Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity.

Most monogenic cases of obesity in humans have been linked to mutations in genes encoding members of the leptin-melanocortin pathway. Specifically, mutations in MC4R, the melanocortin-4 receptor gene, account for 3-5% of all severe obesity cases in humans1-3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity4,5. ADCY3 localizes to the primary cilia of neurons 6 , organelles that function as hubs for select signaling pathways. Mutations that disrupt the functions of primary cilia cause ciliopathies, rare recessive pleiotropic diseases in which obesity is a cardinal manifestation 7 . We demonstrate that MC4R colocalizes with ADCY3 at the primary cilia of a subset of hypothalamic neurons, that obesity-associated MC4R mutations impair ciliary localization and that inhibition of adenylyl cyclase signaling at the primary cilia of these neurons increases body weight. These data suggest that impaired signaling from the primary cilia of MC4R neurons is a common pathway underlying genetic causes of obesity in humans

Changes in cortical bone response to high-fat diet from adolescence to adulthood in mice

UnlabelledDiabetic obesity is associated with increased fracture risk in adults and adolescents. We find in both adolescent and adult mice dramatically inferior mechanical properties and structural quality of cortical bone, in agreement with the human fracture data, although some aspects of the response to obesity appear to differ by age.IntroductionThe association of obesity with bone is complex and varies with age. Diabetic obese adolescents and adult humans have increased fracture risk. Prior studies have shown reduced mechanical properties as a result of high-fat diet (HFD) but do not fully address size-independent mechanical properties or structural quality, which are important to understand material behavior.MethodsCortical bone from femurs and tibiae from two age groups of C57BL/6 mice fed either HFD or low-fat diet (LFD) were evaluated for structural and bone turnover changes (SEM and histomorphometry) and tested for bending strength, bending stiffness, and fracture toughness. Leptin, IGF-I, and non-enzymatic glycation measurements were also collected.ResultsIn both young and adult mice fed on HFD, femoral strength, stiffness, and toughness are all dramatically lower than controls. Inferior lamellar and osteocyte alignment also point to reduced structural quality in both age groups. Bone size was largely unaffected by HFD, although there was a shift from increasing bone size in obese adolescents to decreasing in adults. IGF-I levels were lower in young obese mice only.ConclusionsWhile the response to obesity of murine cortical bone mass, bone formation, and hormonal changes appear to differ by age, the bone mechanical properties for young and adult groups are similar. In agreement with human fracture trends, adult mice may be similarly susceptible to bone fracture to the young group, although cortical bone in the two age groups responds to diabetic obesity differently

Engineering the Melanocortin-4 Receptor to Control Constitutive and Ligand-Mediated Gs Signaling In Vivo

The molecular and functional diversity of G protein–coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein–mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of Gs signaling in vivo. We used naturally occurring human mutations to develop two Gs-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our Gs-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone α-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the Gs pathway in vivo. These RASSLs can be used to activate Gs signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering

Prevalence of Melanocortin-4 Receptor Deficiency in Europeans and Their Age-Dependent Penetrance in Multigenerational Pedigrees

OBJECTIVE— Melanocortin-4 receptor (MC4R) deficiency is the most frequent genetic cause of obesity. However, there is uncertainty regarding the degree of penetrance of this condition, and the putative impact of the environment on the development of obesity in MC4R mutation carriers is unknown

Leptin and Amylin Act in an Additive Manner to Activate Overlapping Signaling Pathways in Peripheral Tissues: In vitro and ex vivo studies in humans

OBJECTIVE: Amylin interacts with leptin to alter metabolism. We evaluated, for the first time, amylin- and/or leptin-activated signaling pathways in human peripheral tissues (hPTs). RESEARCH DESIGN AND METHODS: Leptin and amylin signaling studies were performed in vitro in human primary adipocytes (hPAs) and human peripheral blood mononuclear cells (hPBMCs) and ex vivo in human adipose tissue (hAT) from male versus female subjects, obese versus lean subjects, and subjects with subcutaneous versus omental adipose tissue. RESULTS: The long form of leptin receptor was expressed in human tissues and cells studied in ex vivo and in vitro, respectively. Leptin and amylin alone and in combination activate signal transducer and activator of transcription 3 (STAT3), AMP-activated protein kinase, Akt, and extracellular signal-regulated kinase signaling pathways in hAT ex vivo and hPAs and hPBMCs in vitro; all phosphorylation events were saturable at leptin and amylin concentrations of ∼50 and ∼20 ng/ml, respectively. The effects of leptin and amylin on STAT3 phosphorylation in hPAs and hPBMCs in vitro were totally abolished under endoplasmic reticulum stress and/or in the presence of a STAT3 inhibitor. Results similar to those in the in vitro studies were observed in hAT studied ex vivo. CONCLUSIONS: Leptin and amylin activate overlapping intracellular signaling pathways in humans and have additive, but not synergistic, effects in signaling pathways studied in hPTs in vitro and ex vivo

Variants Near MC4R Are Associated With Obesity and Influence Obesity-Related Quantitative Traits in a Population of Middle-Aged People: Studies of 14,940 Danes

OBJECTIVE— Variants downstream of the melanocortin-4 receptor gene (MC4R) have been reported to associate with obesity. We examined rs17782313, rs17700633, rs12970134, rs477181, rs502933, and rs4450508 near MC4R for association with obesity-related quantitative traits, obesity, and type 2 diabetes in Danish individuals

Insufficiency of Janus Kinase 2–Autonomous Leptin Receptor Signals for Most Physiologic Leptin Actions

OBJECTIVE: Leptin acts via its receptor (LepRb) to signal the status of body energy stores. Leptin binding to LepRb initiates signaling by activating the associated Janus kinase 2 (Jak2) tyrosine kinase, which promotes the phosphorylation of tyrosine residues on the intracellular tail of LepRb. Two previously examined LepRb phosphorylation sites mediate several, but not all, aspects of leptin action, leading us to hypothesize that Jak2 signaling might contribute to leptin action independently of LepRb phosphorylation sites. We therefore determined the potential role in leptin action for signals that are activated by Jak2 independently of LepRb phosphorylation (Jak2-autonomous signals). RESEARCH DESIGN AND METHODS: We inserted sequences encoding a truncated LepRb mutant (LepRb$^{\Delta65c}$, which activates Jak2 normally, but is devoid of other LepRb intracellular sequences) into the mouse Lepr locus. We examined the leptin-regulated physiology of the resulting $\Delta/\Delta$ mice relative to LepRb-deficient $db/db$ animals. RESULTS: The $\Delta/\Delta$ animals were similar to $db/db$ animals in terms of energy homeostasis, neuroendocrine and immune function, and regulation of the hypothalamic arcuate nucleus, but demonstrated modest improvements in glucose homeostasis. CONCLUSIONS: The ability of Jak2-autonomous LepRb signals to modulate glucose homeostasis in $\Delta/\Delta$ animals suggests a role for these signals in leptin action. Because Jak2-autonomous LepRb signals fail to mediate most leptin action, however, signals from other LepRb intracellular sequences predominate

Exposure to Uteroplacental Insufficiency Reduces the Expression of Signal Transducer and Activator of Transcription 3 and Proopiomelanocortin in the Hypothalamus of Newborn Rats

IUGR has been linked to the development of type 2 diabetes. Recent data suggest that some of the molecular defects underlying type 2 diabetes reside in the CNS. Disruption of the signal transducer and activator of transcription 3 (STAT3) in the hypothalamic neurons expressing leptin receptor, results in severe obesity, hyperglycaemia, and hyperinsulinemia. Our aim was to investigate the expression of STAT3 and its downstream effector proopiomelanocortin (POMC) in IUGR rats obtained by uterine artery ligation. On day 19 of gestation, time-dated Sprague-Dawley pregnant rats were anesthetized, and both the uterine arteries were ligated. At birth, hypothalamus was dissected and processed to evaluate the expression of STAT3, its phosphorylated form, and POMC. STAT3 mRNA, STAT3 protein, phosphorylated STAT3, POW mRNA, and POMC protein were significantly reduced in IUGR versus sham animals (p < 0.0001. p < 0.05 and p < 0.001, p < 0.01, p < 0.01 respectively). No significant differences either in serum leptin concentrations or in hypothalamic leptin receptor expression were observed. Our results suggest that an abnormal intrauterine milieu call affect the hypothalamic expression of STAT3 and POW at birth. altering the hypothalamic signaling pathways that regulate the energy homeostasis. (Pediatr Res 66: 208-211, 2009

Weight Loss after Roux-en-Y Gastric Bypass in Obese Patients Heterozygous for MC4R Mutations

BackgroundHeterozygous mutations in melanocortin-4 receptor (MC4R) are the most frequent genetic cause of obesity. Bariatric surgery is a successful treatment for severe obesity. The mechanisms of weight loss after bariatric surgery are not well understood.MethodsNinety-two patients who had Roux-en-Y gastric bypass (RYGB) surgery were screened for MC4R mutations. We compared percent excess weight loss (%EWL) in the four MC4R mutation carriers with that of two control groups: 8 matched controls and with the remaining 80 patients who underwent RYGB.ResultsFour patients were heterozygous for functionally significant MC4R mutations. In patients with MC4R mutations, the %EWL after RYGB (66% EWL) was not significantly different compared to matched controls (70% EWL) and non-matched controls (60% EWL) after 1 year of follow-up.ConclusionsThis study suggests that patients with heterozygous MC4R mutations also benefit from RYGB and that weight loss may be independent of the presence of such mutations

Hypothalamic ERK Mediates the Anorectic and Thermogenic Sympathetic Effects of Leptin

OBJECTIVE—Leptin is an adipocyte hormone that plays a major role in energy balance. Leptin receptors in the hypothalamus are known to signal via distinct mechanisms, including signal transducer and activator of transcription-3 (STAT3) and phosphoinositol-3 kinase (PI 3-kinase). Here, we tested the hypothesis that extracellular signal–regulated kinase (ERK) is mediating leptin action in the hypothalamus