Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.     

Studies on the role of opiate peptides in two forms of genetic obesity: ob/ob mouse and fa/fa rat

Recent reports have indicated that genetically obese hyperinsulinemic mice (ob/ob) and Zucker rats (fa/fa) compared with their lean controls have elevated levels of pituitary and plasma B-endorphins, opiates that can stimulate insulin secretion. In this study we have measured opiate levels by a radio-receptor assay in gastro-intestinal tissues and pancreas in ob/ob and fa/fa animals and their controls. Ob/ob mice showed significantly higher levels than control mice (+/+) in most gastro-intestinal tissues and pancreas. Levels in fa/fa rats did not differ from their controls. Radioimmunoassay of pancreas for B-endorphins, revealed higher levels in ob/ob vs +/+ mice, while there was no difference in the obese and lean rats. Fasting tended to decrease gastro-intestinal opioids in mice, while B-endorphin levels rose. It is concluded that opiates may play a significant role in the obesity of the ob/ob mouse and that this genetic obesity differs from that in Zucker rats.     

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.     

Plasma B-cell tropin (ACTH22-39) concentration in lean and obese (ob/ob) mice and lean and obese (fa/fa) Zucker rats

A method has been developed for the measurement of plasma concentrations of Beta-cell tropin (BCT), which is a potent insulinotropic and lipogenic peptide secreted by the pituitary. The method was employed to compare plasma Beta-cell tropin concentrations between lean and genetically obese (ob/ob) mice and between lean and genetically obese (fa/fa) Zucker rats. The plasma concentration in lean mice was 0.17 +/- 0.02 (5)nmole/l (mean +/- SEM, n = 5), while that in obese (ob/ob) mice was significantly higher, being 2.88 +/- 1.13 (5)nmole/l. The plasma BCT concentration in Zucker rats was 0.14 +/- 0.02 (15)nmole/l, while that in obese Zucker (fa/fa) rats was significantly higher, being 1.69 +/- 0.72 (16)nmole/l. These results explain previously observed differences in the Beta-cell tropin-like biological activity in plasma from lean and obese animals, and support the hypothesis that the peptide has a role in the development of hyperinsulinaemia and obesity.     

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.     

Measurement by radioimmunoassay of the mitochondrial uncoupling protein from brown adipose tissue of obese (ob/ob) mice and Zucker (fa/fa) rats at different ages

The concentration of the 'uncoupling protein' in brown adipose tissue mitochondria has been measured in lean and obese (ob/ob) mice and Zucker (fa/fa) rats at different ages using a specific radioimmunoassay. During the suckling period the concentration of the protein was similar in normal and mutant animals of both types, despite the decrease in mitochondrial GDP binding observed in the obese. The concentration of uncoupling protein was, however, decreased in adult ob/ob mice and adult Zucker rats compared with their respective lean siblings, in parallel with the decrease in GDP binding. It is concluded that there is a 'masked', or inactive, form of uncoupling protein in young ob/ob mice and fa/fa rats.     

Short-term treatment with estrone oleate in liposomes (Merlin-2) does not affect the expression of the ob gene in Zucker obese rats

Young female Zucker fa/fa rats of 370-430 g were implanted with osmotic minipumps releasing 3.5 mol/dayúkg of estrone oleate in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a sustained loss of appetite, and a decrease in body weight of 3.5%, which contrasts with the 8.2% increase in controls during the period studied. Plasma insulin, glucose and urea decreased, and liver glycogen increased with Merlin-2 treatment. Plasma ACTH and corticosterone increased to a maximum at the end of the experiment. The expression of the ob gene in adipose tissue was unchanged, and plasma leptin levels were also unchanged by treatment. Estrone levels increased more than 1500-fold, and estrone oleate rose 100-fold during treatment. The fact that estrone oleate had no effect on the leptin levels or expression in obese rats, in contrast with the marked inhibition observed in the lean suggests that the functionality of the leptin receptor is essential for estrone oleate inhibition of the ob gene. This also suggests that leptin may control ob gene expression in white adipose tissue and that estrone oleate may activate this process. The slimming effect of estrone oleate is, thus, not directly dependent on leptin, since both normoleptinemic and hyperleptinemic animals lose fat following treatment nor are the effects on appetite and energy expenditure mediated by leptin. However, leptin levels and the expression of the ob gene are directly linked with estrone oleate function. A possible involvement of leptin in estrone oleate action is postulated. The results support the participation of estrone oleate in the control of body weight and hint at the complexity of its regulation by leptin and glucocorticoids.     

Endurance training increases lactate transport in male Zucker fa/fa rats

The purpose of this study was to investigate the effect of endurance training (10 weeks) on previously reported alterations of lactate exchange in obese Zucker fa/fa rats. We used sarcolemmal vesicles to measure lactate transport capacity in control sedentary rats, Zucker (fa/fa), and endurance trained Zucker (fa/fa) rats. Monocarboxylate transporter (MCT) 1 and 4 content was measured in sarcolemmal vesicles and skeletal muscle. Training increased citrate synthase activity in soleus and in red tibialis anterior, and improved insulin sensitivity measured by intraperitoneal glucose tolerance test. Endurance training increased lactate influx in sarcolemmal vesicles at 1 mM of external lactate concentration and increased MCT1 expression on sarcolemmal vesicles. Furthermore, muscular lactate level was significantly decreased after training in red tibialis anterior and extensor digitorum longus. This study shows that endurance training improves impairment of lactate transport capacity that is found in insulin resistance state like obesity and type 2 diabetes.     

Leptin effect on intestinal galactose absorption in ob/ob and db/db mice

Our previous works demonstrated that leptin inhibits galactose absorption in rat and mice intestinal rings. Here, we have studied the effect of exogenous leptin on intestinal galactose absorption in the genetically obese db/db (leptin-resistant) and ob/ob (leptin-deficient) mice. Assays were performed by incubating the intestinal rings in saline solution containing 5 mM galactose in the absence or presence of 0.2 or 0.4 nM leptin. Basal galactose uptake was similar in the wild-type and the two obese groups. Contrarily to what happens in wild-type mice, leptin increased galactose uptake in db/db animals; since these mice lack the functional long leptin receptor, the measured effect may be due to the short receptor signaling. In the ob/ob mice, 0.2 nM leptin also increased galactose absorption whereas 0.4 nM did not have any effect, suggesting that in the genetically obese animals the expression and regulation of leptin receptors may be altered.     

Modulation of circulating leptin levels by its soluble receptor

Leptin is an adipocyte-derived hormone with potent weight reducing effects. Genetically obese rodents with mutations of leptin or the leptin receptor are defective in leptin signaling and develop morbid obesity and diabetes. Interestingly, the levels of both leptin mRNA and protein are increased by up to 20-fold in these animals, suggesting the existence of a feedback mechanism controlling the amount of leptin in circulation. In this report, we attempted to determine whether the up-regulation of circulating leptin in Zucker Diabetic Fatty rats, which are nonresponsive to leptin due to a receptor point mutation, is entirely due to increased expression of leptin. We demonstrate that the high level of circulating leptin in these rats is attributable to at least two factors: increased leptin expression by the adipose tissue and delayed clearance of leptin from circulation due to binding to its soluble receptor. The latter conclusion was supported by three lines of evidence: 1) The soluble leptin receptor is up-regulated by about 20-fold in Zucker Diabetic Fatty rats; 2) Adenovirus-mediated overexpression of the soluble leptin receptor results in a similar -fold increase of circulating leptin; 3) In ob/ob mice, which have no endogenous leptin, exogenously administered leptin reaches a higher level when the soluble leptin receptor is overexpressed. The weight-reducing effect of leptin is enhanced in C57Bl/6 ob/ob mice with overexpression of the soluble leptin receptor. Soluble leptin receptor may be a significant factor determining the amount of total leptin in circulation.     

Excessive food intake, obesity and inflammation process in Zucker fa/fa rat pancreatic islets

Inappropriate food intake-related obesity and more importantly, visceral adiposity, are major risk factors for the onset of type 2 diabetes. Evidence is emerging that nutriment-induced β-cell dysfunction could be related to indirect induction of a state of low grade inflammation. Our aim was to study whether hyperphagia associated obesity could promote an inflammatory response in pancreatic islets leading to ?-cell dysfunction. In the hyperphagic obese insulin resistant male Zucker rat, we measured the level of circulating pro-inflammatory cytokines and estimated their production as well as the expression of their receptors in pancreatic tissue and β-cells. Our main findings concern intra-islet pro-inflammatory cytokines from fa/fa rats: IL-1β, IL-6 and TNFα expressions were increased; IL-1R1 was also over-expressed with a cellular redistribution also observed for IL-6R. To get insight into the mechanisms involved in phenotypic alterations, abArrays were used to determine the expression profile of proteins implicated in different membrane receptors signaling, apoptosis and cell cycle pathways. Despite JNK overexpression, cell viability was unaffected probably because of decreases in cleaved caspase3 as well as in SMAC/DIABLO and APP, involved in the induction and amplification of apoptosis. Concerning β-cell proliferation, decreases in important cell cycle regulators (Cyclin D1, p35) and increased expression of SMAD4 probably contribute to counteract and restrain hyperplasia in fa/fa rat islets. Finally and probably as a result of IL-1β and IL-1R1 increased expressions with sub-cellular redistribution of the receptor, islets from fa/fa rats were found more sensitive to both stimulating and inhibitory concentrations of the cytokine; this confers some physiopathological relevance to a possible autocrine regulation of β-cell function by IL-1β. These results support the hypothesis that pancreatic islets from prediabetic fa/fa rats undergo an inflammatory process. That the latter could contribute to β-cell hyperactivity/proliferation and possibly lead to progressive β-cell failure in these animals, deserves further investigations.     

Ablation of ghrelin receptor in leptin-deficient ob/ob mice has paradoxical effects on glucose homeostasis when compared with ablation of ghrelin in ob/ob mice

The orexigenic hormone ghrelin is important in diabetes because it has an inhibitory effect on insulin secretion. Ghrelin ablation in leptin-deficient ob/ob (Ghrelin(-/-):ob/ob) mice increases insulin secretion and improves hyperglycemia. The physiologically relevant ghrelin receptor is the growth hormone secretagogue receptor (GHS-R), and GHS-R antagonists are thought to be an effective strategy for treating diabetes. However, since some of ghrelin's effects are independent of GHS-R, we have utilized genetic approaches to determine whether ghrelin's effect on insulin secretion is mediated through GHS-R and whether GHS-R antagonism indeed inhibits insulin secretion. We investigated the effects of GHS-R on glucose homeostasis in Ghsr-ablated ob/ob mice (Ghsr(-/-):ob/ob). Ghsr ablation did not rescue the hyperphagia, obesity, or insulin resistance of ob/ob mice. Surprisingly, Ghsr ablation worsened the hyperglycemia, decreased insulin, and impaired glucose tolerance. Consistently, Ghsr ablation in ob/ob mice upregulated negative β-cell regulators (such as UCP-2, SREBP-1c, ChREBP, and MIF-1) and downregulated positive β-cell regulators (such as HIF-1α, FGF-21, and PDX-1) in whole pancreas; this suggests that Ghsr ablation impairs pancreatic β-cell function in leptin deficiency. Of note, Ghsr ablation in ob/ob mice did not affect the islet size; the average islet size of Ghsr(-/-):ob/ob mice is similar to that of ob/ob mice. In summary, because Ghsr ablation in leptin deficiency impairs insulin secretion and worsens hyperglycemia, this suggests that GHS-R antagonists may actually aggravate diabetes under certain conditions. The paradoxical effects of ghrelin ablation and Ghsr ablation in ob/ob mice highlight the complexity of the ghrelin-signaling pathway.     

Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents.

Long chain fatty acid transport is selectively up-regulated in adipocytes of Zucker fatty rats, diverting fatty acids from sites of oxidation toward storage in adipose tissue. To determine whether this is a general feature of obesity, we studied [(3)H]oleate uptake by adipocytes and hepatocytes from 1) homozygous male obese (ob), diabetic (db), fat (fat), and tubby (tub) mice and from 2) male Harlan Sprague-Dawley rats fed for 7 weeks a diet containing 55% of calories from fat. V(max) and K(m) were compared with controls of the appropriate background strain (C57BL/6J or C57BLKS) or diet (13% of calories from fat). V(max) for adipocyte fatty acid uptake was increased 5-6-fold in ob, db, fat, and tub mice versus controls (p < 0.001), whereas no differences were seen in the corresponding hepatocytes. Similar changes occurred in fat-fed rats. Of three membrane fatty acid transporters expressed in adipocytes, plasma membrane fatty acid-binding protein mRNA was increased 9-11-fold in ob and db, which lack a competent leptin/leptin receptor system, but was not increased in fat and tub, i.e. in strains with normal leptin signaling capability; fatty acid translocase mRNA was increased 2.2-6.5-fold in tub, ob, and fat adipocytes, but not in db adipocytes; and only marginal changes in fatty acid transport protein 1 mRNA were found in any of the mutant strains. Adipocyte fatty acid uptake is generally increased in murine obesity models, but up-regulation of individual transporters depends on the specific pathophysiology. Leptin may normally down-regulate expression of plasma membrane fatty acid binding protein.     

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.     

AZ 242, a novel PPARalpha/gamma agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats

Abnormalities in fatty acid (FA) metabolism underlie the development of insulin resistance and alterations in glucose metabolism, features characteristic of the metabolic syndrome and type 2 diabetes that can result in an increased risk of cardiovascular disease. We present pharmacodynamic effects of AZ 242, a novel peroxisome proliferator activated receptor (PPAR)alpha/gamma agonist. AZ 242 dose-dependently reduced the hypertriglyceridemia, hyperinsulinemia, and hyperglycemia of ob/ob diabetic mice. Euglycemic hyperinsulinemic clamp studies showed that treatment with AZ 242 (1 micromol/kg/d) restored insulin sensitivity of obese Zucker rats and decreased insulin secretion. In vitro, in reporter gene assays, AZ 242 activated human PPARalpha and PPARgamma with EC(50) in the micro molar range. It also induced differentiation in 3T3-L1 cells, an established PPARgamma effect, and caused up-regulation of liver fatty acid binding protein in HepG-2 cells, a PPARalpha-mediated effect. PPARalpha-mediated effects of AZ 242 in vivo were documented by induction of hepatic cytochrome P 450-4A in mice. The results indicate that the dual PPARalpha/gamma agonism of AZ 242 reduces insulin resistance and has beneficial effects on FA and glucose metabolism. This effect profile could provide a suitable therapeutic approach to the treatment of type 2 diabetes, metabolic syndrome, and associated vascular risk factors.     

Adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferator-activated receptor gamma agonists

Elevated levels of the hormone resistin, which is secreted by fat cells, are proposed to cause insulin resistance and to serve as a link between obesity and type 2 diabetes. In this report we show that resistin expression is significantly decreased in the white adipose tissue of several different models of obesity including the ob/ob, db/db, tub/tub, and KKA(y) mice compared with their lean counterparts. Furthermore, in response to several different classes of antidiabetic peroxisome proliferator-activated receptor gamma agonists, adipose tissue resistin expression is increased in both ob/ob mice and Zucker diabetic fatty rats. These data demonstrate that experimental obesity in rodents is associated with severely defective resistin expression, and decreases in resistin expression are not required for the antidiabetic actions of peroxisome proliferator-activated receptor gamma agonists.     

Leptin receptor-deficient Zucker (fa/fa) rat retards the development of pig serum-induced liver fibrosis with Kupffer cell dysfunction

The aim of this study was to investigate the role of leptin in the development of liver fibrosis with Kupffer cell function using leptin receptor deficient rats. Male Zucker (fa/fa) and control (fa/-) rats received pig serum for 8 weeks. Animals were sacrificed to estimate the degree of liver fibrosis and stellate cell activation with the expression of alpha smooth muscle actin (alphaSMA). Microarray analysis was performed. Isolated Kuppfer cells of Zucker and control rats were treated with LPS. LPS uptake and TNF-alpha production were examined. Stellate cells were also isolated from Zucker and control rats. The expression of procollagen type I mRNAs was examined. Control rats developed liver fibrosis 8 weeks after injection of pig serum and showed an increased liver hydroxyproline content of 348 +/- 34 microg/g (n = 10) compared with Zucker rats (225 +/- 13, n = 10, P < 0.01). The procollagen type I mRNA level and alphaSMA expression of Zucker rats were also significantly reduced. Microarray analysis indicated significantly reduced expression of TNF-alpha, LPS-binding protein, urokinase-type plasminogen activator (uPA), IGF, IGF-binding protein (IGFBP)-3,5, and increased expression of apolipoprotein IV. Isolated Kupffer cells of Zucker rats showed significantly reduced LPS uptake as well as TNF-alpha production compared with control rats. However, no significant change was observed in procollagen type I mRNA levels of isolated stellate cells after 4 days of culture on plastic dishes. These results suggest that leptin receptor deficiency retards the development of liver fibrosis due to the dysfunction of Kuppfer cells.