Glucagon-like peptide-1 and the entero-insular axis in obese hyperglycaemic (ob/ob) mice

The intestines of obese hyperglycaemic (ob/ob) mice contain greatly increased amounts of glucagon-like immunoreactive peptides. To investigate their role in the increased activity of the entero-insular axis of these mice, the insulin-releasing effect of glucagon-like peptide-1 (GLP-1) was examined in 24 hour fasted 12-15 weeks old ob/ob mice under conditions of basal and elevated glycaemia. Compared with glucagon (100 micrograms/kg ip), which produce an approximately 3-fold increase in basal plasma glucose and insulin concentrations, GLP-1 (100 micrograms/kg ip) produce a very small (less than 1 fold) increase in plasma insulin, with no significant change in plasma glucose. The insulin-releasing effect of glucagon, but not GLP-1 was increased by administration in combination with glucose (2 g/kg ip). The results indicate that GLP-1, which exhibits considerable sequence homology with glucagon, exerts only a weak insulin-releasing effect without a significant hyperglycaemic effect in ob/ob mice. Thus GLP-1 is unlikely to be an important endocrine component of the two over-active entero-insular axis in ob/ob mice.     

Dual action of beta-endorphin on insulin release in genetically obese and lean mice

Intraperitoneal administration of beta-endorphin (1 mg/kg) to ob/ob mice doubled fasting plasma insulin concentrations within 30 min, while plasma glucose concentrations were unaltered. In lean mice, beta-endorphin failed to alter plasma insulin or glucose responses. In glucose-loaded ob/ob mice, beta-endorphin (1 mg/kg) reduced insulin levels at 40 min, and delayed glucose disposal. A lower dose of beta-endorphin (0.1 mg/kg) decreased plasma insulin at 90 min, with no effect on plasma glucose disposal. In lean mice, only the higher dose of beta-endorphin suppressed the glucose-stimulated rise in plasma insulin concentrations, without affecting plasma glucose. Beta-endorphin's actions were blocked by naltrexone and could not be mimicked by N-acetyl-beta-endorphin. Beta-endorphin (10(-8)M) enhanced insulin release from isolated ob/ob and lean mouse islets incubated in medium containing 6 mM glucose, but inhibited release when 20 mM glucose was present. These effects were naloxone reversible. The results indicate that 1) ob/ob mice display a greater magnitude of response in vivo to beta-endorphin's actions on insulin release compared with lean mice, 2) high concentrations of beta-endorphin exacerbate glucose disposal in ob/ob mice. 3) the prevailing glucose concentration is an important determinant of whether beta-endorphin's effects on insulin release will be stimulatory or inhibitory and 4) these actions are mediated via opiate receptors.     

Influence of cycloheximide on acute diabetogenic effects of S-carboxymethylated human growth hormone in obese (ob/ob) mice

Acute treatment of ob/ob mice with S-carboxymethylated hGH (RCM-hGH), a diabetogenic derivative of GH which lacks significant insulin-like and growth-promoting activities, results in an increase in fasting plasma insulin and blood glucose levels and enhanced peripheral tissue insulin resistance. Plasma insulin level increases within 3 h after RCM-hGH is administered, whereas increased blood glucose concentration and enhanced peripheral tissue insulin resistance became evident 6 h after the hormone derivative is given. The lag period seen in the manifestation of these diabetogenic effects of RCM-hGH is consistent with the time required for gene expression. Therefore, the present study was undertaken to determine whether the above acute responses to the diabetogenic action of RCM-hGH would be expressed in ob/ob mice in which protein synthesis was blocked with cycloheximide. Female ob/ob mice were given either saline or cycloheximide (0.1 mg/g BW) ip and 1 h later were fasted and treated with either saline or 200 micrograms RCM-hGH ip. The mice were given a second injection of cycloheximide during the middle of the hormone treatment period to insure that protein synthesis remained blocked for the entire 6 h. In the animals not receiving cycloheximide, fasting plasma insulin level and blood glucose concentration were markedly elevated 6 h after the injection of RCM-hGH. Also, the GH derivative attenuated the ability of insulin added in vitro to stimulate glucose oxidation by adipose tissue segments isolated from the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Degradation and glycemic effects of His(7)-glucitol glucagon-like peptide-1(7-36)amide in obese diabetic ob/ob mice

Glucagon-like peptide-1(7-36)amide (tGLP-1) has attracted considerable potential as a possible therapeutic agent for type 2 diabetes. However, tGLP-1 is rapidly inactivated in vivo by the exopeptidase dipeptidyl peptidase IV (DPP IV), thereby terminating its insulin releasing activity. The present study has examined the ability of a novel analogue, His(7)-glucitol tGLP-1 to resist plasma degradation and enhance the insulin-releasing and antihyperglycemic activity of the peptide in 20-25-week-old obese diabetic ob/ob mice. Degradation of native tGLP-1 by incubation at 37 degrees C with obese mouse plasma was clearly evident after 3 h (35% intact). After 6 h, more than 87% of tGLP-1 was converted to GLP-1(9-36)amide and two further N-terminal fragments, GLP-1(7-28) and GLP-1(9-28). In contrast, His(7)-glucitol tGLP-1 was completely resistant to N-terminal degradation. The formation of GLP-1(9-36)amide from native tGLP-1 was almost totally abolished by addition of diprotin A, a specific inhibitor of DPP IV. Effects of tGLP-1 and His(7)-glucitol tGLP-1 were examined in overnight fasted obese mice following i.p. injection of either peptide (30 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Plasma glucose was significantly lower and insulin response greater following administration of His(7)-glucitol tGLP-1 as compared to glucose alone. Native tGLP-1 lacked antidiabetic effects under the conditions employed, and neither peptide influenced the glucose-lowering action of exogenous insulin (50 units/kg). Twice daily s.c. injection of ob/ob mice with His(7)-glucitol tGLP-1 (10 nmol/kg) for 7 days reduced fasting hyperglycemia and greatly augmented the plasma insulin response to the peptides given in association with feeding. These data demonstrate that His(7)-glucitol tGLP-1 displays resistance to plasma DPP IV degradation and exhibits antihyperglycemic activity and substantially enhanced insulin-releasing action in a commonly used animal model of type 2 diabetes.     

Development of glucose intolerance and impaired plasma insulin response to glucose in obese hyperglycaemic (ob/ob) mice

The development of glucose intolerance in Aston ob/ob mice showed a gross exaggeration of the age-related changes of glucose tolerance in lean (+/+) mice. Intraperitoneal glucose tolerance in ob/ob mice was poor at 5 weeks, improved by 10 weeks, but markedly worsened by 20 weeks. A 24 hour fast further exaggerated the glucose intolerance of ob/ob mice. Unlike lean mice, tolerance improved in ob/ob mice at 40 weeks. Alterations of insulin sensitivity and the plasma insulin response to glucose accounted in part for these observations. Insulin sensitivity deteriorated until 20 weeks, but improved at 40 weeks in both fed and 24 hour fasted ob/ob mice. A positive plasma insulin response to glucose was lost after 5 weeks in fed ob/ob mice. The severity of this abnormality corresponded with the extent of the basal hyperinsulinaemia. A 24 hour fast reduced plasma insulin concentrations and restored a positive plasma insulin response to glucose in ob/ob mice. The results suggest that the plasma insulin response to glucose in ob/ob mice is related to the secretory activity of the B-cells prior to stimulation. Furthermore, it is evident that factors in addition to insulin insensitivity and the impaired plasma insulin response to glucose contribute to the development of glucose intolerance in these mice.     

Long-term administration of PACAP receptor antagonist, PACAP(6-27), impairs glucose tolerance and insulin sensitivity in obese diabetic ob/ob mice

Pituitary adenylate cyclase-activating peptide (PACAP) is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the PACAP receptor antagonist, PACAP(6-27) to evaluate the role of endogenous PACAP in genetic obesity-related diabetes and related metabolic abnormalities using ob/ob mice. Acute in vivo antagonistic potency of PACAP(6-27) was confirmed in ob/ob mice by blockade of the insulin-releasing action but not hyperglycaemia. In longer-term studies, ob/ob mice were given once daily injections of PACAP(6-27) or vehicle for 14 days. Feeding activity, body weight, basal plasma glucose and plasma insulin concentrations were not significantly affected by chronic PACAP(6-27) treatment. However, PACAP(6-27) treatment impaired glucose tolerance, insulin sensitivity and the glycaemic response to feeding. Plasma glucagon and lipids were unchanged. These observations indicate a role of endogenous PACAP for normal glucose homeostasis, but indicate a minor involvement in the regulation of insulin secretion in ob/ob mice.     

Immunoreactive C-peptide in spontaneous syndromes of obesity and diabetes in mice

Immunoreactive C-peptide was evaluated in the plasma and pancreas of Aston ob/ob and C57BL/KsJ db/db mice in relation to disturbances in pancreatic B-cell function. At 18-24 weeks of age, ob/ob and db/db mice displayed hyperglycaemia (1.6 and 3.8 fold increases respectively) and hyperinsulinaemia (10.8 and 5.1 fold increases respectively) despite a similar pancreatic insulin content to their respective non-diabetic lean control mice. Immunoreactive C-peptide concentrations in the plasma and pancreas of the mutants corresponded with the degree of hyperinsulinaemia and pancreatic insulin content, and the insulin: C-peptide molar ratios in both mutants were similar to lean controls. In ob/ob mice parenteral glucose administration decreased plasma insulin and C-peptide concentrations, despite markedly raised glucose concentrations. However, administration of a low dose of insulin (5 U/kg) to lean mice and much higher doses of insulin (50 and 120 U/kg) to ob/ob mice markedly decreased plasma glucose and C-peptide concentrations. When the rate and extent of insulin-induced glucose suppression observed in ob/ob mice was mimicked in lean mice, an almost complete (95%) inhibition of C-peptide was achieved compared with a 57% decrease in the ob/ob mutant. Injection of ob/ob mice with glucose to counter the insulin-induced hypoglycaemia failed to affect the fall of C-peptide concentrations. The data suggest that the metabolic processing of insulin and C-peptide are undisturbed in obese-diabetic mice, and that the impaired suppression of circulating C-peptide by insulin-hypoglycaemia in ob/ob mice predominantly reflects impaired feedback inhibition by insulin.     

Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean and ob/ob mice

Liver X receptor (LXR) agonists have been proposed to act as anti-diabetic drugs. However, pharmacological LXR activation leads to severe hepatic steatosis, a condition usually associated with insulin resistance and type 2 diabetes mellitus. To address this apparent contradiction, lean and ob/ob mice were treated with the LXR agonist GW-3965 for 10 days. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Hepatic glucose production (HGP) and metabolic clearance rate (MCR) of glucose were determined with stable isotope techniques. Blood glucose and hepatic and whole body insulin sensitivity remained unaffected upon treatment in lean mice, despite increased hepatic triglyceride contents (61.7 +/- 7.2 vs. 12.1 +/- 2.0 nmol/mg liver, P < 0.05). In ob/ob mice, LXR activation resulted in lower blood glucose levels and significantly improved whole body insulin sensitivity. GW-3965 treatment did not affect HGP under normo- and hyperinsulinemic conditions, despite increased hepatic triglyceride contents (221 +/- 13 vs. 176 +/- 19 nmol/mg liver, P < 0.05). Clamped MCR increased upon GW-3965 treatment (18.2 +/- 1.0 vs. 14.3 +/- 1.4 ml x kg(-1) x min(-1), P = 0.05). LXR activation increased white adipose tissue mRNA levels of Glut4, Acc1 and Fasin ob/ob mice only. In conclusion, LXR-induced blood glucose lowering in ob/ob mice was attributable to increased peripheral glucose uptake and metabolism, physiologically reflected in a slightly improved insulin sensitivity. Remarkably, steatosis associated with LXR activation did not affect hepatic insulin sensitivity.     

Effects of zinc supplementation on the plasma glucose level and insulin activity in genetically obese (ob/ob) mice

The effects of zinc supplementation (20 mM ZnCl₂ from the drinking water for eight weeks) on plasma glucose and insulin levels, as well as its in vitro effect on lipogenesis and lipolysis in adipocytes were studied in genetically obese (ob/ob) mice and their lean controls (+/?). Zinc supplementation reduced the fasting plasma glucose levels in both obese and lean mice by 21 and 25%, respectively (p < 0.05). Fasting plasma insulin levels were significantly decreased by 42% in obese mice after zinc treatment. In obese mice, zinc supplementation also attenuated the glycemic response by 34% after the glucose load. The insulin-like effect of zinc on lipogenesis in adipocytes was significantly increased by 80% in lean mice. However, the increment of 74% on lipogenesis in obese mice was observed only when the zinc plus insulin treatment was given. This study reveals that zinc supplementation alleviated the hyperglycemia of ob/ob mice, which may be related to its effect on the enhancement of insulin activity.     

Decreased clearance of serum retinol-binding protein and elevated levels of transthyretin in insulin-resistant ob/ob mice

Serum retinol-binding protein (RBP4) is secreted by liver and adipocytes and is implicated in systemic insulin resistance in rodents and humans. RBP4 normally binds to the larger transthyretin (TTR) homotetramer, forming a protein complex that reduces renal clearance of RBP4. To determine whether alterations in RBP4-TTR binding contribute to elevated plasma RBP4 levels in insulin-resistant states, we investigated RBP4-TTR interactions in leptin-deficient ob/ob mice and high-fat-fed obese mice (HFD). Gel filtration chromatography of plasma showed that 88-94% of RBP4 is contained within the RBP4-TTR complex in ob/ob and lean mice. Coimmunoprecipitation with an RBP4 antibody brought down stoichiometrically equal amounts of TTR and RBP4, indicating that TTR was not more saturated with RBP4 in ob/ob mice than in controls. However, plasma TTR levels were elevated approximately fourfold in ob/ob mice vs. controls. RBP4 injected intravenously in lean mice cleared rapidly, whereas the t(1/2) for disappearance was approximately twofold longer in ob/ob plasma. Urinary fractional excretion of RBP4 was reduced in ob/ob mice, consistent with increased retention. In HFD mice, plasma TTR levels and clearance of injected RBP4 were similar to chow-fed controls. Hepatic TTR mRNA levels were elevated approximately twofold in ob/ob but not in HFD mice. Since elevated circulating RBP4 causes insulin resistance and glucose intolerance in mice, these findings suggest that increased TTR or alterations in RBP4-TTR binding may contribute to insulin resistance by stabilizing RBP4 at higher steady-state concentrations in circulation. Lowering TTR levels or interfering with RBP4-TTR binding may enhance insulin sensitivity in obesity and type 2 diabetes.     

Dietary copper supplementation increases the catecholamine levels in genetically obese (ob/ob) mice

The interactive relationship between Cu deficiency and depressed synthesis of certain neurotransmitters has been recognized. To investigate the effects of dietary Cu supplementation on the catecholamine levels in genetically obese mice, male obese (ob/ob) mice and their lean (+/?) counterparts were administered either a control diet (4.0 mg/kg) or a Cu-supplemented diet (50 mg/kg) for 4 wk. The ob/ob mice that were fed a control diet showed lower liver and higher plasma levels of Cu. Depressed levels of plasma and brain catecholamines were also found in ob/ob mice that were fed the control diet. The ob/ob mice that received a Cu-supplemented diet showed significant increases in the levels of catecholamine in the plasma and brain. This study showed that catecholamine levels in ob/ob mice can be increased by dietary Cu supplementation. However, the interaction between Cu and sympathetic nervous activity in obesity was not elucidated in this study.     

Anti-diabetic effect of ginsenoside Re in ob/ob mice

We evaluated the anti-diabetic effects of ginsenoside Re in adult male C57BL/6J ob/ob mice. Diabetic ob/ob mice with fasting blood glucose levels of approximately 230 mg/dl received daily intraperitoneal injections of 7, 20 and 60 mg/kg ginsenoside Re for 12 consecutive days. Dose-related effects of ginsenoside Re on fasting blood glucose levels were observed. After the 20 mg/kg treatment, fasting blood glucose levels were reduced to 188+/-9.2 and 180+/-10.8 mg/dl on Day 5 and Day 12, respectively (both P<0.01 compared to vehicle group, 229+/-9.5 and 235+/-13.4 mg/dl, respectively). The EC(70) of ginsenoside Re was calculated to be 10.3 mg/kg and was used for subsequent studies. Consistent with the reduction in blood glucose, there were significant decreases in both fed and fasting serum insulin levels in mice treated with ginsenoside Re. With 12 days of ginsenoside treatment, glucose tolerance of ob/ob mice increased significantly, and the area under the curve for glucose decreased by 17.8% (P<0.05 compared to vehicle treatment). The hypoglycemic effect of the ginsenoside persisted even at 3 days of treatment cessation (blood glucose levels: 198+/-13.1 with ginsenoside treatment vs. 253+/-20.3 mg/dl with vehicle, P<0.01). There were no significant changes in body weight or body temperature. Preliminary microarray analysis revealed differential expression of skeletal muscle genes associated with lipid metabolism and muscle function. The results suggest that ginsenoside Re may prove to be useful in treating type 2 diabetes.     

Insulin receptor tyrosine kinase activity is unaltered in ob/ob and db/db mouse skeletal muscle membranes

Insulin binding and insulin receptor tyrosine kinase activity were examined in two rodent models with genetic insulin resistance using partially-purified skeletal muscle membrane preparations. Insulin binding activity was decreased about 50% in both 12-week (219 +/- 184 vs 1255 +/- 158 fmoles/mg, p less than 0.01) and 24-week old (2120 +/- 60 vs 1081 +/- 60 fmoles/mg, p less than 0.01) ob/ob mice. In contrast, insulin binding to membrane derived from 24-week old db/db mice was not significantly different from lean controls (1371 +/- 212 vs 1253 +/- 247 fmoles/mg). Insulin-associated tyrosine kinase activity of membranes from ob/ob skeletal muscle was decreased, compared to its normal lean littermate, when compared on a per mg of protein basis in both 12-week (37 +/- 3 vs 21 +/- 3 pmoles/min/mg, p less than 0.05) and 24-week old (71 +/- 5 vs 37 +/- 6 pmoles/min/mg, p less than 0.01) mice. However, no significant differences in kinase activities were observed when the data were normalized and compared on a per fmole of insulin-binding activity basis for the 12-week (12 +/- 1 vs 11 +/- 2) and 24-week (27 +/- 2 vs 20 +/- 3) age groups. Insulin receptor tyrosine kinase activity of db/db skeletal muscle membranes was not different than its normal lean littermate whether expressed on a protein (34 +/- 7 vs 30 +/- 3) or fmole of insulin-binding activity (21 +/- 4 vs 18 +/- 4) basis. These data suggest that insulin receptor tyrosine kinase is not associated with the insulin resistance observed in ob/ob and db/db mice and demonstrate differences in receptor regulation between both animal models.     

Insulin stimulation of particulate, low Km adenosine 3',5'-monophosphate phosphodiesterase activity in fat cells of the obese-hyperglycemic (ob/ob) mouse.

Cyclic AMP phosphodiesterase activity was examined in particulate (30,000 X g for 30 min sediment) and supernatant subcellular fractions of epididymal fat cells isolated from obese-hyperglycemic (ob/ob) mice and their lean (+/?) LITTERMATES. The activity of the enzyme(s) was measured during both the early onset phase (5-6 weeks of age) and the static (5 months of age) of the obese-hyperglycemia syndrome. Fat cell particulate and supernate cyclic AMP phosphodiesterase activity of obese-hyperglycemic mice and their lean littermates at both ages displayed nonlinear Lineweaver-Burk kinetic plots. The maximum velocities of the fat cell particulate cyclic AMP phosphodiesterase activity of the obese mice were 67% and 84% lower than those of their lean littermates at 5-6 weeks and 5 months of age, respectively. Incubating fat cells obtained from either lean or obese mice of both age groups with 30 to 240 microunits of insulin per ml for 15 min increased the activity of the particulate, low Km cyclic AMP phosphodiesterase. This increase in activity was manifest as an increase in the maximum velocity of the enzyme(s) with no significant alteration of the affinity of the enzyme(s) for cyclic AMP.     

Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, β-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the β-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.     

Adiponectin is stimulated by adrenalectomy in ob/ob mice and is highly correlated with resistin mRNA

Plasma levels of the adipocyte product adiponectin, a putative insulin-sensitizing agent, are reduced in obesity, whereas plasma levels of resistin, an agent that some believe to confer insulin resistance, are thought to increase with obesity. Because adrenalectomy can increase insulin sensitivity, we hypothesized that adrenalectomy would increase expression of adiponectin and decrease expression of resistin. Therefore, we measured adiponectin mRNA, adiponectin peptide, and resistin mRNA in adrenalectomized ob/ob mice. Adrenalectomy restored adiponectin expression in ob/ob mice to wild-type levels and stimulated adiponectin peptide to above wild-type levels. Surprisingly, expression of adiponectin and resistin was highly positively correlated even after statistical removal of effects of insulin, glucose, and adiposity. In addition, adiponectin and resistin expression were also highly correlated in diet-induced obese mice. The data support a role for adiponectin in mediating some effects of adrenalectomy on insulin sensitivity.     

S-adenosyl methionine protects ob/ob mice from CYP2E1-mediated liver injury

Pyrazole treatment to induce cytochrome P-450 2E1 (CYP2E1) was recently shown to cause liver injury in ob/ob mice but not in lean mice. The present study investigated the effects of S-adenosyl-l-methionine (SAM) on the CYP2E1-dependent liver injury in ob/ob mice. Pyrazole treatment of ob/ob mice for 2 days caused necrosis, steatosis, and elevated serum transaminase and triglyceride levels compared with saline ob/ob mice. Administration of SAM (50 mg/kg body wt ip every 12 h for 3 days) prevented the observed pathological changes as well as the increase of apoptotic hepatocytes, caspase 3 activity, and serum TNF-alpha levels. SAM administration inhibited CYP2E1 activity but not CYP2E1 content. The pyrazole treatment increased lipid peroxidation, 4-hydroxynonenal and 3-nitrotyrosine protein adducts, and protein carbonyls. These increases in oxidative and nitrosative stress were prevented by SAM. Treatment of ob/ob mice with pyrazole lowered the endogenous SAM levels, and these were elevated after SAM administration. Mitochondrial GSH levels were very low after pyrazole treatment of the ob/ob mice; this was associated with elevated levels of malondialdehyde and 4-hydroxynonenal and 3-nitrotyrosine protein adducts in the mitochondria. All these changes were prevented with SAM administration. SAM protected against pyrazole-induced increase in serum transaminases, necrosis, triglyceride levels, caspase-3 activity, and lipid peroxidation even when administered 1 day after pyrazole treatment. In the absence of pyrazole, SAM lowered the slightly elevated serum transaminases, triglyceride levels, caspase-3 activity, and lipid peroxidation in obese mice. In conclusion, SAM protects against and can also reverse or correct CYP2E1-induced liver damage in ob/ob mice.     

Dose-related involvement of CCK in bombesin-induced pancreatic growth.

We examined the role of CCK in bombesin-induced pancreatic growth in rats using the CCK receptor antagonist L-364,718. Rats (155 +/- 1 g, 8-10 per group) received subcutaneous injections every 8 h for 5 days with bombesin (0.6, 1.7 and 5 nmol/kg) or bombesin in combination with L-364,718 (1 mg/kg). After 5 days the pancreas was removed and pancreatic weight, protein content, DNA, amylase and chymotrypsin contents were determined. Bombesin produced a significant increase (48-475%) of pancreatic weight, tissue contents of protein, DNA, amylase and chymotrypsinogen (F = 82, P less than 0.001). When a large dose of bombesin (5 nmol/kg) was combined with L-364,718 a significant inhibition (up to 70%) of all tissue parameters was observed (P less than 0.001). L-364,718 did not affect the growth response to a small dose of bombesin (0.6 nmol/kg). Plasma CCK levels 15 min after a single injection of bombesin (0.6, 1.7 and 5 nmol/kg) were significantly increased in response to the 5 nmol/kg dose (2.0 +/- 0.7 to 3.4 +/- 0.8 pM, F = 6.9, P less than 0.01). No increases of CCK plasma levels were found in response to the 0.6 and 1.7 nmol/kg doses of bombesin, corresponding to the lack of effects of L-364,718 on growth parameters at these doses. Measuring the time-course of CCK plasma levels after a single injection of 5 nmol/kg bombesin revealed an increase from basal values of 1.4 +/- 0.3 pM to maximal levels of 3.5 +/- 0.5 pM after 15 min (F = 7.1, P less than 0.001). Values returned to basal after 60 min. These results suggest that low doses of bombesin act directly at the acinar cell or through release of non-CCK growth factors whereas high doses of bombesin act in part through CCK release.     

Half life of injected 125I-insulin in control and ob/ob mice

The t-1/2 of moniodo 125I-insulin in ob/ob mice and their lean litter mates is 10 min. No difference was found between the two groups. Further, 48 hr of fasting did not alter the t-1/2 in ob/ob mice. In view of the markedly enlarged insulin pool in ob/ob mice, one must conclude that the mass of insulin degraded in unit time is increased. However, these findings indicate that the cause of hyperinsulinism in ob/ob mice is unrelated to rates of insulin degradation.