Pioglitazone ameliorates insulin resistance and diabetes by both adiponectin-dependent and -independent pathways

Thiazolidinediones have been shown to up-regulate adiponectin expression in white adipose tissue and plasma adiponectin levels, and these up-regulations have been proposed to be a major mechanism of the thiazolidinedione-induced amelioration of insulin resistance linked to obesity. To test this hypothesis, we generated adiponectin knock-out (adipo-/-) ob/ob mice with a C57B/6 background. After 14 days of 10 mg/kg pioglitazone, the insulin resistance and diabetes of ob/ob mice were significantly improved in association with significant up-regulation of serum adiponectin levels. Amelioration of insulin resistance in ob/ob mice was attributed to decreased glucose production and increased AMP-activated protein kinase in the liver but not to increased glucose uptake in skeletal muscle. In contrast, insulin resistance and diabetes were not improved in adipo-/-ob/ob mice. After 14 days of 30 mg/kg pioglitazone, insulin resistance and diabetes of ob/ob mice were again significantly ameliorated, which was attributed not only to decreased glucose production in the liver but also to increased glucose uptake in skeletal muscle. Interestingly, adipo-/-ob/ob mice also displayed significant amelioration of insulin resistance and diabetes, which was attributed to increased glucose uptake in skeletal muscle but not to decreased glucose production in the liver. The serum-free fatty acid and triglyceride levels as well as adipocyte sizes in ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were significantly reduced to a similar degree after 30 mg/kg pioglitazone. Moreover, the expressions of TNFalpha and resistin in adipose tissues of ob/ob and adipo-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were decreased after 30 mg/kg pioglitazone. Thus, pioglitazone-induced amelioration of insulin resistance and diabetes may occur adiponectin dependently in the liver and adiponectin independently in skeletal muscle.     

gAd-globular head domain of adiponectin increases fatty acid oxidation in newborn rabbit hearts

Adiponectin is an adipocyte-derived hormone that has a number of metabolic effects in the body, including the control of both glucose and fatty acid metabolism. The globular head domain of adiponectin, gAd, has also been shown to increase fatty acid oxidation in skeletal muscle. Within days after birth, a rapid increase in fatty acid oxidation occurs in the heart. We examined whether adiponectin or gAd plays a role in this maturation of cardiac fatty acid oxidation. Plasma adiponectin increased in newborn rabbits following birth: 1.2 +/- 0.3 microg/ml in 1-day-old, 6.8 +/- 1.8 microg/ml in 7-day-old, and 45 +/- 5 microg/ml in 6-week-old rabbits. Because plasma insulin levels decrease and remain low throughout the suckling period, and because this decrease may contribute to the maturation of fatty acid oxidation, we examined the effects of adiponectin and gAd on fatty acid oxidation in isolated perfused 1-day-old rabbit hearts in the presence or absence of 100 microunits/ml insulin. Adiponectin (10 microg/ml) did not alter fatty acid oxidation in the presence of insulin. In the absence of insulin, the addition of recombinant gAd (1.5 microg/ml) increased fatty acid oxidation compared with control (129 +/- 18 versus 66 +/- 11 nmol.g dry weight(-1).min(-1), respectively (p < 0.05). In 7-day-old hearts, where fatty acid oxidation rates were 5-fold higher than 1-day-old hearts, gAd did not alter fatty acid oxidation rates. The increase in fatty acid oxidation in 1-day-old hearts occurred independently of changes in 5'-AMP-activated protein kinase, acetyl-CoA carboxylase, or malonyl-CoA. The effect of gAd on fatty acid oxidation was reversed in the presence of 100 microunits/ml insulin. These results suggest that a decrease in plasma insulin and increase in gAd are involved in the increase of cardiac fatty acid oxidation in the immediate newborn period.     

MKR mice are resistant to the metabolic actions of both insulin and adiponectin: discordance between insulin resistance and adiponectin responsiveness

Most rodent models of insulin resistance are accompanied by decreased circulating adiponectin levels. Adiponectin treatment improves the metabolic phenotype by increasing fatty acid oxidation in skeletal muscle and suppressing hepatic glucose production. Muscle IGF-I receptor (IGF-IR)-lysine-arginine (MKR) mice expressing dominant-negative mutant IGF-IRs in skeletal muscle are diabetic with insulin resistance in muscle, liver, and adipose tissue. Adiponectin levels are elevated in MKR mice, suggesting an unusual discordance between insulin resistance and adiponectin responsiveness. Therefore, we investigated the metabolic actions of adiponectin in MKR mice. MKR and ob/ob mice were treated both acutely (28 microg/g) and chronically (for 2 wk) with full-length adiponectin. Acute hypoglycemic effects of adiponectin were evident only in ob/ob mice but not in MKR mice. Chronic adiponectin treatment significantly improved both insulin sensitivity and glucose tolerance in ob/ob but not in MKR mice. Adiponectin receptor mRNA levels and adiponectin-stimulated phosphorylation of AMPK in skeletal muscle and liver were similar among MKR, wild-type, and ob/ob mice. Thus MKR mice are adiponectin resistant despite normal expression of adiponectin receptors and normal AMPK phosphorylation in muscle and liver. MKR mice may be a useful model for dissecting relationships between insulin resistance and adiponectin action in regulation of glucose homeostasis.     

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.     

Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice

Conjugated linoleic acid (CLA) induces insulin resistance preceded by rapid depletion of the adipokines leptin and adiponectin, increased inflammation, and hepatic steatosis in mice. To determine the role of leptin in CLA-mediated insulin resistance and hepatic steatosis, recombinant leptin was coadministered with dietary CLA in ob/ob mice to control leptin levels and to, in effect, negate the leptin depletion effect of CLA. In a 2 x 2 factorial design, 6 week old male ob/ob mice were fed either a control diet or a diet supplemented with CLA and received daily intraperitoneal injections of either leptin or vehicle for 4 weeks. In the absence of leptin, CLA significantly depleted adiponectin and induced insulin resistance, but it did not increase hepatic triglyceride concentrations or adipose inflammation, marked by interleukin-6 and tumor necrosis factor-alpha mRNA expression. Insulin resistance, however, was accompanied by increased macrophage infiltration (F4/80 mRNA) in adipose tissue. In the presence of leptin, CLA depleted adiponectin but did not induce insulin resistance or macrophage infiltration. Despite this, CLA induced hepatic steatosis. In summary, CLA worsened insulin resistance without evidence of inflammation or hepatic steatosis in mice after 4 weeks. In the presence of leptin, CLA failed to worsen insulin resistance but induced hepatic steatosis in ob/ob mice.     

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.     

Palmitate acutely induces insulin resistance in isolated muscle from obese but not lean humans

Exposure to high fatty acids (FAs) induces whole body and skeletal muscle insulin resistance. The globular form of the adipokine, adiponectin (gAd), stimulates FA oxidation and improves insulin sensitivity; however, its ability to prevent lipid-induced insulin resistance in humans has not been tested. The purpose of this study was to determine 1) whether acute (4 h) exposure to 2 mM palmitate would impair insulin signaling and glucose transport in isolated human skeletal muscle, 2) whether muscle from obese humans is more susceptible to the effects of palmitate, and 3) whether the presence of 2 mM palmitate + 2.5 mug/ml gAd (P+gAd) could prevent the effects of palmitate. Insulin-stimulated (10 mU/ml) glucose transport was not different, relative to control, following exposure to palmitate (-10%) or P+gAd (-3%) in lean muscle. In obese muscle, the absolute increase in glucose transport from basal to insulin-stimulated conditions was significantly decreased following palmitate (-55%) and P+gAd (-36%) exposure (control vs. palmitate; control vs. P+gAd, P < 0.05). There was no difference in the absolute increase in glucose transport between palmitate and P+gAd, indicating that in the presence of palmitate, gAd did not improve glucose transport. The palmitate-induced reduction in insulin-stimulated glucose transport in muscle from obese individuals may have been due to reduced Ser Akt (control vs. palmitate; P+gAd, P < 0.05) and Akt substrate 160 (AS160) phosphorylation (control vs. palmitate; P+gAd, P < 0.05). FA oxidation was significantly increased in muscle of lean and obese individuals in the presence of gAd (P < 0.05), suggesting that the stimulatory effects of gAd on FA oxidation may not be sufficient to entirely prevent palmitate-induced insulin resistance in obese muscle.     

Novel qualitative aspects of tissue fatty acids related to metabolic regulation: lessons from Elovl6 knockout

Insulin resistance, often associated with obesity, precipitates metabolic syndrome, type 2 diabetes, and finally, atherosclerosis. Sources of excess energy cause abnormal accumulation of tissue lipids leading to cellular dysfunction through cellular stress and inflammation. This process is often referred to as lipotoxicity. Until date, effective approaches that aim to overcome insulin resistance involve amelioration of obesity by caloric restriction and/or exercise. Quantitative control of lipids, especially triglycerides and fatty acids in adipose and other tissues, and plasma can be addressed using these measures. However, altering tissue lipid composition may provide another strategy to prevent or control lipotoxicity. Endogenous fatty acid synthesis plays a crucial role in determining tissue energy states. As a target gene of SREBP-1 that controls lipogenesis we identified a unique enzyme, Elovl6, which is responsible for the final step in endogenous saturated fatty acid synthesis, thereby controlling tissue fatty acid composition. Elovl6-deficient mice become obese and develop hepatosteatosis when fed a high-fat diet or when mated to leptin-deficient ob/ob mice. However, the mice exhibited marked protection from hyperinsulinemia, hyperglycemia, and hyperleptinemia. Hepatic fatty acid composition is a novel determinant of insulin sensitivity independent of cellular energy balance. Inhibiting Elovl6 activity may provide a novel therapeutic approach for treating insulin resistance, diabetes, metabolic syndrome, and cardiovascular risks by circumventing obesity problems. In this review, we consider fatty acid metabolism and lipotoxicity, and discuss the role of Elovl6 in newly recognized aspects of metabolic regulation.     

Effects of ciglitazone on insulin resistance and thermogenic responsiveness to acute cold in brown adipose tissue of genetically obese (ob/ob) mice

Genetically obese (ob/ob) mice develop a marked insulin resistance in brown adipose tissue soon after weaning, and this is paralleled by a fall in the acute activation of the mitochondrial proton conductance pathway in the tissue on cold exposure. Treatment of ob/ob mice with ciglitazone, a new oral hypoglycaemic, led to a restoration of insulin sensitivity in brown adipose tissue. The amelioration of insulin resistance was accompanied by a normalization of the acute, cold-induced increase in mitochondrial GDP binding. These results support the hypothesis that the development of insulin resistance in brown adipose tissue is an important factor in the impaired thermogenic responsiveness of obese mice.     

L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1beta and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1beta and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes.     

Disruption of adiponectin causes insulin resistance and neointimal formation

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.     

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.     

Plasma proteome analysis for anti‐obesity and anti‐diabetic potentials of chitosan oligosaccharides in ob/ob mice

Altered levels of adipokines, derived as a result of distorted adipocytes, are the major factors responsible for changing biochemical parameters in obesity that leads to the development of metabolic disorders such as insulin resistance and atherosclerosis. In our previous reports, chitosan oligosaccharides (CO) were proved to inhibit the differentiation of 3T3‐L1 adipocytes. In the present study, an attempt was made to investigate the anti‐obesity and anti‐diabetic effect of CO on ob/ob mice, by means of differential proteomic analysis of plasma. This was followed by immunoblotting, and gene expression in adipose tissue to clarify the molecular mechanism. CO treatment showed reduced diet intake (13%), body weight gain (12%), lipid (29%) and glucose levels (35%). 2‐DE results showed differential levels of five proteins namely RBP4, apoE, and apoA‐IV by >2‐fold down‐regulation and by >2‐fold of apoA‐I and glutathione peroxidase (GPx) up‐regulation after CO treatment. Immunoblotting studies of adiponectin and resistin showed amelioration in their levels in plasma. Furthermore, the results of gene expressions for adipose tissue specific TNF‐α, and IL‐6 secretary molecules were also down‐regulated by CO treatment. Gene expressions of PPARγ in adipose tissue were in good agreement with the ameliorated levels of adipokines, thereby improving the pathological state. Taken together, CO might act as a potent down‐regulator of obesity‐related gene expression in ob/ob mice that may normalize altered plasma proteins to overcome metabolic disorders of obesity.     

Isolated adipocytes from growth hormone-treated obese (ob/ob) mice exhibit insulin resistance

The genetically obese (ob/ob) mouse is a useful model for the study of the diabetogenic action of growth hormone (GH), because treatment of these animals with GH results in decreased responsiveness of their adipose tissue to insulin in vitro. Studies of the mechanisms involved in GH-induced insulin resistance using isolated adipocytes of ob/ob mice have not been possible, however, because of their extreme fragility and the lack of an adequate system for the maintenance of these cells. This study describes a new method for the isolation of ob/ob mouse adipocytes. The isolated cells are stable, viable and metabolically responsive to insulin. In addition, these adipocytes have been maintained in primary culture, in serum-free medium, for up to 3 days. During culture, the cells exhibit large increases in 125I-hGH binding (10-20-fold) and porcine 125I-insulin binding (5-10-fold). The induction of insulin resistance by GH has also been demonstrated in these freshly isolated ob/ob mouse adipocytes. The studies to date indicate that the ob/ob mouse adipocyte system should provide a useful model for detailed studies of the cellular and molecular mechanisms of GH induced insulin resistance.     

Adiponectin modulates carnitine palmitoyltransferase-1 through AMPK signaling cascade in rat cardiomyocytes

Adiponectin, an adipocyte-derived polypeptide hormone, plays an important role in regulating fatty acid oxidation. beta-oxidation of fatty acids supplies most of the cardiac energy and carnitine palmitoyltransferase (CPT)-1 serves as a key regulator during this process. To characterize the potential effects of adiponectin on CPT-1, we incubated rat neonatal cardiomyocytes with globular adiponectin (gAd). Results showed that gAd promoted the activity and mRNA expression of CPT-1. The underlying signal pathway involved in this modulatory effect was further investigated. Inhibition of AMP-activated protein kinase (AMPK) with adenine 9-beta-d-arabinofuranoside (AraA) completely abrogated gAd-mediated AMPK and acetyl coenzyme A carboxylase (ACC) phosphorylation and suppressed the promotion of CPT-1 activity. gAd also induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor (PPAR)-alpha, which was inhibited by AraA. SB202190, a p38MAPK inhibitor, blocked gAd-stimulated PPAR-alpha phosphorylation. When AMPK and/or p38MAPK was inhibited, gAd-enhanced mRNA expression of CPT-1 was partially reduced. In conclusion, our study suggests that the activation of AMPK signaling cascade participates in the promotion effect of gAd on CPT-1.     

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.     

Partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice

Partial leptin deficiency is not uncommon in the general population. We hypothesized that leptin insufficiency could favor obesity, nonalcoholic steatohepatitis (NASH), and other metabolic abnormalities, particularly under high calorie intake. Thus, mice partially deficient in leptin (ob/+) and their wild-type (+/+) littermates were fed for 4 mo with a standard-calorie (SC) or a high-calorie (HC) diet. Some ob/+ mice fed the HC diet were also treated weekly with leptin. Our results showed that, when fed the SC diet, ob/+ mice did not present significant metabolic abnormalities except for elevated levels of plasma adiponectin. Under high-fat feeding, increased body fat mass, hepatic steatosis, higher plasma total cholesterol, and glucose intolerance were observed in +/+ mice, and these abnormalities were further enhanced in ob/+ mice. Furthermore, some metabolic disturbances, such as blunted plasma levels of leptin and adiponectin, reduced UCP1 expression in brown adipose tissue, increased plasma liver enzymes, beta-hydroxybutyrate and triglycerides, and slight insulin resistance, were observed only in ob/+ mice fed the HC diet. Whereas de novo fatty acid synthesis in liver was decreased in +/+ mice fed the HC diet, it was disinhibited in ob/+ mice along with the restoration of the expression of several lipogenic genes. Enhanced expression of several genes involved in fatty acid oxidation was also observed only in ob/+ animals. Leptin supplementation alleviated most of the metabolic abnormalities observed in ob/+ fed the HC diet. Hence, leptin insufficiency could increase the risk of obesity, NASH, glucose intolerance, and hyperlipidemia in a context of calorie overconsumption.