Increased metabolic rate and insulin sensitivity in male mice lacking the carcino-embryonic antigen-related cell adhesion molecule 2

Abstract

Aims/hypothesis: The carcino-embryonic antigen-related cell adhesion molecule (CEACAM)2 is produced in many feeding control centres in the brain, but not in peripheral insulin-targeted tissues. Global Ceacam2 null mutation causes insulin resistance and obesity resulting from hyperphagia and hypometabolism in female Ceacam2 homozygous null mutant mice (Cc2 [also known as Ceacam2](-/-)) mice. Because male mice are not obese, the current study examined their metabolic phenotype.

Methods: The phenotype of male Cc2(-/-) mice was characterised by body fat composition, indirect calorimetry, hyperinsulinaemic-euglycaemic clamp analysis and direct recording of sympathetic nerve activity.

Results: Despite hyperphagia, total fat mass was reduced, owing to the hypermetabolic state in male Cc2(-/-) mice. In contrast to females, male mice also exhibited insulin sensitivity with elevated β-oxidation in skeletal muscle, which is likely to offset the effects of increased food intake. Males and females had increased brown adipogenesis. However, only males had increased activation of sympathetic tone regulation of adipose tissue and increased spontaneous activity. The mechanisms underlying sexual dimorphism in energy balance with the loss of Ceacam2 remain unknown.

Conclusions/interpretation: These studies identified a novel role for CEACAM2 in the regulation of metabolic rate and insulin sensitivity via effects on brown adipogenesis, sympathetic nervous outflow to brown adipose tissue, spontaneous activity and energy expenditure in skeletal muscle.

Fig. 1

Peripheral insulin action. Overnight-fasted, awake wild-type Cc2^+/+ and Cc2^−/− mice (5 months old; n≥8 per group) were subjected to a 2 h hyperinsulinaemic–euglycaemic clamp. a Whole blood glucose at basal (white Cc2^+/+, black Cc2^−/− all panels) and clamp (striped Cc2^+/+, checked Cc2^−/− all panels) states, and b at time 0 and up to 120 min of clamp (white, Cc2^+/+; black Cc2^−/−). c Steady-state glucose infusion rate during clamp. d Hepatic glucose production as above (a). e–g Glucose uptake as indicated; WAT, white adipose tissue and Gastro., gastrocnemius muscle. h Whole-body glucose turnover (R_d). i Glycolysis during clamp. j Whole-body glycogen synthesis. Values are all expressed as mean±SEM in milligrams per kilogram per minute, except for blood glucose levels (mmol/l). *p<0.05 vs basal. k Ex vivo analysis of insulin signalling. Liver was excised from 5 months old male mice, lysed, and treated with insulin (+) or buffer alone (−) prior to immunoprecipitating (Ip) with an antibody against α-IR_β and immunoblotting (Ib) with anti-phosphotyrosine (α-pTyr) antibody to assess the amount of tyrosine phosphorylated insulin receptor. Gels were reprobed (reIb) with α-IR_β to normalise to the amount of insulin receptor loaded. The gel is representative of three experiments, performed on one pair of mice from each group in each experiment. The band density was scanned and analysed relative to loaded protein, and is quantified in arbitrary units (AU); *p<0.05 vs untreated

Fig. 2

Biochemical analysis of lipid metabolism in skeletal muscle of male mice. Mixed skeletal muscle was isolated (gastrocnemius and soleus) from overnight-fasted 5 to 7 months old male mice (white bars, Cc2^+/+; black bars, Cc2^−/− all panels). a mRNA expression of genes involved in fatty acid uptake and oxidation was determined by semi-quantitative real-time PCR and normalised to Gapdh. b FATP1 abundance by western blot analysis, using α-FATP1 antibody to immunoblot (Ib) and α-actin antibody to reprobe (reIb) and normalise against the amount of protein loaded. c Ex vivo palmitate oxidation. d triacylglycerol content. Values are expressed as mean±SEM; n=4–8 per group; *p<0.05 vs Cc2^+/+

Fig. 3

Characterisation of the metabolic state of male mice. Cc2^+/+ (white bars) and Cc2^−/− (black bars) mice were individually caged, given free access to food and subjected to: a analysis of daily food intake over seven consecutive days (n>6 per group) and b–f indirect calorimetry analysis (performed over a 24 h period at age 4 months, and values as indicated (b–f) were measured). T, total light–dark cycle; D, dark period; L, light period; n=5–6 mice per group. Values are expressed as mean±SEM; *p<0.05 and ^†p<0.005 vs Cc2^+/+

Fig. 4

Elevated brown adipogenesis and SNA in male Cc2^−/− mice. a mRNA content was assessed by semi-quantitative real-time PCR and normalised to Gapdh in BAT from 3-month-old Cc2^+/+ (white bars) and Cc2^−/− (black bars) mice; n≥6 per group. b Immunohistochemical analysis of WAT from four of each mouse group at age 3 months. Tissues were fixed and immunostained with α-UCP1 antibody (brown). Representative images from three sections per mouse are shown. Magnification ×20. Scale bar, 100 μm. c, d Baseline SNA, recorded directly from the nerve subserving (c) WAT or (d) BAT, was compared between male (M) and female (F) mouse groups as above (a), but at 5 months of age; n=9–10 per group. Values (a, c, d) are expressed as mean±SEM; *p<0.05 vs Cc2^+/+ and **p<0.01 vs Cc2^+/+

Fig. 5

Comparable hypothalamic CEACAM2 levels in male and female Cc2^+/+ mice. Male (M) and female (F) Cc2^+/+ mice aged 5 months (n≥4 per group) were fasted overnight and their hypothalami removed to analyse (a) Ceacam2 mRNA levels by semiquantitative real-time PCR (Values are expressed as mean±SEM) and (b) CEACAM2 abundance by immunoblotting (Ib) of CEACAM2 immunopellet (Ip) with α-CEACAM2 (CC2) polyclonal antibody