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

Abstract

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 β-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.

Keywords: CEACAM1; Energy homeostasis; fatty acid oxidation; fatty-acid synthase (FAS); hyperleptinemia; hyperphagia; insulin resistance; leptin; leptin resistance NIH [, and (to S.M. Najjar), (to J.W. Hill) and; nuclear magnetic resonance (NMR).

FIGURE 1.

Intraperitoneal glucose and insulin tolerance tests. 6-Month-old male Cc1^+/+ and Cc1^−/− mice (n >5 per treatments per genotype) were challenged with an intraperitoneal injection of glucose (1.5 g/kg BW) (A) or insulin (0.75 units/kg BW) (B) to assess blood glucose levels at 0–180 min post-injection. Insulin tolerance in B, was measured as percentage of basal blood glucose level. *, p < 0.05 versus Cc1^+/+ mice (open circles).

FIGURE 2.

Indirect calorimetry analysis. Six-month-old male Cc1^+/+ and Cc1^−/− mice (n = 5 per genotype) were individually housed and subjected to indirect calorimetry analysis to determine daily food intake (A), spontaneous physical activity (B), and oxygen consumption (VO₂) (C). Hourly measurements over the full 24-h period (starting at 7 am) were plotted. Graphs show average hourly data over each 24-h period during the last 3 consecutive days. C, data are expressed as means ± S.E. *, p < 0.05 in Cc1^−/− (solid circles and bars) versus Cc1^+/+ mice (open circles and bars). Graphs are representatives of different experiments done on at least four different sets of mice.

FIGURE 3.

Fatty acid oxidation in skeletal muscle. A, semi-quantitative RT-PCR analysis was used to determine mRNA transcripts of genes involved in fatty acid oxidation in the skeletal (Sk.) muscle of 6-month-old male Cc1^+/+ (open bar) and Cc1^−/− (solid bar) mice. Genes examined are as follows: uncoupled protein 3 (Ucp3, panel i), carnitine palmitoyltransferase 1B (Cpt1b, panel ii), cyclooxygenase-2 (Cox2, panel iii), and pyruvate dehydrogenase kinase isozyme 4 (Pdk4, panel iv). mRNA levels were analyzed in triplicate per mouse and normalized to Gapdh content. Data are expressed as means ± S.E. *, p < 0.05 versus Cc1^+/+ (open bars). B, fatty acid oxidation was assayed in skeletal muscle isolated from fasted 6-month-old Cc1^+/+ (open bar) and Cc1^−/− (solid bar) mice (n >7 per genotype). Data are expressed as mean ± S.E. *, p < 0.05 versus Cc1^+/+ (open bars).

FIGURE 4.

Hypothalamic leptin signaling. A, 4-month-old, and B, 6-month-old male Cc1^+/+ and Cc1^−/− mice (n = 3–5 per treatments per genotype) received an i.c.v. injection of vehicle (Veh, open or solid bars) or leptin (Lep, gray- and black-striped bars) 45 min prior to tissue isolation. Coronal sections from the medial hypothalamus were subjected to immunohistochemical analysis with phospho-STAT3. The mean number of stained hypothalamic neurons of the medial hypothalamus is shown as ± S.E. *, p < 0.05 versus vehicle in each mouse group.

FIGURE 5.

Effect of leptin on food intake. Mice (n >6 per group) were individually housed, fasted during the light cycle, and injected with saline vehicle (Veh, open bars) or leptin (Lep, gray- and black-striped bars) before being fed a pre-weighed amount of food overnight. Food intake was measured in the morning, and the experiment was repeated for 5 days. Food intake was calculated as the weight of food provided to each mouse minus the weight of food remaining in the cage. Data are mean ± S.E. *, p < 0.05 versus vehicle in each mouse group.

FIGURE 6.

Hypothalamic FASN activity. FASN activity was assessed in triplicate in the hypothalamus of overnight fasted 6-month-old mice (n = 5 per genotype). Data are expressed as means ± S.E. *, p < 0.05 versus Cc1^+/+ (open bar).

FIGURE 7.

Immunofluorescence analysis of CEACAM1 in hypothalamic POMC cells. Coronal brain sections of POMC-GFP mice were sectioned, and the arcuate nucleus was analyzed by immunofluorescence to detect nuclei in DAPI (A, E, and I). GFP-labeled POMC neurons are in green (B and F), and CEACAM1 is shown in red (C). D, merged image of POMC-GFP and CEACAM1. G shows a negative control (Neg. ct) for secondary antibody in the absence of primary antibodies. Sections from Cc1^−/− mouse (I and J) show negative control for primary antibody with a magnified view of the arcuate (arc) nucleus in the inset (J). Arrows point to neurons magnified in the inset (B–D) or cells positive for GFP (F and H). Abbreviations used are as follows: v, lateral ventricle; hy, hypothalamus; th, thalamus; and hp, hippocampus. Scale bar, 100 μm.