Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese Zucker rats.

The effect of subchronic metformin treatment on food intake, weight gain and plasma and tissue hormone levels was investigated in genetically obese male Zucker rats and in their lean controls. Metformin hydrochloride (320 mg/kg/day for 14 days in the drinking water) significantly reduced 24 hour food intake both after one and two weeks treatment in obese rats. In contrast, metformin had only a transient effect on food intake in lean animals. The reduced food intake was associated with body weight decrease, particularly in obese rats. Metformin markedly reduced also the hyperinsulinemia of the obese animals without altering their plasma glucose or pancreatic insulin content which may reflect an improved insulin sensitivity after metformin treatment. Metformin did not change plasma corticosterone levels or insulin and somatostatin concentrations in the pancreas. Metformin reduced pyloric region somatostatin content in lean rats. It is concluded that metformin has an anorectic effect and reduces body weight and hyperinsulinemia in genetically obese Zucker rat.     

CCK-resistance in Zucker obese versus lean rats

Obese Zucker rats are less sensitive to the satiety effect of CCK than lean litter mates. The present studies further characterised this CCK resistance. Subcutaneous injection of the CCK agonist caerulein dose-dependently decreased food intake in Zucker obese and lean rats whereas the CCK-B agonist gastrin-17 did not. Caerulein at 4 μg/kg, which resulted in CCK plasma bioactivity slightly above postprandial levels, decreased food intake in lean rats but not in obese rats. The decrease in food intake was also more marked at higher caerulein doses (20–100 μg/kg) in lean versus obese rats. In lean animals the satiety effects of the “near physiological” 4 μg/kg caerulein dose was abolished after blockade of vagal afferents with capsaicin, whereas the effects of higher caerulein doses were not. CCK-stimulated amylase secretion from pancreatic acini and binding capacity of ¹²⁵I- labelled CCK-8 were decreased in obese versus lean rats. The CCK-A antagonist loxiglumide at 20 mg/kg, a dose which abolished the action of all caerulein doses on food intake, failed to alter the food intake either in obese or in lean rats when given without an agonist. The results suggest that the satiety effects of “near physiological” doses of caerulein in lean rats are mediated by vagal afferents whereas pharmacological doses act via non-vagal mechanisms. The differences in CCK's satiety effect between lean and obese rats may be due to differences in CCK-receptor binding and action at peripheral vagal sites. However, the failure of the CCK-A antagonist to increase food intake questions whether any of the effects of exogenous CCK are of physiological relevance.     

Insulin resistance is accompanied by impairment of amylase-gene expression in the exocrine pancreas of the obese Zucker rat.

Insulin plays a major role in the control of pancreatic amylase biosynthesis. In this study we determined glucose metabolism by pancreatic acini as well as the pancreatic content of both amylase protein and amylase mRNA during development of insulin resistance in the obese Zucker rat. At age 4 weeks there were no abnormalities detected in the above parameters, although the obese animals were already hyperinsulinaemic. At 6 weeks glucose metabolism was decreased by 50% in acini from obese rats, whereas pancreatic amylase-gene expression was only slightly impaired. At 22 weeks glucose metabolism was decreased by 50%, amylase content by 55% and amylase mRNA by 60% in acinar tissue of obese rats. As expected, hyperinsulinaemia increased markedly with age. Thus development of severe insulin resistance was associated with impairment of amylase-gene expression. To decrease insulin resistance, one group of adult obese rats was treated with Ciglitazone for 4 weeks. A lowered plasma insulin concentration without alteration of food intake was taken as evidence of decreased insulin resistance. This was associated with normalization of glucose metabolism and a marked increase of both amylase content of pancreatic tissue and amylase mRNA. In conclusion, both the increase of insulin resistance with age and its partial reversal by Ciglitazone treatment appear to modulate pancreatic amylase-gene expression in the obese Zucker rat.     

Role of adrenal glands in the development of abnormal glucose and insulin homeostasis in genetically obese (ob/ob) mice

This study evaluates the role of adrenal hormones in the development of hyperinsulinaemia and impaired glucose homeostasis in genetically obese hyperglycaemic C57BL/6J ob/ob mice. Lean (+/?) and obese mice were bilaterally adrenalectomised or sham operated at 5 weeks of age, and glucose tolerance was examined after 7 and 14 days. Adrenalectomy temporarily reduced food intake and body weight gain in lean mice, and improved glucose tolerance without a significant change in plasma insulin concentrations at both intervals studied. In obese mice adrenalectomy permanently reduced body weight gain and food intake to values comparable with lean mice. Glucose tolerance was improved in adrenalectomised obese mice at both intervals studied, resulting in plasma glucose concentrations similar to adrenalectomised lean mice. Plasma insulin concentrations during the tolerance tests were reduced in adrenalectomised obese mice, but remained higher than in lean mice. Adrenalectomy did not improve the poor insulin response to parenteral glucose in obese mice. The results indicate that adrenal hormones play an important role in the development of glucose intolerance and contribute to the hyperinsulinaemia in obese (ob/ob) mice, in part by promoting hyperphagia.     

Neotomodon alstoni mice present sex differences between lean and obese in daily hypothalamic leptin signaling

This article compared the effects of spontaneous obesity on the daily profile in the relative amount of the leptin receptor (LepRb), and its output. That is the precursor Pro-opiomelanocortin (POMC) over a 24-hour period and compared with differences in locomotion and food intake in periods of artificial light. Differences between lean and obese mice were examined, as were sex differences. Body weight, food intake and locomotor activity were monitored in freely moving lean and obese mice. Hypothalamic tissue was collected at 5 h, 10 h, 15 h, 19 h and 24 h. Samples were analyzed by western blotting to determine the relative presence of protein for LepRb, STAT3 phosphorylation (by pSTAT3/STAT3 ratio) and POMC. Obese mice were 60% less active in locomotion than lean mice during the night. While both locomotor activity and food intake were noticeably greater during the day in obese mice than in lean mice, the hypothalamus in obese mice showed a lower relative abundance of POMC and reduced pSTAT3/STAT3 ratio and leptin receptors. Behavioral and biochemical differences were more evident in obese females than in obese males. These results indicate that obesity in N. alstoni affects hypothalamic leptin signaling according to sex.     

Factors influencing insulin and glucagon secretion in lean and genetically obese mice.

The control of insulin and glucagon secretion from isolated pancreatic islets of lean and genetically obese mice has been compared. The enlarged islets of obese mouse pancreas and islets of obese mouse pancreas and islets of obese mice maintained on a restricted diet manifested a greater response to glucose stimulation of insulin secretion than the lean mice islets. The glucagon content of the islets, the secretion of glucagon in a medium containing 150 mg% glucose and the stimulation of glucagon secretion by arginine did not differ significantly in the two groups. Adrenaline stimulated glucagon secretion in vitro from obese mice but not from lean mice. Antinsulin serum injections into obese mice increased the plasma glucagon levels about twofold and had no effect on glucagon levels in lean mice, although the level of hyperglycaemia was the same in both groups. It is suggested that the suppression of glucagon release by glucose requires a higher concentration of insulin in the obese mouse pancreas than in lean mice.     

A sex difference in the effect of CCK-8 on food and water intake in obese (ob/ob) and lean (+/+) mice

Male and female ob/ob and +/+ mice were tested with CCK-8 (1, 2, 4 and 8 micrograms/kg) administered 15 min prior to 30-min access to solid food after 17.5 hr food deprivation and 15 min prior to 30-min access to water after 17.5 hr water deprivation. The threshold dose for suppressing 30-min food intake was 2 micrograms/kg for all mice. Larger doses of CCK-8 inhibited food intake in male obese and lean mice in a linear fashion. The dose-response relationship for female mice, however, was not linear: lean females failed to suppress food intake following 4 or 8 micrograms/kg and obese females did not suppress food intake at 4 micrograms/kg. There was also a sex difference in the effect on water intake. No dose of CCK-8 changed water intake in lean males and only the 8 micrograms/kg dose decreased water intake in obese males. In contrast CCK-8 (1, 4 and 8 micrograms/kg) increased water intake in obese females, CCK-8 (1 and 8 micrograms/kg) increased water intake in lean females and no dose of CCK-8 decreased water intake in females. These data demonstrate significant sex differences in the effect of CCK-8 on food and water intake in mice.     

Pancreatic islet hypertrophy in spontaneous maturity onset obese-diabetic CBA/Ca mice

Mature male CBA/Ca mice develop a spontaneous mild diabetes-obesity syndrome which is characterized by hyperglycaemia, hyperinsulinaemia and insulin resistance, and resembles human Type II diabetes mellitus. Immunocytochemical staining of pancreas sections for insulin showed that the pancreas from mature obese mice possessed significantly enlarged islets compared to those from age-matched control (lean) mice. The pancreatic insulin content was significantly greater in 24-week-old obese mice (1.78 ± 0.14mU/mg) compared with lean controls (0.92 ± 0.09 mU/mg). This increase was still apparent at 48 weeks of age. We conclude that, unlike most other rodent models of Type II diabetes, there is no chronic degeneration of beta cells in these mice, so that circulating insulin levels remain high throughout their life. We suggest, therefore, that the male CBA/Ca mouse represents a valuable model for investigating maturity onset diabetes.     

Polyamines in pancreatic islets of obese-hyperglycemic (ob/ob) mice of different ages

To further evaluatethe role of polyamines in insulin production and cell replication indiabetic pancreatic islets, we have studied hyperplastic islets ofobese-hyperglycemic mice of different ages and normal islets of thesame strain. The aims of the study were to investigate the impact ofthe diabetic state and aging on polyamine contents and requirements inthese islets. Cultured islets from lean and obese animals containedsignificantly less polyamines than freshly isolated islets.Spermine-to-spermidine ratio was elevated in freshly isolated isletsfrom young obese mice compared with those from lean mice. In isletsfrom old obese animals, spermidine content was decreased, whereas thecontent of spermine was not different from that of young obese mice.The physiological significance of polyamines was investigated byexposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets butfailed to affect polyamine content of cell nuclei. Insulin content wasnot affected in polyamine-deficient islets of obese mice, irrespectiveof age, in contrast to decreased islet insulin content inpolyamine-depleted young lean animals. Polyamine depletion depressedDNA synthesis rate in obese mouse islets; in lean mice it actuallystimulated DNA synthesis. We concluded that important qualitative andquantitative differences exist between islets from obese-hyperglycemicand normal mice with respect to polyamine content and requirements ofpolyamines for regulation of insulin content and cell proliferation.The results suggest that spermine may be involved in mediating therapid islet cell proliferation noted early in obese-hyperglycemicsyndrome, but changes in spermine concentration do not seem to accountfor the decline in islet cell DNA synthesis in aged normoglycemic animals.

     

Subdiaphragmatic vagal deafferentation affects body weight gain and glucose metabolism in obese male Zucker (fa/fa) rats

We investigated the effect of subdiaphragmatic vagal deafferentation (SDA) on food intake, body weight gain, and metabolism in obese (fa/fa) and lean (Fa/?) Zucker rats. Before and after recovery from surgery, food intake and body weight gain were recorded, and plasma glucose and insulin were measured in tail-prick blood samples. After implantation of a jugular vein catheter, an intravenous glucose tolerance test (IVGTT) was performed, followed by minimal modeling to estimate the insulin sensitivity index. Food intake relative to metabolic body weight (g/kg(0.75)) and daily body weight gain after surgery were lower (P < 0.05) in SDA than in sham obese but not lean rats. Before surgery, plasma glucose and insulin concentrations were lower (P < 0.05) in lean than in obese rats but did not differ between surgical groups within both genotypes. Four weeks after surgery, plasma glucose and insulin were still similar in SDA and sham lean rats but lower (P < 0.05) in SDA than in sham obese rats. IVGTT revealed a downward shift of the plasma insulin profile by SDA in obese but not lean rats, whereas the plasma glucose profile was unaffected. SDA decreased (P < 0.05) area under the curve for insulin but not glucose in obese rats. The insulin sensitivity index was higher in lean than in obese rats but was not affected by SDA in both genotypes. These results suggest that elimination of vagal afferent signals from the upper gut reduces food intake and body weight gain without affecting the insulin sensitivity index measured by minimal modeling in obese Zucker rats.     

The effect of acarbose on the food intake, weight gain, and adiposity of LA/N-cp rats.

1. Groups of lean and obese LA/N-cp rats were administered the intestinal glucosidase inhibitor acarbose at 150 or 300 mg/kg diet from 7 until 17 weeks of age and the effects of the drug on food intake patterns and adiposity determined. 2. Dose related effects on body weights, adiposity and feed efficiency ratio were observed (control greater than 150 mg greater than 300 mg drug/kg diet) following treatment in both phenotypes, with the greatest differences observed in the obese phenotype. 3. Acarbose at both dosages was associated with phenotype-specific alterations in food intake amount and feeding pattern, resulting in an attenuation of age-associated increases in food intake. The feed efficiency ratio decreased in both phenotypes, and approached normally fed lean controls in obese rats administered the greater dosage. 4. These results indicate that patterns of food intake and weight gain differ markedly between lean and obese rats of this strain, and acarbose brings about a dose-related attenuation of developing food intake patterns in both phenotypes and which are associated with decreases in weight gain and adiposity. Thus, this drug may have therapeutic potential as an adjunct agent in the treatment of obesity and/or other disorders of carbohydrate intolerance.     

The activity of the pyruvate dehydrogenase complex in heart muscle in the previously obese mouse model

Obese gold thioglucose injected mice were reduced to lean control weight by food restriction. When pair fed with lean controls these animals then gained weight (were metabolically more efficient). Serum glucose was also elevated in this group (14.5±0.4 (14)vs 12.1±0.3 mmol/L, p<0.001). If previously obese animals were weight maintained with lean controls (by mild food restriction), serum glucose remained at control levels. The activity of the pyruvate dehydrogenase complex in heart muscle was decreased in both obese and pair fed previously obese, whilst it was similar to that of lean controls in the weight maintained previously obese and in obese mice actually dieted. In all obese and previously obese animals serum insulin was elevated. In hearts from control animals subjected to mild food restriction the pyruvate dehydrogenase complex was activated (11.53±1.80 (5)vs 3.34±0.62 (9) U/g dry weight), despite a reduced serum insulin level (42±2vs 74±10 U/ml, p<0.01). These diverse changes in the proportion of the pyruvate dehydrogenase complex in the active form and insulin levels argue for a persistent alteration in the sensitivity of the pyruvate dehydrogenase complex to insulin in obesity, as well as indicating that glucose metabolism in obese animals is altered by both body weight and diet amount.To whom correspondence should be addressed.     

Constitutive and inducible expression of hepatic CYP2E1 in leptin-deficient ob/ob mice

In this study we have analyzed the inducible as well as constitutive hepatic expression of Cyp2e1 in a genetic model of obesity and non-insulin dependent (type II) diabetes, the leptin-deficient ob/ob mouse. In obese mice, Cyp2e1 levels were decreased compared to lean littermates. Treatment with leptin increased hepatic Cyp2e1 in obese mice to the levels observed in lean animals, but failed to alter Cyp2e1 expression in lean animals. As expected, leptin also reduced food intake in treated mice compared to saline-treated controls. In obese mice pair-fed the reduced amount of food, there was a significant increase in Cyp2e1 mRNA but no increase in Cyp2e1 protein or enzyme activity. Fasting and administration of acetone and 4-methylpyrazole increased Cyp2e1 mRNA as well as protein and activity in both obese and lean mice. The present data indicate that while Cyp2e1 is still inducible in obese mice by xenobiotics and fasting, full constitutive expression of Cyp2e1 requires leptin to be present. This effect of leptin appears to be at least partly independent of the hypothalamic control of food intake.     

Diurnal variations of food consumption, plasma glucose and plasma insulin concentrations in lean and obese hyperglycaemic mice.

Diurnal variations in food consumption and plasma concentrations of glucose and insulin were determined at 3-hourly intervals in obese hyperglycaemic mice (C57BL/6J ob/ob) and lean mice (C57BL/6J+/+). In lean mice, food consumption and plasma insulin concentrations increased during the light period and were reduced during the dark period, whereas plasma glucose concentrations were maximal at the beginning of the light period and declined to a minimum during the early dark period. In ob/ob mice, the plasma glucose concentration declined temporarily at the beginning of both the light and the dark period and became elevated towards the ends of these periods, but there were no significant diurnal variations of food consumption or plasma insulin concentrations. These observations indicate differences in the diurnal regulation of glucose homeostasis in lean and ob/ob mice.     

On the characteristics of mitochondrial monoamine oxidase in pancreas and adipose tissues from genetically obese mice.

The substrate specificity of mitochondrial monoamine oxidase (MAO) in pancreatic and adipose tissues of obese mice and their lean counterparts was determined. The pancreatic MAO of obese mice had a greater specific activity than that of the lean mice. The white adipose tissue MAO was found to be more active than the brown adipose MAO in both groups of mice. While there was no appreciable difference in the MAO activities of brown adipose tissues between obese and lean mice, the enzyme from the white adipose tissue of obese mice had a higher specific activity than that of the lean mice. The higher MAO activity in white adipose tissue was observed when tyramine or serotonin was employed as substrate but not with benzylamine. Examination of mitochondrial MAO from epididymal adipocytes revealed marked differences in the properties of the enzyme between whole adipose tissue and isolated adipocytes. The inhibition characteristics of MAO from these tissues were studied with the specific inhibitors clorgyline and deprenyl.     

Adrenalectomy increases norepinephrine turnover in brown adipose tissue of obese (ob/ob) mice

The hyperphagia and rapid body weight gain normally observed in young obese (ob/ob) mice were abolished by removal of their adrenal glands, whereas food intake and weight gain of lean mice were not significantly affected by adrenalectomy. Adrenalectomy lowered body energy density (kcal/g carcass) in obese mice more than could be attributed to reduced food intake per se, suggesting that their energy expenditure was also increased. In control obese mice, low stimulation of brown adipose tissue by the sympathetic nervous system, as indicated by the low fractional rates of norepinephrine (NE) turnover in their brown adipose tissue may have contributed to the reduced energy expenditure in these animals. Adrenalectomy increased the rates of NE turnover in brown adipose tissue of obese mice to rates nearly equal to those observed in lean mice without affecting NE turnover in this tissue of lean mice. Likewise, removal of the adrenals normalized the low rates of NE turnover in hearts of obese mice without affecting lean mice. Rates of NE turnover in two other organs, white adipose tissue and pancreas, of control and adrenalectomized obese mice were similar to rates observed in lean counterparts. The adrenal may thus contribute to both the hyperphagia and the low energy expenditure by brown adipose tissue that together cause gross obesity in ob/ob mice.     

Effects of insulin on lipolysis and lipogenesis in adipocytes from genetically obese (ob/ob) mice.

A method for the preparation of isolated adipocytes from obese mice is described. Similar yields of adipocytes (50--60%), as judged by several criteria, are obtained from obese mice and lean controls. Few fat-globules and no free nuclei were observed in cell preparations, which are metabolically active, respond to hormonal control and appear to be representative of intact adipose tissue. Noradrenaline-stimulated lipolysis was inhibited by insulin, equally in adipocytes from lean and obese mice. Inhibition in obese cells required exogenous glucose, and the insulin dose--response curve was shifted to the right. Basal lipogenesis from glucose was higher in adipocytes from obese mice, and the stimulatory effect of insulin was greater in cells from obese mice compared with lean controls. A rightward shift in the insulin dose--response curve was again observed with cells from obese animals. This suggests that adipose tissue from obese mice is insulin-sensitive at the high blood insulin concentrations found in vivo. The resistance of obese mice to the hypoglycaemic effect of exogenous insulin and their impaired tolerance to glucose loading appear to be associated with an impaired insulin response by muscle rather than by adipose tissue.     

The regulation of hepatic stearoyl-coenzyme A desaturase in obese-hyperglycaemic (ob/ob) mice by food intake and the fatty acid composition of the diet.

1. The effects of food intake and the fatty acid composition of the diet on the hepatic stearoyl-CoA desaturase activity of obese-hyperglycaemic (ob/ob) mice were investigated. 2. Obese mice fed on a commercial mouse diet, ad libitum, had 6.5-fold more activity per liver cell than had lean mice. 3. On a diet containing 14% corn oil the activity was 65% less in obese mice and 62% less in lean mice compared with animals fed on the commercial diet. 4. Feeding with 14% saturated fat in the diet doubled the activity in lean mice compared with those on the commercial diet, but had no effect on the activity in obese mice. 5. Obese mice fed on the corn-oil diet contained a higher proportion of linoleic acid in the liver lipids than did lean mice fed on the commercial diet, but the acyl-CoA desaturase activity was 125% higher than in the lean mice. 6. Limiting the food intake of obese mice by pair-feeding with lean mice decreased their acyl-CoA desaturase activity when the animals were fed on the saturated-fat diet, but the activity remained 75% higher than in lean mice, whereas in obese mice pair-fed on the corn-oil diet the activity was the same as in lean mice. 7. During starvation the acyl-CoA desaturase activity in livers from obese mice decreased more slowly and proportionately less than in livers from lean mice. 8. It is concluded that increased substrate supply as a result of hyperphagia and not low concentration of linoleic acid is the main factor causing high acyl-CoA desaturase activity in obese mice.     

Studies with crystalline insulin from obese and lean mice of the BL/6J strain.

Crystalline insulin was extracted and purified from the pancreases of obese (BL/6J/-ob/ob) and lean mice (BL/6J and BL/6J-ob/+). The two insulin preparations were compared with respect to their radioimmunologic properties as well as their ability to stimulate glucose metabolism in rat epididymal adipocytes and epididymal adipose tissue from obese and lean mice. No significant differences could be seen between the two insulin preparations and thus an insulin of altered biological properties is not likely to be an adequate explanation for the symptoms observed in the obese mouse.     

Amylin improves the effect of leptin on insulin sensitivity in leptin-resistant diet-induced obese mice

Leptin enhances insulin sensitivity in addition to reducing food intake and body weight. Recently, amylin, a pancreatic β-cell-derived hormone, was shown to restore a weight-reducing effect of leptin in leptin-resistant diet-induced obesity. However, whether amylin improves the effect of leptin on insulin sensitivity in diet-induced obesity is unclear. Diet-induced obese (DIO) mice were infused with either saline (S), leptin (L; 500 μg·kg?1·day?1), amylin (A; 100 μg·kg?1·day?1), or leptin plus amylin (L/A) for 14 days using osmotic minipumps. Food intake, body weight, metabolic parameters, tissue triglyceride content, and AMP-activated protein kinase (AMPK) activity were examined. Pair-feeding and weight-matched calorie restriction experiments were performed to assess the influence of food intake and body weight reduction. Continuous L/A coadministration significantly reduced food intake, increased energy expenditure, and reduced body weight, whereas administration of L or A alone had no effects. L/A coadministration did not affect blood glucose levels during ad libitum feeding but decreased plasma insulin levels significantly (by 48%), suggesting the enhancement of insulin sensitivity. Insulin tolerance test actually showed the increased effect of insulin in L/A-treated mice. In addition, L/A coadministration significantly decreased tissue triglyceride content and increased AMPKα2 activity in skeletal muscle (by 67%). L/A coadministration enhanced insulin sensitivity more than pair-feeding and weight-matched calorie restriction. In conclusion, this study demonstrates the beneficial effect of L/A coadministration on glucose and lipid metabolism in DIO mice, indicating the possible clinical usefulness of L/A coadministration as a new antidiabetic treatment in obesity-associated diabetes.