Bromocriptine/SKF38393 treatment ameliorates dyslipidemia in ob/ob mice.

Our previous studies have shown that the dopaminergic D1 receptor agonist SKF38393 (SKF) plus the D2 receptor agonist bromocriptine (BC) act synergistically to reduce obesity in obese C57BL/6J (ob/ob) mice. The present study investigated the effects of this combination on dyslipidemia in ob/ob mice. Female ob/ob mice were treated daily with vehicle or SKF (20 mg/kg body weight [BW]) plus BC (16 mg/kg BW [BC/SKF]) for 14 days. The animals were then used for the characterization of plasma lipoprotein profiles, hepatic triacylglycerol synthesis and secretion, adipocyte lipolysis, adipose and muscle lipoprotein lipase (LPL) activity, and muscle triglyceride (TG) content. The treatment significantly reduced plasma glucose 54%, TG 41%, cholesterol 21%, phospholipid 20%, and free fatty acid (FFA) 36% (P < .01). Hepatic triacylglycerol synthesis was 55% lower in treated mice versus control mice (P < .01). The cell size of isolated adipocytes was significantly reduced (41%) by treatment. LPL activity was increased in soleus skeletal muscle (25%, P < .05) but was sharply reduced in adipose tissue (91%, P < .01) in treated versus control mice. The TG content of hindlimb muscle was about 49% lower in treated versus control mice (P < .05). The basal and isoproterenol-stimulated lipolytic rate was decreased (approximately 53%) in adipocytes from treated animals compared with the control (P < .01). In conclusion, BC/SKF normalized the hypertriglyceridemia likely via its simultaneous antilipogenic action in liver tissue and antilipolytic action in adipose tissue. Decreased plasma flux of FFA partially contributed to the reduced hepatic lipogenesis, plasma very-low-density lipoprotein (VLDL)-TG, and TG in skeletal muscle. The above-described effects of BC/SKF treatment are largely independent of its effect to normalize hyperphagia in ob/ob mice.     

Fed and fasting thermoregulation in ob/ob mice

The body temperatures of mature lean and obese C57BL/6J mice were measured just after feeding, during ad libitum access to food, or every 24 h throughout a 3-day fast. Obese mice had body temperatures 1.0-1.4 degrees C lower than lean mice in the postprandial state and during ad libitum feeding. During food deprivation, obese mice became more hypothermic than lean sex-matched controls. A 5 degrees C fall in body temperature was observed in mutant females in the first 24 h of starvation, about twice that seen in any other experimental group. Over the same period the temperature changes of obese males and lean females were similar and both groups had larger hypothermic responses than lean males. The present results indicate that both genotype and gender affect thermoregulation in these mice. Under normal colony room conditions (ad libitum feeding, 23 degrees C) the ob/ob mutation is expressed by lower body temperatures which along with hypoactivity and hyperphagia account for the high rates of energy storage. When food availability is limited, females of both phenotypes display an increased capacity to reduce their maintenance energy requirements by lowering body temperatures. This hypothermia may be responsible for both the increased conservation of body mass seen during starvation and the slightly greater (5%) fat stores observed in female mice.     

Systemic treatment with sympatholytic dopamine agonists improves aberrant beta-cell hyperplasia and GLUT2, glucokinase, and insulin immunoreactive levels in ob/ob mice.

Sympatholytic dopamine agonist treatment utilizing bromocriptine and SKF38393 (BC/SKF) significantly lowers basal plasma insulin levels and normalizes basal and glucose-induced insulin secretion of the pancreatic beta cell in ob/ob mice. While BC/SKF has no significant effect on pancreatic islet cells directly, drug action is mediated via alterations in the hypothalamic-neuroendocrine axis, which drives metabolic changes in peripheral tissues leading to a marked reduction in hyperglycemia and hyperlipidemia and corrects autonomic control of islet function. To elucidate the nature of the functional response of islets to systemic BC/SKF treatment in ob/ob mice, we investigated the relative changes in the levels of functionally important beta-cell proteins in situ, as well as differences in the beta-cell turnover rate, following a 2-week drug treatment. Isolated islets from treated mice exhibit a 3.5-fold increase in insulin content (P <.01) that correlated with a 51% reduction in basal plasma insulin levels (P <.01) compared with vehicle-treated controls. Using quantitative immunofluorescence microscopy on pancreatic tissue sections, insulin and GLUT2 immunoreactivity of islet beta cells of BC/SKF-treated mice were significantly increased (approximately 2.3-fold and approximately 4.4-fold, respectively; P <.002) to the levels observed in islets of their lean littermates. Glucokinase (GK) immunoreactivity was greatly (75%) reduced in beta cells from ob/ob versus lean mice (P <.0001). A modest increase in GK immunoreactivity in beta cells of drug-treated mice was observed (approximately 1.6-fold; P <.05). Isolated islets from BC/SKF-treated mice exhibit a 42% reduction in DNA content compared with vehicle-treated controls (P <.01) to levels observed in lean mice, but without notable differences in islet size. In situ assays for mitosis and apoptosis, using 5-bromodeoxyuridine (BrdU) and terminal deoxyribotransferase (TdT)-UTP nick end labeling (TUNEL) staining techniques, respectively, were performed in pancreas of these mice to determine if beta cells show a reduction in hyperplasia following BC/SKF treatment. Accordingly, a pronounced decrease in replicating, BrdU-positive beta cells in the drug-treated mice compared with the control group was observed, but without differences in their TUNEL-staining patterns. Collectively, these data suggest that systemic sympatholytic dopaminergic therapy that attenuates hyperglycemia and hyperlipidemia improves islet function in ob/ob mice by improving aberrations in the beta cell's glucose-sensing apparatus, enhancing insulin storage and/or retention, and stabilizing hyperplasia, thus reducing basal insulin levels.     

Dopaminergic agonists normalize elevated hypothalamic neuropeptide Y and corticotropin-releasing hormone, body weight gain, and hyperglycemia in ob/ob mice

Hypothalamic neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) influence feeding and levels of plasma glucose, insulin, free fatty acids, and triglycerides. Treatment of genetically obese, ob/ob mice, with dopamine receptor D(1)/D(2) agonists normalizes hyperphagia, body weight gain, hyperglycemia, and hyperlipidemia. We therefore examined whether levels of NPY and CRH immunoreactivity in discrete hypothalamic nuclei are altered in ob/ob mice, and whether dopaminergic treatment reverses this alteration. Female ob/ob mice were treated daily at 1 h after light onset with the D(1)/D(2) agonists, SKF-38393 (20 mg/kg) and bromocriptine (15 mg/kg), respectively or vehicle for 2 weeks. Such treatment, while normalizing body weight gain and hyperglycemia, also significantly reduced elevated NPY immunoreactivity in the suprachiasmatic (by 39%), intergeniculate (by 43%), paraventricular (PVN; by 31%), and arcuate (by 41%) nuclei in obese mice to levels observed in lean mice. This treatment also caused a 45-50% decline in levels of CRH in the PVN and dorsomedial hypothalamus compared to obese controls to levels observed in lean mice. Taken together, these findings suggest that dopaminergic D(1)/D(2) receptor coactivation may improve hyperphagia, hyperglycemia, and obesity in the ob/ob mouse, in part, by normalizing elevated levels of both NPY and CRH.     

Elevated serum and antral gastrin in obese (ob/ob) mice

Elevations in serum gastrin concentration of six to seven times were found in postabsorptive 5- and 10-wk-old, but not 30- and 40-wk-old, obese mice in comparison with the appropriate lean controls. At 10 wk of age a fourfold hypergastrinemia was also evident in (ob/ob) mice denied food for 48 h. In 10-wk-old (ob/ob) mice that had eaten the same amount of food as lean mice from weaning, serum gastrin was six times that of lean controls. Antral gastrin concentration was 54% higher in fed 10-wk-old (ob/ob) mice than in lean mice. No relationship was found between alterations in serum gastrin and measures of gastrointestinal size or proliferative status. Maximal gastric acid output, expressed with respect to oxyntic mucosal dry weight, was reduced by 52% in 10-wk-old (ob/ob) mice compared with lean controls. It is concluded that the hypergastrinemia of 10-wk-old (ob/ob) mice is not caused by hyperphagia, but may be a consequence of reduced acid inhibition of gastrin release.     

Age dependent effects of oxytetracycline in ob/ob mice

Summary The effects of daily oxytetracycline injections on the metabolism and growth of prepubertal, 10 and 24 week old ob/ob and lean mice were examined. Drug treatment (100 mg/kg) between 17 and 28 days of age similarly reduced body weight gain and skeletal growth of both ob/ob and lean mice, but had little effect on obesity, serum glucose, or IRI levels. In older obese mice, 6 days of oxytetracycline led to substantial reductions of serum glucose, serum IRI, and obesity when compared with either ad libitum fed obese mice or food-restricted ob/ob mice serving as weight loss controls. In lean mice oxytetracycline also significantly reduced body weight and serum glucose, while no effects on obesity or IRI levels were noted. The present data show that oxytetracycline reduces the severity of many of the diabetic symptoms of ob/ob mice, but that only the ameliorative effects on obesity and hyperinsulinaemia appear to be specific to the ob/ob genotype.     

Increased protease activity in muscles of obese- (ob/ob) mice

Muscles of genetically-obese animals exhibit decreased binding of and metabolic responses to insulin. Muscle protein catabolism was investigated by measuring the activity of alkaline, myofibril-bound protease in male (ob/ob) mice, fed ad libitum, or fasted for 5 d. Enzyme activity in the isolated myobrillar fraction was determined by the degradation of 14C-globin. Compared to the lean siblings, protease activity in the obese mice was 2.0, 1.5 and 1.3-fold higher in gastrocnemius, diaphragm and soleus muscle respectively, but without change in heart. The higher protease activity in gastrocnemius, diaphragm and soleus was associated with a parallel decrease in the weight and protein mass of the muscles. The muscles of obese mice also showed a 3 to 4-fold increase in triglyceride and a 2-fold increase in glycogen content. After 5-d starvation, the activity of protease rose in the gastrocnemius of obese mice only 1.5 fold, while it increased as much as 4 and 2 fold in gastrocnemius and diaphragm, respectively, in the lean mice. There was no significant change in heart enzyme activity. After 5-d starvation, serum insulin in obese mice fell markedly but remained still higher than that in ad libitum fed lean mice. Insulin-dependent serum metabolites, as well as adipose tissue lipoprotein lipase and hepatic enzymes related to lipogenesis and gluconeogenesis were consequently much less affected in obese mice and the prevalence of adequate insulin supply appeared to be the cause for lack of significant effect on muscle protease activity in fasting obese mice. It is suggested, therefore, that the induction of myofibrillar protease in obesity is linked to the decrease in cellular responsiveness to insulin and may also be interrelated with the intracellular metabolic adjustments to the enhanced muscle lipid availability.     

Adrenalectomy reduces but does not reverse obesity in ob/ob mice

Bilateral adrenalectomy (ADX) of 4-month-old ob/ob mice led to reduced rates of body weight gain, a complete cessation of fat deposition and increased percentage carcass protein and ash during a 2-month observation period after surgery. However, ADX obese mice were still heavier and had more body fat and lower concentrations of carcass protein and ash than intact sex-matched littermate lean mice at the end of the experiment. When adrenalectomy was performed in younger obese mice before the syndrome was fully expressed (23 +/- 2 days of age), body weight gain was reduced by 40 per cent and fat deposition by 50 per cent during the next 3.5 months, but each was still greater than that of littermate lean mice. Despite the lower rate of weight gain after adrenalectomy, the skeletal and lean body growth of the early ADX obese mice equalled that of both obese and lean mice fed ad libitum. When the carcass composition of early ADX obese mice was compared with that of intact obese mice which were calorically restricted to the same rate of body weight gain, the ADX group had significantly less carcass fat (28 per cent) and more protein (50 per cent) and ash (20 per cent) than the dieted obese mice. In both experiments adrenalectomy led to reduced circulating immunoreactive insulin levels, although hyperinsulinemia persisted. The present results show that adrenalectomy is an effective tool for ameliorating the severity of many aspects of the ob/ob syndrome, particularly when compared with caloric restriction, but the procedure does not entirely reverse the deranged metabolism or abnormal carcass composition of these mice.     

Effects of long-term restricted insulin production in obese-hyperglycemic (genotype ob/ob) mice

Summary Primary hypersecretion of insulin has been suggested as one possibility for the genetic fault of ob/ob mice. To test this hypothesis, streptozotocin (SZO) was used to reduce permanently insulin secretion in young lean and obese mice. After establishment of hyperglycaemia and weight reduction in treated obese mice (obese-SZO), daily insulin replacement was begun in some (obese-SZO-Ins). Obese-SZO mice maintained insulin levels and body weights similar to lean controls, though they were shorter and fatter, while food intake and blood sugar levels exceeded lean values. Obese-SZO-Ins mice with reduced islet hyperplasia, but great insulin resistance, gained more weight than obese-SZO mice; had high serum insulin and controlled blood glucose; and exhibited hyperphagia. These results suggest that primary hypersecretion of insulin cannot be the genetic defect, as ob/ob mice are hyperphagic, hyperglycaemic, insulin resistant, and obese even when insulin levels are restricted.     

Glycerol release in vitro from adipose tissue of obese (ob/ob) mice treated with thyroid hormones.

Glycerol release from adipose tissue of adult genetically obese (ob/ob) mice was greater than that from adipose tissue of lean litter-mates. However, a higher concentration of adrenaline was required to stimulate lipolysis in ob/ob adipose tissue. Since oxygen consumption and the uptake of radioactive iodine by the thyroid were reduced and the biological half-life of thyroidal radio-iodine was lengthened, it is suggested that ob/ob mice are hypothyroid. The poor sensitivity of ob/ob adipose tissue to low doses of adrenaline was improved by treatment with either tri-iodothyronine or thyroxine. The lipolytic responses to both dibutyryl 3':5'-cyclic adenosine monophosphate and aminophylline were enhanced in thyroid hormone-treated lean and ob/ob mice. A higher dose of tri-iodothyronine was required to enhance lipolysis in ob/ob mice than in lean controls. The possibility that hypothyroidism is responsible for the change in response of ob/ob adipose tissue to lipolytic hormones is discussed.     

Regulation of plasma immunoreactive glucagon in obese hyperglycaemic (ob/ob) mice

This study examines the role of glucagon in the pathogenesis of the obese hyperglycaemic (ob/ob) syndrome in mice. Plasma C-terminal immunoreactive glucagon concentrations were measured in fed and fasted ob/ob mice at different ages between 5-40 weeks, and in 20-week-old mice after the administration of established stimulators and inhibitors of glucagon secretion. Plasma glucagon concentrations were inappropriately raised irrespective of age, nutritional status and the accompanying prominent changes in plasma glucose and insulin concentrations. Glucose suppressed plasma glucagon in the fed but not the fasted state, suggesting a dependence on the marked hyperinsulinaemia associated with feeding. Administration of 0.25 units insulin/kg to fasted mice failed to affect plasma glucagon and glucose concentrations. Increasing the dose to 100 units/kg restored the normal suppressive actions of insulin. Fasted mice showed an exaggerated glucagon response to arginine but not to the parasympathomimetic agent pilocarpine. Fed mice displayed normal plasma glucagon responses to the sympathomimetic agents noradrenaline and adrenaline. Administration of insulin antiserum or 2-deoxy-D-glucose raised plasma glucagon concentrations of fed mice. Contrary to the lack of suppression by glucose in the fasted state, heparin-induced increase in free fatty acids reduced plasma glucagon concentrations. This study demonstrates inappropriate hyperglucagonaemia and defective A-cell function in ob/ob mice. The extent of the abnormality is exacerbated by fasting and appears to result from insensitivity of the A-cell to the normal suppressive action of insulin.     

Lipogenesis, lipolysis and feeding rhythms

Recordings of rat's diurnal ad libitum feeding patterns and responses to short term food deprivation suggested that analysis of diurnal feeding rhythms could provide a clue for understanding mechanisms involved in control and regulation of food intake. A simultaneous recording of free feeding patterns and of respiratory exchanges showed that a nocturnal positive energy balance and hyperphagia were mainly due to a diversion of ingested nutrients to fat deposition while negative energy balance and hypophagia during daytime were an effect of fat mobilization and oxidation. Further it was demonstrated that a neuroendocrine diurnal cycle underlined the lipogenesis-lipolysis cycle with hyper-insulinism and glucose tolerance at night, hypoinsulinism and glucose intolerance during the day. Similar phenomena were found along with the human scheduled feeding. Negative correlations between nocturnal lipogenesis and subsequent daytime lipolysis and the diurnal cycle of free fed rats compared to experimentally induced and reversible obesity and to seasonal cycles of hibernators give evidence on the nature of the lipostatic mechanism and its monitoring by hypothalamic ventromedial nuclei.     

Effect of food restriction, dehydroepiandrosterone, or obesity on the binding of 3H-7,12-dimethylbenz(a)anthracene to mouse skin DNA

This study was undertaken to determine whether a reduction in body weight in laboratory mice by regimens that appear to delay the rate of aging (i.e., food restriction and chronic dehydroepiandrosterone (DHEA) treatment), or a production of obesity by the presence of the ob (obese) gene or by gold thioglucose-induced hyperphagia alter the rate of binding of 3H-7,12-dimethylbenz(a)anthracene (3H-DMBA) to mouse skin deoxyribonucleic acid (DNA). We have found that treating A/J mice with food containing .6% DHEA for 10 weeks or reducing the food intake of non-DHEA treated mice to 60% of ad libitum fed animals significantly reduces the amount of 3H-DMBA bound to mouse skin DNA 12 hours after a topical application of the carcinogen. Conversely, A/J mice made obese by a gold thioglucose-induced hyperphagia and C57BL/6J mice with the ob mutation bind significantly increased amounts of 3H-DMBA to skin DNA when compared to their nonobese counterparts.     

Increased responsiveness to the hyperglycemic, hyperglucagonemic and hyperinsulinemic effects of circulating norepinephrine in ob/ob mice

OBJECTIVE: Several studies have implicated increased sympathetic tone as a contributing factor to the hyperglycemia and hyperglucagonemia of ob/ob mice. However, the responsiveness of plasma glucose, insulin and glucagon to circulating norepinephrine (NE) in ob/ob vs normal lean mice has never been described. Therefore, the present study investigated the effect of a 15 min intravenous NE infusion (1 pmol/min/g) on plasma glucose, insulin and glucagon in anesthetized lean, ob/ob, ob/ob-concurrent yohimbine (alpha(2) antagonist) treated, and ob/ob-chronically sympatholytic dopamine agonist treated (for 14 days prior to infusion) mice. In an effort to gain insight into a possible relation between norepinephrine, hyperglucagonemia and hyperinsulinemia in ob/ob mice, this study also examined the isolated islet responses to NE and glucagon in lean, ob/ob and ob/ob-sympatholytic dopamine agonist treated mice. RESULTS: Basal humoral values of glucose, insulin and glucagon were all elevated in ob/ob vs lean mice (by 63, 1900 and 63%, respectively, P<0.01). However, NE infusion further increased levels of glucose, insulin and glucagon in ob/ob (by 80, 90 and 60%, respectively, P<0.05) but not in lean mice (between group difference for all parameters P<0.05). Acute concurrent yohimbine treatment as well as chronic prior sympatholytic dopamine agonist treatment (bromocriptine plus SKF38393) simultaneously strongly aborgated or abolished all these humoral hypersensitivity responses to intravenous NE in ob/ob mice (P<0.05). Clamping the plasma glucose level in untreated ob/ob mice at a high level (30 mM) established by NE infusion did not significantly alter the plasma insulin level, suggesting that some other influence of NE was responsible for this insulin effect. Direct NE administration at 1 microM to islets from lean and ob/ob mice inhibited 15 mM glucose-stimulated insulin secretion in both groups, but at 0.1 microM it was inhibitory only in islets from ob/ob mice. However, glucagon (10 nM) increased 15 mM glucose-stimulated insulin secretion in ob/ob (by 170%, P<0.05) but not lean mice (between group difference P<0.05). CONCLUSION: These findings suggest that hypersensitivity to circulating NE may potentiate hyperglycemia and hyperglucagonemia in ob/ob mice, and the subsequent hyperglucagonemia coupled with increased islet beta-cell insulin secretory responsiveness to glucagon in ob/ob mice may support hyperinsulinemia, thus explaining the increased plasma insulin level response to intravenous NE in these animals. These findings further support a role for increased peripheral noradrenergic activities in the development and maintenance of the hyperglycemic, hyperglucagonemic and hyperinsulinemic state, characteristic of type 2 diabetes.     

Effects of phentermine and phenformin on biomarkers of aging in rats

BACKGROUND: Caloric restriction (CR) is the only treatment known to substantially prolong both average and maximal life span in experimental animals. Interventions that mimic certain effects of CR could be potential anti-aging treatments in humans. Drugs which reduce appetite (anorexiants) represent one class of candidate treatments. Agents that reduce the glucose utilization by the organism could also represent another class of candidate CR mimetics. OBJECTIVE: In our study, we addressed the following questions: (1) Does treatment with an anorexiant reduce caloric intake and body weight of experimental animals comparable to that caused by CR? (2) Does treatment with an antidiabetic agent influence caloric intake and body weight? (3) Does treatment with any of these drugs affect metabolic parameters of an organism in the way similar to that observed with CR? Methods: One hundred and twenty 6-month-old female Wistar-derived LIO rats were randomly subdivided into four groups and exposed to: (1) ad libitum feeding with placebo (controls); (2) the antidiabetic drug phenformin (2 mg/kg); (3) the anorectic drug phentermine (1 mg/kg), and (4) the same amount of food as the group with the least food intake during the previous week (pair-fed controls). Food and water intake, body weight, and rectal temperature were measured weekly during 16 weeks. At the end of the 16th week of the experiment, serum levels of glucose, total beta-lipoprotein and pre-beta-lipoprotein fractions, cholesterol, triglycerides, insulin, total triiodothyronine, and free thyroxine were estimated. The contents of diene conjugates and Schiff's bases, total antioxidant activity, the activities of Cu/Zn superoxide dismutase, glutathione S-transferase, and glutathione peroxidase, and the generation of reactive oxygen species (ROS) were studied in brain and liver homogenates and in the serum. RESULTS: The controls exposed to pair feeding had a significantly reduced food consumption (about 20%) as compared with the ad libitum fed controls and thus exhibited a moderate CR. Treatment with phentermine reduced the caloric intake by about 12% as compared with the ad libitum fed controls. Body weight and water intake in this group were only slightly decreased (by about 2 and 5%, respectively) as compared with the controls. The mean rectal temperature in the phentermine group (38 degrees C) was significantly higher than in the ad libitum fed (37.8 degrees C) and pair-fed (37.6 degrees C) controls. Treatment with phentermine also resulted in significantly reduced ROS levels in all tissues studied, while the highest ROS production was found in ad libitum (blood serum) and pair-fed (brain) controls. Treatment with phenformin did not significantly influence food and water consumption, body weight, and temperature when compared with the ad libitum fed controls. Rats treated with this antidiabetic drug showed intermediate values of ROS generation. Differences among the groups in total antioxidant activity were not obvious. CONCLUSIONS: Treatment with phentermine reduces caloric intake slightly less than is commonly observed in CR studies. CR due to forcibly reduced feeding and CR due to substance-suppressed appetite appear to act through different metabolic mechanisms and thus may affect aging and longevity in different ways.     

Dual CCR2/CCR5 antagonist treatment attenuates adipose inflammation,but not microvascular complications in ob/ob mice

Diabetic peripheral neuropathy (DPN) and diabetic kidney disease (DKD) are common diabetic complications with limited treatment options. Experimental studies show that targeting inflammation using chemokine receptor (CCR) antagonists ameliorates DKD, presumably by reducing macrophage accumulation or activation. As inflammation is implicated in DPN development, we assessed whether CCR2 and CCR5 antagonism could also benefit DPN. Five‐week‐old ob/ob mice were fed a diet containing MK‐0812, a dual CCR2‐CCR5 receptor antagonist, for 8 weeks; DPN, DKD and metabolic phenotyping were then performed to determine the effect of CCR inhibition. Although MK‐0812 reduced macrophage accumulation in adipose tissue, the treatment had largely no effect on metabolic parameters, nerve function or kidney disease in ob/ob mice. These results conflict with published data that demonstrate a benefit of CCR antagonists for DKD and hyperglycaemia. We conclude that CCR signaling blockade is ineffective in ob/ob mice and suspect that this is explained by the severe hyperglycaemia found in this model. It remains to be determined whether MK‐0812 treatment, alone or in combination with improved glycaemic control, is useful in preventing diabetic complications in alternate animal models.     

Decreased deformability of erythrocytes in hyperglycaemic non-inbred ob/ob mice

Summary The deformability of erythrocytes from non-inbred ob/ob mice and lean controls was analyzed by filtration through Nuclepore polycarbonate under constant pressure. At the age of 1–2 months there was no difference in erythrocyte filtrability between the two types of mice, whereas from 3 months the ob/ob mouse erythrocytes exhibited a markedly decreased deformability. The filtrability of erythrocytes was sensitive to osmotic pressure (NaCl or glucose). However, the difference between normal and ob/ob mouse of erythroyctes was not due to acute osmotic effects of the hyperglycaemia in the ob/ob mice. When filtration was performed in the same glucose concentration as that recorded in the blood of the erythrocyte-donor animal, the difference in filtrability between adult normal and ob/ob mice remained large and significant (p < 0.01). Moreover, the most pronounced hyperglycaemia occurred in young ob/ob mice with normal erythrocyte filtrability. It is suggested that non-inbred ob/ob mice are a useful model for studying the damaging influence of diabetes on erythrocyte deformability.     

5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice

Aims/hypothesis: The 5'AMP-activated protein kinase is an important mediator of muscle contraction-induced glucose transport and a target for pharmacological treatment of Type II (non-insulin-dependent) diabetes mellitus. The 5'AMP-activated protein kinase can be activated by 5-aminoimidazole-4-carboxamide ribonucleoside. We hypothesised that 5-aminoimidazole-4-carboxamide ribonucleoside treatment could restore glucose homeostasis in ob/ob mice. Methods: Lean and ob/ob mice were given 5-aminoimidazole-4-carboxamide ribonucleoside (1 mg · g body wt^–1· day^–1 s.c) or 0.9 % NaCl (vehicle) for 1–7 days. Results: Short-term 5-aminoimidazole-4-carboxamide ribonucleoside treatment normalised glucose concentrations in ob/ob mice within 1 h, with effects persisting over 4 h. After 1 week of daily injections, 5-aminoimidazole-4-carboxamide ribonucleoside treatment corrected hyperglycaemia, improved glucose tolerance, and increased GLUT4 and hexokinase II protein expression in skeletal muscle, but had deleterious effects on plasma non-esterified fatty acids and triglycerides. Treatment with 5-aminoimidazole-4-carboxamide ribonucleoside increased liver glycogen in fasted and fed ob/ob mice and muscle glycogen in fasted, but not fed ob/ob and lean mice. Defects in insulin-stimulated phosphatidylinositol 3-kinase and glucose transport in skeletal muscle from ob/ob mice were not corrected by 5-aminoimidazole-4-carboxamide ribonucleoside treatment. While ex vivo insulin-stimulated glucose transport was reduced in isolated muscle from ob/ob mice, the 5-aminoimidazole-4-carboxamide ribonucleoside stimulated response was normal. Conclusion/interpretation: The 5-aminoimidazole-4-carboxamide ribonucleoside mediated improvements in glucose homeostasis in ob/ob mice can be explained by effects in skeletal muscle and liver. Due to the apparently deleterious effects of 5-aminoimidazole-4-carboxamide ribonucleoside on the blood lipid profile, strategies to develop tissue-specific and pathway-specific activators of 5'AMP-activated protein kinase should be considered in order to improve glucose homeostasis. [Diabetologia (2002) 45: 56–65] Received: 21 June 2001 and in revised form: 20 July 2001     

Insulin releasing effects of adrenocorticotropin (ACTH 1-39) and ACTH fragments (1-24 and 18-39) in lean and genetically obese hyperglycaemic (ob/ob) mice

An excessive insulin releasing effect of adrenocorticotropic hormone (ACTH) and ACTH fragments has been considered as a possible factor contributing to the hyperinsulinaemia of genetically obese hyperglycaemic (ob/ob) mice. To investigate this possibility, plasma glucose and insulin responses of 11- to 14-week-old fed lean and ob/ob mice were examined after intraperitoneal administration of ACTH 1-39, ACTH 1-24 and ACTH 18-39, each at a dose of 25 nmol/mouse (50-115 micrograms/mouse). ACTH 1-39 produced a marked and rapid increase of plasma insulin in both lean and ob/ob mice, the effect being much greater in the ob/ob mutant (maximum increases of 5.5 +/- 1.5 and 46.1 +/- 4.1 ng/ml at 10 min in lean and ob/ob mice respectively, P less than 0.001). In lean mice plasma glucose concentrations showed a protracted decreased (maximum decrease of 3.7 +/- 0.5 mmol/l at 120 min), whereas glucose concentrations were increased (maximum increase of 4.2 +/- 1.3 mmol/l at 60 min) in ob/ob mice. ACTH 1-24 produced qualitatively similar but generally smaller effects than ACTH 1-39, while ACTH 18-39 did not significantly affect glucose and insulin concentrations. In 24 h fasted mice, ACTH 1-39 produced similar but generally smaller effects than in fed mice. The results suggest that the effects of ACTH on glucose and insulin homoeostasis are conferred by the N-terminal 1-24 sequence, and ACTH may exert acute effects which contribute to the hyperinsulinaemia and hyperglycaemia of ob/ob mice.     

Current biochemical studies of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis suggest a new therapeutic approach

The study population in this report by Lin et al. was ob/ob mice that have an inherited genetic deficiency of the appetite-suppressing hormone leptin. These mice develop hyperinsulinemia, insulin resistance, and fatty livers. Compared with their lean littermates and wild-type C57BL-6 mice, ob/ob mice have hepatomegaly. In this study, the authors compared three different groups of adult mice (aged 8-10 wk), including male ob/ob C57BL-6 mice, their lean littermates, and wild-type C57BL-6 mice of the same age and sex.The primary purpose of this study was to test the efficacy of metformin for treatment of fatty liver disease in obese, ob/ob mice that develop hyperinsulinemia or insulin resistance and fatty livers. Metformin therapy was found to eliminate fatty liver disease in this model. The potential mechanisms of the action of metformin were the inhibition of hepatic tumor necrosis factor (TNF)alpha and several TNF-inducible responses, which are likely to promote hepatic steatosis and necrosis.In these experiments, ob/ob mice were divided into three treatment groups. Group 1 consisted of eight mice that were treated with metformin and permitted to consume a nutritiously replete liquid mouse diet ad libitum. Mice in group 2 (n = 8) did not receive metformin but were pair-fed the same volume of liquid diet that the mice in the metformin-treated group had consumed on the previous day. Obese ob/ob mice in group 3 (n = 4) and lean mice received no metformin, as with the mice in group 2, but were permitted to consume the liquid diet ad libitum. Liquid diet was given to facilitate accurate daily comparison of food intake among the various treatment groups. All mice were weighed at the beginning of the study and weekly thereafter until killed and then sera, fat, and liver tissues were collected. Tissues were either fixed in buffered formalin and processed from the deceased mice for histology or snap frozen in liquid nitrogen and stored until RNA and proteins were isolated.The feeding protocol was repeated with a second group of 18 ob/ob mice. After 4 wk, hepatocytes were obtained by in situ liver perfusion with collagenase and assayed for cellular adenosine triphosphate (ATP) content. In each experiment, hepatocytes isolated from 3 mice from each treatment group were suspended in a medium and pooled for subsequent analysis to evaluate cell viability, determine the number of obtained cells, and to assay cellular ATP content. These experiments were repeated using another 3 mice from each treatment group, so that analysis of hepatocytes took place from six ob/ob mice in each feeding group.Hepatic steatosis was decreased significantly only in the metformin-treated group. The authors found that metformin's beneficial effect on the fatty liver disease of mice was not due to its ability to constrain hyperphagia, nor due to decreased caloric ingestion, because the daily caloric intakes of the metformin-treated mice and the pair-fed control mice were virtually identical. These caloric intakes were consistently approximately 20% less than that of another obese control group that was permitted to consume diet ad libitum. The authors also observed no significant effect of metformin on serum glucose concentration from fed, ob/ob mice. Metformin is known to reduce hyperinsulinemia by about 40% in both of these obese hyperinsulinemic and insulin-resistant rodent strains. In conclusion, Lin et al. documented that metformin improves fatty liver disease and reverses hepatomegaly, steatosis, and aminotransferase abnormalities in mice. In addition, the authors suggest that metformin might inhibit dieting-induced redistribution of lipid from the liver to adipose tissue depots. In summary, this study identifies a potential treatment for fatty liver disease in humans.