Suppressor of cytokine signaling-3 (Socs-3) negatively regulates the action of various cytokines, as well as the metabolic hormones leptin and insulin. Mice with haploinsufficiency of Socs-3, or those with neuronal deletion of Socs-3, are lean and more leptin and insulin sensitive. To examine the role of Socs-3 within specific neurons critical to energy balance, we created mice with selective deletion of Socs-3 within pro-opiomelanocortin (POMC)-expressing cells. These mice had enhanced leptin sensitivity, measured by weight loss and food intake after leptin infusion. On chow diet, glucose homeostasis was improved despite normal weight gain. On a high-fat diet, the rate of weight gain was reduced, due to increased energy expenditure rather than decreased food intake; glucose homeostasis and insulin sensitivity were substantially improved. These studies demonstrate that Socs-3 within POMC neurons regulates leptin sensitivity and glucose homeostasis, and plays a key role in linking high-fat diet to disordered metabolism.

Peroxisome proliferator-activated receptor-␥ (PPAR␥) is a nuclear hormone receptor that is critical for adipogenesis and insulin sensitivity. Ligands for PPAR␥ include some polyunsaturated fatty acids and prostanoids and the synthetic high affinity antidiabetic agents thiazolidinediones. However, the identity of a biologically relevant endogenous PPAR␥ ligand is unknown, and limited insight exists into the factors that may regulate production of endogenous PPAR␥ ligands during adipocyte development. To address this question, we created a line of 3T3-L1 preadipocytes that carry a ␤-galactosidase-based PPAR␥ ligand-sensing vector system. In this system, induction of adipogenesis resulted in elevated ␤-galactosidase activity that signifies activation of PPAR␥ via its ligand-binding domain (LBD) and suggests generation and/or accumulation of a ligand moiety. The putative endogenous ligand appeared early in adipogenesis in response to increases in cAMP, accumulated in the medium, and dissipated later in adipogenesis. Organically extracted and high pressure liquid chromatography-fractionated conditioned media from differentiating cells, but not from mature adipocytes, were enriched in this activity. One or more components within the organic extract activated PPAR␥ through interaction with its LBD, induced lipid accumulation in 3T3-L1 cells as efficiently as the differentiation mixture, and competed for binding of rosiglitazone to the LBD of PPAR␥. The active species appears to be different from other PPAR␥ ligands identified previously. Our findings suggest that a novel biologically relevant PPAR␥ ligand is transiently produced in 3T3-L1 cells during adipogenesis. Peroxisome proliferator-activated receptor-␥ (PPAR␥)1 is a nuclear hormone receptor that forms obligate heterodimers with the retinoid X receptor (RXR) (1) and binds to direct repeat 1-type motifs found in the promoter sites of target genes (2). PPAR␥ is a major modulator of several aspects of development and homeostasis. It is expressed in breast, colon, prostate, macrophages, and adipose tissue (3-6) and has been shown to play a critical role in glucose and lipid metabolism (7), macrophage function (8), and adipogenesis (9).As a member of the nuclear receptor superfamily, PPAR␥ is activated through ligand binding, which results in allosteric changes in receptor conformation, recruitment of coactivators, assembly of a transcriptional complex, and regulated transcription of target genes (10). Among known PPAR␥ agonists are the synthetic high affinity antidiabetic drugs thiazolidinediones (11) and a number of natural substances. These include the dehydration product of prostaglandin D 2 , 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15-dPGJ 2 ) (12, 13); derivatives of linoleate, 9-and 13-hydroxyoctadeca-9Z,11E-dienoic acids, both found in oxidized low density lipoprotein (14); certain polyunsaturated fatty acids (15,16); and oxidized alkyl phospholipids such as lysophosphatidic acid (17). Most of these natural ligands bind PPAR␥ with relatively low affinity compared...

The melanocortin-4 receptor (MC4R) is well recognized as an important mediator of body weight homeostasis. Activation of MC4R causes dramatic weight loss in rodent models, and mutations in human are associated with obesity. This makes MC4R a logical target for pharmacological therapy for the treatment of obesity. However, previous studies in rodents and humans have observed a broad array of side effects caused by acute treatment with MC4R agonists, including increased heart rate and blood pressure. We demonstrate that treatment with a highly-selective novel MC4R agonist (BIM-22493 or RM-493) resulted in transient decreases in food intake (35%), with persistent weight loss over 8 weeks of treatment (13.5%) in a diet-induced obese nonhuman primate model. Consistent with weight loss, these animals significantly decreased adiposity and improved glucose tolerance. Importantly, we observed no increases in blood pressure or heart rate with BIM-22493 treatment. In contrast, treatment with LY2112688, an MC4R agonist previously shown to increase blood pressure and heart rate in humans, caused increases in blood pressure and heart rate, while modestly decreasing food intake. These studies demonstrate that distinct melanocortin peptide drugs can have widely different efficacies and side effects.

Low levels of the adipocyte hormone leptin are considered to be the key signal contributing to inhibited gonadotrophin-releasing hormone (GnRH) release and reproductive acyclicity during negative energy balance. Hypoleptinaemia-induced inhibition of GnRH may be initiated with upstream inhibition of the secretagogue kisspeptin (Kiss1) because GnRH neurones do not express leptin receptors. The present study aimed to determine whether eliminating the hypoleptinaemia associated with caloric restriction (CR), by restoring leptin to normal basal levels, could reverse the suppression of the reproductive neuroendocrine axis. Fifty percent CR resulted in significant suppression of anteroventral periventricular Kiss1 mRNA, arcuate nucleus (ARH) Kiss1 and neurokinin B (NKB) mRNA levels and serum luteinising hormone (LH). Restoring leptin to normal basal levels did not restore Kiss1 or NKB mRNA or LH levels. Surprisingly, leptin did not activate expression of phosphorylated signal-transducer and activator of transcription-3 in ARC Kiss1 neurones, indicating that these neurones may not relay leptin signalling to GnRH neurones. Previous work in fasting models showing restoration of LH used a pharmacological dose of leptin. Therefore, in a 48-h fast study, replacement of leptin to pharmacological levels was compared with replacement of leptin to normal basal levels. Maintaining leptin at normal basal levels during the fast did not prevent inhibition of LH. By contrast, pharmacological levels of leptin did maintain LH at control values. These results suggest that, although leptin may be a permissive signal for reproductive function, hypoleptinaemia is unlikely to be the critical signal responsible for ARC Kiss1 and LH inhibition during negative energy balance.

BACKGROUND The intake of a Western diet enriched in animal fat has been shown to be a major risk factor for Type 2 diabetes and obesity. Previous rodent studies have indicated that these conditions may be triggered by the accumulation of the sphingolipid ceramide in insulin-sensitive tissues. However, data are lacking in this regard from both humans and non-human primates. OBJECTIVE Here we have investigated the relationship between plasma ceramides and metabolic syndrome in Rhesus macaques fed a high-fat and high-fructose (HFFD) ‘western’ diet. METHODS We investigated this relationship in cohorts of monkeys fed a HFFD for a period of 8 months to 5 years. Animals were classified as control, pre-diabetic or diabetic based on fasting plasma parameters and insulin sensitivity. RESULTS HFFD treatment produced significant increases in body weight and body fat and also resulted in a decline in insulin sensitivity. In parallel to the reduction in insulin sensitivity, significant increases in both plasma ceramide and dihydroceramide levels were observed, which further increased as animals progressed to the diabetic state. Plasma levels of the rare sphingolipid C18:0 deoxysphinganine, a marker of increased metabolic flux through serine palmitoyl transferase (SPT), were also elevated in both pre- and diabetic animals. Furthermore, plasma serine levels were significantly elevated in diabetic monkeys, which may indicate a shift in SPT substrate selectivity from serine to alanine or glycine. In contrast, branch chain amino acids were unchanged in pre-diabetic non-human primates, and only plasma valine levels were elevated in diabetic animals. CONCLUSION Together, these data indicate that HFFD induces de novo synthesis of ceramides in non-human primates, and that increased production of plasma ceramides is significantly correlated with the decline in insulin sensitivity.

Objective To utilize a nonhuman primate model to examine the impact of maternal high-fat diet (HFD) consumption and pre-pregnancy obesity on offspring intake of palatable food. We will also examine whether maternal HFD consumption impaired development of the dopamine system, critical for the regulation of hedonic feeding. Methods The impact of exposure to maternal HFD and obesity on offspring consumption of diets of varying composition was assessed after weaning. We also examined the influence of maternal HFD consumption on the development of the prefrontal cortex-dopamine system at 13 months of age. Results During a preference test, offspring exposed to maternal obesity and HFD consumption displayed increased intake of food high in fat and sugar content relative to offspring from lean control mothers. Maternal HFD consumption suppressed offspring dopamine signaling (as assessed by immunohistochemistry) relative to control offspring. Specifically, there was decreased abundance of dopamine fibers and of dopamine receptor 1 and 2 protein. Conclusion Our findings reveal that offspring exposed to both maternal HFD consumption and maternal obesity during early development are at increased risk for obesity due to overconsumption of palatable energy-dense food, a behavior that may be related to reduced central dopamine signaling.

Background Inflammation and insulin resistance (IR) are associated processes that potentiate risk for cardiovascular disease in obesity. The temporal relation between IR and inflammation is not completely characterized. We hypothesized that endothelial cell adhesion molecule (ECAM) expression in large arteries is an early event that coincides with diet-induced obesity and IR in primates. Methods and Results Ten adult male rhesus macaques were studied at baseline and every 4-6 months on high-fat diet (HFD) for 2 years. Truncal fat, carotid intima-media thickness (IMT), plasma inflammatory biomarkers, and carotid P-selectin and VCAM-1 expression by contrast-enhanced ultrasound molecular imaging were assessed. Intravenous glucose tolerance test (IVGTT) was performed at baseline, 4 and 18 months. HFD produced a rapid increase (p<0.01) in weight, truncal fat, and degree of IR indicated by the insulin area-under-the-curve and glucose disappearance rate on IVGTT; all of which worsened minimally thereafter. Molecular imaging detected a progressive increase in ECAM expression over time (5-7-fold greater than control agent signal at 2 yrs, p<0.01). Changes in IMT were not detected until 2 years and, while there was a trend toward an increase in plasma markers of inflammation (MCP-1, CRP), the pattern of increase varied considerably over time. Conclusions In primates with diet-induced obesity, endothelial inflammatory activation is an early event that occurs coincident with the development of IR and long before any measurable change carotid IMT. Endothelial activation is more related to the duration rather than severity of IR and is not mirrored by changes in plasma biomarkers.

ObjectiveNon-alcoholic fatty liver disease (NAFLD) risk begins in utero in offspring of obese mothers. A critical unmet need in this field is to understand the pathways and biomarkers underlying fetal hepatic lipotoxicity and whether maternal dietary intervention during pregnancy is an effective countermeasure.MethodsWe utilized a well-established non-human primate model of chronic, maternal, Western-style diet induced obesity (OB-WSD) compared with mothers on a healthy control diet (CON) or a subset of OB-WSD mothers switched to the CON diet (diet reversal; OB-DR) prior to and for the duration of the next pregnancy. Fetuses were studied in the early 3rd trimester.ResultsFetuses from OB-WSD mothers had higher circulating triglycerides (TGs) and lower arterial oxygenation suggesting hypoxemia, compared with fetuses from CON and OB-DR mothers. Hepatic TG content, oxidative stress (TBARs), and de novo lipogenic genes were increased in fetuses from OB-WSD compared with CON mothers. Fetuses from OB-DR mothers had lower lipogenic gene expression and TBARs yet persistently higher TGs. Metabolomic profiling of fetal liver and serum (umbilical artery) revealed distinct separation of CON and OB-WSD groups, and an intermediate phenotype in fetuses from OB-DR mothers. Pathway analysis identified decreased tricarboxylic acid cycle intermediates, increased amino acid (AA) metabolism and byproducts, and increased gluconeogenesis, suggesting an increased reliance on AA metabolism to meet energy needs in the liver of fetuses from OB-WSD mothers. Components in collagen synthesis, including serum protein 5-hydroxylysine and hepatic lysine and proline, were positively correlated with hepatic TGs and TBARs, suggesting early signs of fibrosis in livers from the OB-WSD group. Importantly, hepatic gluconeogenic and arginine related intermediates and serum levels of lactate, pyruvate, several AAs, and nucleotide intermediates were normalized in the OB-DR group. However, hepatic levels of CDP-choline and total ceramide levels remained high in fetuses from OB-DR mothers.ConclusionsOur data provide new metabolic evidence that, in addition to fetal hepatic steatosis, maternal WSD creates fetal hypoxemia and increases utilization of AAs for energy production and early activation of gluconeogenic pathways in the fetal liver. When combined with hyperlipidemia and limited antioxidant activity, the fetus suffers from hepatic oxidative stress and altered intracellular metabolism which can be improved with maternal diet intervention. Our data reinforce the concept that multiple “first hits” occur in the fetus prior to development of obesity and demonstrate new biomarkers with potential clinical implications for monitoring NAFLD risk in offspring.