRF promoted a rapid (peak at 1 min) transient doubling of basal insulin levels without a concomitant change in glycemia. The dose-dependency of this early insulin response was bell-shaped in both lean and obese animals, with a maximal response at 500 pmol oCRF. After this early rise in insulinemia, glycemia increased in a dose-dependent manner in both lean and obese rats. It was accompanied by a bell-shaped insulin response in lean rats, while such a response was linear in obese rats. The early transient stimulatory effect of CRF on plasma insulin levels could not be prevented by the prior administration of an anti-CRF serum or the alpha-helical CRF-(9-41) antagonist, although administration of either one of these compounds was effective in preventing the CRF-induced changes in the pituitary-adrenal axis. The effect of CRF on the early insulin response was, however, completely suppressed by an acute cholinergic blockade (i.v. injection of atropine). It is suggested that i.v. CRF administration mimics the reflex, cephalic phase insulin secretion. Such cephalic phase insulin output is known to play a role in oral glucose tolerance and may be of physiopathological importance in the Zucker rat strain.     

Acute effects of fat and carbohydrate on metabolic rate in normal, cold-acclimated and lean and obese (fa/fa) Zucker rats

The rise in metabolic rate after intragastric feeding with fat and carbohydrate was enhanced in cold-acclimated (5 degrees C) rats and diminished in warm-acclimated (30 degrees C) rats compared to controls (24 degrees C), but the response was largest in cold-acclimated animals intubated with fat. These acute effects of nutrients were almost completely abolished by beta-adrenergic blockade with propranolol in all groups, while the parasympathetic antagonist atropine sulphate enhanced the responses in control rats, but had little effect in cold-acclimated animals. Feeding carbohydrate produced similar increases in interscapular brown adipose tissue (BAT) temperature in control and cold-acclimated rats, but fat caused a much greater rise in the latter group. The thermic effects of both nutrients were lower in genetically obese Zucker rats than in their lean littermates. Atropine slightly increased the thermic responses to fat and carbohydrate in the lean Zucker rats and caused marked potentiation in obese rats intubated with fat, but did not alter the effect of carbohydrate in the obese animals These data suggest that the size of the acute rise in metabolic rate after fat and carbohydrate is dependent on the thermogenic capacity of the animal. The response to fat was particularly large in cold-acclimated rats, where BAT activity is high, possibly due to a direct action of fat on the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

The parasympathetic nervous system and glucocorticoid-mediated hyperinsulinaemia in the genetically obese (fa/fa) Zucker rat

Lean (Fa/-) and genetically obese (fa/fa) Zucker rats were adrenalectomized at 18 days of age (3 days before weaning) before the onset of hyperinsulinaemia. At 40-41 days of age, basal and glucose-stimulated insulin concentrations did not differ significantly between lean and obese rats. Plasma insulin and glucose concentrations were higher in both phenotypes 24 h after administration of corticosterone (2.0 mg at 12-h intervals). Corticosterone-treated obese rats had higher basal and glucose-stimulated insulin levels than similarly treated lean animals, although plasma glucose concentrations did not differ between phenotypes. The basal plasma insulin concentration of obese rats treated with corticosterone for 24 h was reduced 15, 30 and 45 min after injection of atropine (0.3 mg) without any significant change in the plasma glucose level. Injection of atropine (0.3 mg) 20 min before a glucose load prevented the greater increment in plasma insulin concentration of corticosterone-treated obese rats compared with similarly treated lean animals. Atropine administration (0.3 mg) to intact obese rats at 40 days of age reduced, but did not abolish, their hyperinsulinaemia compared with intact lean animals. It is concluded that (1) pre-weaning adrenalectomy prevents the development of hyperinsulinaemia in genetically obese rats, (2) corticosterone replacement for only 24 h restores the hyperinsulinaemia of obese rats, (3) the differential effects of corticosterone on insulin secretion by lean and obese rats are mediated by the parasympathetic nervous system and (4) the parasympathetic nervous system contributes to, but is not the only cause of, hyperinsulinaemia in intact obese rats.     

Vagal mediation of corticotropin-releasing-factor-induced increase in insulinemia in lean and genetically obese fa/fa rats

The effects of intracerebroventricular (i.c.v.) administration of corticotropin-releasing factor (CRF) on plasma glucose and insulin levels were investigated in lean Zucker (FA/FA) rats; i.c.v. CRF induced a rapid (within 1 min), marked, but transient increase in insulinemia that was not accompanied by any change in glycemia. At a time when insulinemia already returned toward basal values, glycemia started to increase. The effect of i.c.v. CRF in stimulating plasma insulin levels was dose-dependent and could be blocked by pretreatment of the animals with atropine methylnitrate. Similar results were obtained when studying the acute effects of i.c.v. CRF in genetically obese (fa/fa) rats. Although, in absolute values, the amount of insulin released in response to i.c.v. CRF was higher in obese than in lean animals, it was similar in both groups of rats, when expressed as fold increase over basal insulin levels. In summary, i.c.v. CRF elicits a rapid, vagally mediated stimulatory effect on insulin secretion both in lean and genetically obese fa/fa rats with no qualitative or quantitative difference between the two groups of animals. The site(s) of action of this CRF effect on insulinemia remains to be elucidated.     

Lifelong sequential changes in glucose tolerance and insulin secretion in genetically obese Zucker rats (fa/fa) fed a diabetogenic diet

Life-long sequential changes in glucose tolerance and insulin secretion were investigated in genetically obese Zucker rats (fa/fa) fed a diabetogenic diet rich in lard and sucrose. Comparisons were made with lean littermates (Fa/-) receiving normal chow diet. At 3-month intervals, seven to nine lean and obese rats had two permanent venous catheters implanted, allowing stress- and pain-free sampling of blood before, during, and after substrate administration. Intravenous glucose, iv arginine, and oral glucose tolerance were tested. The obese rats progressively developed hyperglycemia and severe hyperinsulinemia; their basal glycemia reached 8.8 +/- 1.1 vs. 5.8 +/- 0.2 mmol/liter in the lean rats at 46 weeks of age; respective insulinemia was 287.7 +/- 61.9 and 18.1 +/- 2.8 mU/liter (mean +/- SD). In the obese rats a distinct loss in glucose tolerance was seen with progression of age in spite of rising stimulated insulin secretion, which suggests progressive development of insulin resistance without exhaustion of B-cell secretory capacity. Absence of insulin deficiency was also suggested by immunohistochemical staining of pancreatic tissue specimens from obese rats, which showed large populations of insulin-containing cells. Like the obese animals, lean rats exhibited a decrease in insulin sensitivity with age. Relating basal individual glycemia and insulinemia, a rise by 1 mmol/liter in glycemia was associated with a 8.8-fold rise in basal insulinemia in lean rats, but only with a 1.8-fold increase in obese rats. Similar correlations for stimulated glycemia and insulinemia suggest impaired glucose sensitivity of pancreatic B-cells in obese vs. lean rats. In conclusion, hyperglycemia and hyperinsulinemia in insulin-resistant obese Zucker rats on a diabetogenic diet are not characterized by quantitatively deficient B-cell secretory capacity, but, rather, by impaired B-cell sensitivity to glucose with qualitatively intact regulation of glycemia and insulinemia at elevated plasma concentrations.     

Pancreatic hypersensitivity to glucose by young obese Zucker rats (fa/fa)

Insulin secretory response to glucose was investigated in 5- to 6-week-old male Zucker obese (fa/fa) and lean (Fa/Fa) rats using a pancreatic perfusion procedure. Blood glucose response to fasting was studied in lean and obese animals over 24 hours. Plasma glucose was slightly elevated in pentobarbital-anesthetized obese rats. However, plasma insulin was 4.6 times greater than that of leans. A hypoglycemic glucose stimulus (75 mg/dL) caused pancreata from obese animals to release 6 times more insulin than lean animals. Stimuli of 125 mg/dL (normoglycemic) and 600 mg/dL (hyperglycemic) caused hypersecretion of 8 and 5 times, respectively. Hypersecretion was not accounted for solely by the twofold increase in pancreatic insulin content. Obese animals had steeper decreases in plasma glucose than lean controls during seven to 13 hours of fasting. Hypersecretion by pancreata from young obese rats to physiological levels of glucose may result in hyperphagia in order to maintain normoglycemia.     

Central corticotropin-releasing factor administration prevents the excessive body weight gain of genetically obese (fa/fa) rats

The genetic obesity of the fa/fa rat is due to or accompanied by perturbances in the autonomic nervous control of different target tissues (e.g. endocrine pancreas, brown adipose tissue). These disorders are likely to be secondary to central dysregulation(s), which could lie somewhere within or in relationship with the hypothalamus. In view of the reported effects of CRF in stimulating sympathetic nerve-mediated mechanisms, while inhibiting vagus nerve-mediated ones, ovine CRF (oCRF) was administered for 7 days into the cerebral ventricles of fa/fa rats at a dose (5 micrograms/day) that did not affect the pituitary-adrenal axis. oCRF treatment stopped the excessive weight gain of the obese animals; oCRF-treated animals gained only 1 g over 6 days, while the vehicle-treated ones gained 29 g (P = 0.044). The oCRF effect was unrelated to changes in food intake, as the two groups were pair-fed. oCRF-treated obese rats were characterized by a decrease in basal hyperinsulinemia, increases in brown adipose tissue weight and activity, and decreases in hepatic glycogen content and epididymal fat pad weight. It is suggested that intracerebroventricular oCRF administration to obese fa/fa rats prevents the 10-15% increase in body weight observed in vehicle-infused obese rats within 1 week by modulating the impaired autonomic nervous control of different target tissues. This does not occur in lean rats.     

Decreased insulin binding to pancreatic islets of genetically obese (fa/fa) rats

Binding of insulin and insulin secretion were studied in isolated pancreatic islets of homozygous obese fa/fa rats, their lean littermates (Fa/?) and Wistar rats. Despite normoglycemia fa/fa rats exhibit hyperinsulinemia. Glucose-induced insulin secretion from pancreatic islets in vitro was increased by more than 50% in fa/fa rats compared with islets of lean littermates and normal Wistar rats when calculated per microgram islet protein. Exogenous insulin inhibited glucose (16.7 mM)-induced insulin secretion in islets of either of these rats, and maximum inhibition was rather the same (secretion was reduced by 62.3-65.6%). However, the EC50 (half-maximal effective concentration) for inhibition was increased in fa/fa rats being 1.4 +/- 0.1 nM compared with 0.6 +/- 0.2 and 0.5 +/- 0.2 nM in lean littermates and Wistar rats, respectively (P less than 0.05 vs. fa/fa rats). Islets of fa/fa rats found 24% less [125I]insulin (P less than 0.01) than islets of lean littermates and of Wistar rats. Scatchard analysis of data of displacement of [125I]insulin binding by native insulin showed 2 binding sites; a decrease in the number of high affinity insulin binding sites (Bmax) from 4.2 +/- 1.3 and 4.7 +/- 1.6 fmol/mg protein to 2.6 +/- 0.7 fmol/mg protein was calculated when islets of lean littermates and normal Wistar rats were compared to islets of fa/fa rats. The Kd of the high affinity binding site was not changed (0.77 +/- 0.06, 0.78 +/- 0.11 and 0.61 +/- 0.14 nM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipogenesis in situ in the genetically obese Zucker fatty rat (fa/fa): Role of hyperphagia and hyperinsulinaemia

Summary In situ fatty acid synthesis has been measured with ³H₂O in anaesthetised lean and obese Zucker (fa/fa) rats. The accumulation of fatty acids was increased in both the liver and adipose tissue of young fa/fa rats as a result of both an increased rate of lipogenesis and an increase in tissue mass. Whereas total hepatic lipogenesis increased with age, total adipose tissue lipogenesis decreased in older fa/fa rats. Experiments with hepatectomized rats showed that the liver was the major site of the excess fatty acid synthesis in fa/fa rats. The enhanced rate of lipogenesis in fa/fa rats was abolished by either pairfeeding or streptozotocin treatment. The results suggest that the increased fatty acid synthesis in fa/fa rats is secondary to the hyperphagia, hpyerinsulinaemia, and increased mass of hepatic and adipose tissues.The authors are grateful to the M. R. C. for award of a project grant.     

Effect of pertussis toxin on islet insulin secretion in obese (fa/fa) Zucker rats.

We used isolated islets of lean and obese Zucker rats to determine whether inhibitory pathways mediated by pertussis toxin-sensitive guanyl nucleotide-binding (Gi) proteins contribute to hyperinsulinemia in obese rats. Epinephrine (10(-4) M) and somatostatin (10(-7) M) inhibited insulin secretion by +/- 75% in lean and fa/fa rats. Overnight culture of islets with pertussis toxin (300 ng/ml) enhanced insulin release more in lean (+/- 120%) than obese (+/- 60%) rats. In lean rats incubation of pertussis toxin-treated islets with epinephrine resulted in lower immunoreactive insulin release (p = 0.0005) than pertussis toxin-treated islets without epinephrine. However, in obese rats pertussis toxin treatment reversed this inhibition. Pertussis toxin completely reversed inhibition by somatostatin in both phenotypes. Galanin had no effect on insulin secretion. Cellular cAMP content was similar in lean and obese rats. Inhibitory hormones had no effect on cAMP production. We conclude that islets of obese rats respond normally to inhibitors of insulin release. Reversal of somatostatin-induced inhibition by pertussis toxin indicates normal function of Gi in obese rats. A subtle difference in sensitivity to pertussis toxin between lean and obese islets was noted.     

Dysregulation of glucose transport and transporters in perfused hearts of genetically obese (fa/fa) rats

Summary The regulation of glucose transport in normal and insulin-resistant obese rat hearts have been studied by measuring glucose transport via the efflux of labelled 3-0-methyl-D-glucose. Glucose transporters in obese rat hearts were also investigated using the labelled cytochalasin B-binding assay. Basal, and insulin — or increasing workload-induced stimulation of glucose transport was decreased in obese rat hearts compared to those of normal ones. Total number of glucose transporters (plasma membrane plus microsomal ones) was about half that previously reported for normal rat hearts. Insulin or workload favoured the translocation of glucose transporters from an intercellular pool (microsomes) to the plasma membrane, as they do in normal rats. Due to the measured decrease in total number of transporters of obese rat hearts, those present in the plasma membrane (under basal conditions, or following stimulation by insulin or workload) were less than those previously found in normal rat hearts tested under identical conditions. In obese rat hearts, regulation of plasma membrane transporters was perturbed. The Hill coefficient (an index of positive cooperativity amongst glucose transporters) was paradoxically decreased by insulin while leaving affinity values unaltered. The Hill coefficient was unaltered by workload, although the affinity values were increased compared to respective controls. To sum up, obese rat hearts have decreased total transporter number, and although the two stimuli studied favour the translocation of available transporters, they fail to activate them adequately once present in the plasma membrane.     

Severe hepatic and peripheral insulin resistance as evidenced by euglycemic clamps in genetically obese fa/fa rats

The action of insulin on glucose metabolism and hepatic glucose production was studied in vivo over a wide range of insulin concentrations in lean and genetically obese (fa/fa) rats, using the euglycemic clamp technique. While total glucose metabolism was stimulated 3-fold by insulin in lean animals (half-maximal stimulation at 400 microU/ml insulin), the hormone had no significant effect on glucose metabolism in obese animals, whatever the concentration used. In lean rats, the endogenous (i.e. hepatic) glucose production was completely suppressed at a steady state insulin concentration of about 360 microU/ml. In obese rats, an insulin concentration as high as 10,000 microU/ml was needed to suppress the hepatic glucose production. These results suggest that, in obese rats 1) basal plasma insulin levels appear to maximally stimulate peripheral glucose metabolism, and the presence of postreceptor defects prevents any further stimulatory effect of the hormone on glucose metabolism; 2) grave impairments of the action of insulin on hepatic glucose production are present, despite a normal responsiveness obtained at pharmacological concentrations of the hormone. These hepatic alterations could be due to postbinding and/or intracellular defects, as well as to defects, yet to be defined, of the homeostasis of insulin counterregulatory hormones.     

The effects of gonadectomy on glucose tolerance of genetically obese (fa/fa) rats: influence of sex and genetic background.

In both humans and rodents the occurrence and severity of obesity-associated non-insulin dependent diabetes mellitus (NIDDM) may be influenced by both gonadal hormones and genetic background. Early gonadectomy (at 3-5 days of age) of female and male Wistar diabetic fatty (WDF) rats and of male Zucker rats allowed us to examine these effects in genetically obese rats carrying the fatty (fa) gene. Impairment of glucose tolerance and insulin sensitivity by obesity, and amelioration or exacerbation (in the case of female rats) of this impairment by gonadectomy were assessed by intragastric glucose tolerance tests when the rats reached adulthood. Both glucose tolerance and insulin sensitivity were significantly deranged in obese WDF rats of both sexes and in obese male Zucker rats compared to lean controls of the same sex and strain. Obese male WDF rats were less glucose tolerant and insulin sensitive than were obese male Zucker rats. Glucose intolerance was not ameliorated by castration in lean or obese male WDF or Zucker rats. Insulin sensitivity was significantly improved by castration in obese male rats of both strains, as fasting plasma insulin levels and total areas under the insulin curves were significantly reduced compared to obese sham-operated controls. This effect was greater in the Zucker than in the WDF male rats. Castration significantly decreased the insulin response areas in obese male Zucker rats, but did not alter those of the obese male WDF rats. Ovariectomy did not alter glucose homeostasis of obese female WDF rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dysregulation of glucose transport in hearts of genetically obese (fa/fa) Rats

Overall D-glucose metabolism and 3-0-methylglucose transport were measured in the perfused heart preparation of lean and genetically obese (fa/fa) rats. Absolute values of basal and insulin-stimulated glucose metabolism were decreased in hearts of 15-week-old obese rats when compared to lean age-matched controls. Basal and maximally stimulated (i.e., by the combined addition of insulin and increasing perfusion pressure) 3-0-methylglucose transport was normal in hearts from young obese rats (5-week-old). However, when only one stimulus was used (insulin or increasing perfusion pressure alone), 3-0-methylglucose transport was stimulated to values that were lower than those of lean rats. Basal 3-0-methylglucose transport was four times lower in hearts from older obese rats (15-week-old) than in lean ones of the same age. At this age, stimulation of 3-0-methylglucose transport by insulin alone, by increasing perfusion pressure alone or by the combination of both stimuli, reached values in obese rats that were only half those of lean animals. It is concluded that: (a) in the early phase of the syndrome, the basal glucose transport system in hearts of obese rats is normal, but its response to stimulation becomes abnormal and; (b) at a later phase of obesity, the glucose transport system becomes abnormal even under basal conditions and its responsiveness to various stimuli is markedly impaired.     

Evaluation of inhibitory guanine nucleotide regulatory protein Gi function in hepatocyte and liver membranes from obese Zucker (fa/fa) rats and their lean (Fa/?) littermates

Summary Previous studies have shown that hepatocyte and liver membranes from insulin resistant animals exhibit an impairment of inhibitory guanine nucleotide binding regulatory protein, G_i function, such that a G_i defect may contribute towards the diabetic syndrome. In the current studies, it is shown that the demonstration of G_i activity in liver and hepatocyte membranes is dependent critically on the membrane preparation technique. A technique is defined that allows functional G_i activity to be demonstrated in liver and hepatocyte membranes from both lean (Fa/?) and obese (fa/fa) Zucker rats. Consequently, previous reports on the loss of G_i function in insulin resistant states require revaluation.     

Ocular complications in the old and glucose-intolerant genetically obese (fa/fa) rat

Summary Genetically obese fatty (fa/fa) male rats with abnormal oral glucose tolerance associated with initial hyperinsulinaemia as well as control lean (FA/FA) rats were investigated for the development of retinal microangiopathies. The animals were kept on a standard or sucrose supplemented diet. When tested at 60 weeks, the glucose intolerance of fa/fa rats was accompanied by an insulin response that was now either comparable to that of lean rats (standard diet) or close to nil (sucrose supplemented diet). At killing (68 weeks of age), retinal vasculature was examined by electron microscopy and morphological changes were quantitatively assessed by ultrastructural morphometry. A retinal microangiopathy was observed in all mutant animals which was more pronounced in the sucrose fed group, and which was characterized by: (1), an increase in focal thickenings and in nodules of the basement membrane adjacent to the perivascular glial cells; (2), a decrease in the number of pericyte nulei with concomitant signs of early degenerative cytoplasmic changes of pericytes; (3), an increase in the pinocytic activity of endothelial cells, indicative of presumptive changes in vascular permeability; (4), an increase in the number of intercellular endothelial junctions; (5), the presence of numerous stimulated platelets within capillaries. The fa/fa rat may thus be considered as a suitable model for studying the pathophysiology of ocular complications, in particular retinopathy accompanying non-insulin-dependent diabetes.