Leptin Resistance Contributes to Obesity in Mice with Null Mutation of Carcinoembryonic Antigen-related Cell Adhesion Molecule 1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance. Consistently, mice with null mutation of Ceacam1 (Cc1(-/-)) exhibit impaired insulin clearance with increased lipid production in liver and redistribution to white adipose tissue, leading to visceral obesity at 2 months of age. When the mutation is propagated on the C57/BL6J genetic background, total fat mass rises significantly with age, and glucose intolerance and systemic insulin resistance develop at 6 months of age. This study was carried out to determine the mechanisms underlying the marked increase in total fat mass in 6-month-old mutants. Indirect calorimetry analysis showed that Cc1(-/-) mice develop hyperphagia and a significant reduction in physical activity, in particular in the early hours of the dark cycle, during which energy expenditure is only slightly lower than in wild-type mice. They also exhibit increased triglyceride accumulation in skeletal muscle, due in part to incomplete fatty acid ß-oxidation. Mechanistically, hypothalamic leptin signaling is reduced, as demonstrated by blunted STAT3 phosphorylation in coronal sections in response to an intracerebral ventricular injection of leptin. Hypothalamic fatty-acid synthase activity is also elevated in the mutants. Together, the data show that the increase in total fat mass in Cc1(-/-) mice is mainly attributed to hyperphagia and reduced spontaneous physical activity. Although the contribution of the loss of CEACAM1 from anorexigenic proopiomelanocortin neurons in the arcuate nucleus is unclear, leptin resistance and elevated hypothalamic fatty-acid synthase activity could underlie altered energy balance in these mice.

PPARα (Peroxisome Proliferator-activated Receptor α) Activation Reduces Hepatic CEACAM1 Protein Expression to Regulate Fatty Acid Oxidation during Fasting-refeeding Transition.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed at high levels in the hepatocyte, consistent with its role in promoting insulin clearance in liver. CEACAM1 also mediates a negative acute effect of insulin on fatty acid synthase activity. Western blot analysis reveals lower hepatic CEACAM1 expression during fasting. Treating of rat hepatoma FAO cells with Wy14,643, an agonist of peroxisome proliferator-activated receptor α (PPARα), rapidly reduces Ceacam1 mRNA and CEACAM1 protein levels within 1 and 2 h, respectively. Luciferase reporter assay shows a decrease in the promoter activity of both rat and mouse genes by Pparα activation, and 5'-deletion and block substitution analyses reveal that the Pparα response element between nucleotides -557 and -543 is required for regulation of the mouse promoter activity. Chromatin immunoprecipitation analysis demonstrates binding of liganded Pparα toCeacam1promoter in liver lysates ofPparα(+/+), but notPparα(-/-)mice fed a Wy14,643-supplemented chow diet. Consequently, Wy14,643 feeding reduces hepatic Ceacam1 mRNA and CEACAM1 protein levels, thus decreasing insulin clearance to compensate for compromised insulin secretion and maintain glucose homeostasis and insulin sensitivity in wild-type mice. Together, the data show that the low hepatic CEACAM1 expression at fasting is mediated by Pparα-dependent mechanisms. Changes in CEACAM1 expression contribute to the coordination of fatty acid oxidation and insulin action in the fasting-refeeding transition.

Differentiation of hypothalamic-like neurons from human pluripotent stem cells.

The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. Insights into the complex cellular physiology of this region are critical to the understanding of obesity pathogenesis; however, human hypothalamic cells are largely inaccessible for direct study. Here, we developed a protocol for efficient generation of hypothalamic neurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenetic forms of obesity. Combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition in human ESCs/iPSCs resulted in efficient conversion into hypothalamic NKX2.1+ precursors. Application of a NOTCH inhibitor and brain-derived neurotrophic factor (BDNF) further directed the cells into arcuate nucleus hypothalamic-like neurons that express hypothalamic neuron markers proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), somatostatin, and dopamine. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles defined by a hypothalamic-specific gene expression signature that lacked pituitary markers. Importantly, these cells displayed hypothalamic neuron characteristics, including production and secretion of neuropeptides and increased p-AKT and p-STAT3 in response to insulin and leptin. Our results suggest that these hypothalamic-like neurons have potential for further investigation of the neurophysiology of body weight regulation and evaluation of therapeutic targets for obesity.

Preserved energy balance in mice lacking FoxO1 in neurons of Nkx2.1 lineage reveals functional heterogeneity of FoxO1 signaling within the hypothalamus.

Transcription factor forkhead box O1 (FoxO1) regulates energy expenditure (EE), food intake, and hepatic glucose production. These activities have been mapped to specific hypothalamic neuronal populations using cell type-specific knockout experiments in mice. To parse out the integrated output of FoxO1-dependent transcription from different neuronal populations and multiple hypothalamic regions, we used transgenic mice expressing Cre recombinase from the Nkx2.1 promoter to ablate loxP-flanked Foxo1 alleles from a majority of hypothalamic neurons (Foxo1KO(Nkx2.1) mice). This strategy resulted in the expected inhibition of FoxO1 expression, but only produced a transient reduction of body weight as well as a decreased body length. The transient decrease of body weight in male mice was accompanied by decreased fat mass. Male Foxo1KO(Nkx2.1) mice show food intake similar to that in wild-type controls, and, although female knockout mice eat less, they do so in proportion to a reduced body size. EE is unaffected in Foxo1KO(Nkx2.1) mice, although small increases in body temperature are present. Unlike other neuron-specific Foxo1 knockout mice, Foxo1KO(Nkx2.1) mice are not protected from diet-induced obesity. These studies indicate that, unlike the metabolic effects of highly restricted neuronal subsets (proopiomelanocortin, neuropeptide Y/agouti-related peptide, and steroidogenic factor 1), those of neurons derived from the Nkx2.1 lineage either occur in a FoxO1-independent fashion or are compensated for through developmental plasticity.

Blunted refeeding response and increased locomotor activity in mice lacking FoxO1 in synapsin-Cre-expressing neurons.

Successful development of antiobesity agents requires detailed knowledge of neural pathways controlling body weight, eating behavior, and peripheral metabolism. Genetic ablation of FoxO1 in selected hypothalamic neurons decreases food intake, increases energy expenditure, and improves glucose homeostasis, highlighting the role of this gene in insulin and leptin signaling. However, little is known about potential effects of FoxO1 in other neurons. To address this question, we executed a broad-based neuronal ablation of FoxO1 using Synapsin promoter-driven Cre to delete floxed Foxo1 alleles. Lineage-tracing experiments showed that NPY/AgRP and POMC neurons were minimally affected by the knockout. Nonetheless, Syn-Cre-Foxo1 knockouts demonstrated a catabolic energy homeostatic phenotype with a blunted refeeding response, increased sensitivity to leptin and amino acid signaling, and increased locomotor activity, likely attributable to increased melanocortinergic tone. We confirmed these data in mice lacking the three Foxo genes. The effects on locomotor activity could be reversed by direct delivery of constitutively active FoxO1 to the mediobasal hypothalamus, but not to the suprachiasmatic nucleus. The data reveal that the integrative function of FoxO1 extends beyond the arcuate nucleus, suggesting that central nervous system inhibition of FoxO1 function can be leveraged to promote hormone sensitivity and prevent a positive energy balance.

Carcinoembryonic antigen-related cell adhesion molecule 2 controls energy balance and peripheral insulin action in mice.

BACKGROUND & AIMS: The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein with pleotropic functions, including clearance of hepatic insulin. We investigated the functions of the related protein CEACAM2, which has tissue-specific distribution (kidney, uterus, and crypt epithelia of intestinal tissues), in genetically modified mice. METHODS: Ceacam2-null mice (Cc2-/-) were generated from a 129/SvxC57BL/6J background. Female mice were assessed by hyperinsulinemic-euglycemic clamp analysis and indirect calorimetry and body fat composition was measured. Cc2-/- mice and controls were fed as pairs, given insulin tolerance tests, and phenotypically characterized. RESULTS: Female, but not male Cc2-/- mice exhibited obesity that resulted from hyperphagia and reduced energy expenditure. Pair feeding experiments showed that hyperphagia led to peripheral insulin resistance. Insulin action was normal in liver but compromised in skeletal muscle of female Cc2-/- mice; the mice had incomplete fatty acid oxidation and impaired glucose uptake and disposal. The mechanism of hyperphagia in Cc2-/- mice is not clear, but appears to result partly from increased hyperinsulinemia-induced hypothalamic fatty acid synthase levels and activity. Hyperinsulinemia was caused by increased insulin secretion. CONCLUSIONS: In mice, CEACAM2 is expressed by the hypothalamus. Cc2-/- mice develop obesity from hyperphagia and reduced energy expenditure, indicating its role in regulating energy balance and insulin sensitivity.