An OLTAM system for analysis of brown/beige fat thermogenic activity.

BACKGROUND/OBJECTIVES: Thermogenic fat is present in humans and emerging evidence indicates that increasing the content and activity of these adipocytes may lead to weight loss and improved metabolic health. Multiple reporter systems have been developed to assay thermogenic fat activity based on the transcriptional and translational activation of Ucp1, the key molecule that mediates nonshivering thermogenesis. Our study aims to develop a much-needed tool to monitor thermogenic fat activity through a mechanism independent of Ucp1 regulation, therefore effectively assaying not only canonical ß-adrenergic activation but also various non-UCP1-mediated thermogenic pathways that have been increasingly appreciated. METHODS: We detected increased luciferase activity upon thermogenic activation in interscapular brown and inguinal subcutaneous fat in ODD-Luc mice, a hypoxia reporter mouse model. We then developed an OLTAM (ODD-Luc based Thermogenic Activity Measurement) system to assay thermogenic fat cell activity. RESULTS: In both primary murine and human adipocytes and an immortalized adipose cell line that were transduced with the OLTAM system, luciferase activity can be readily measured and visualized by bioluminescence imaging in response to a variety of stimuli, including UCP1-independent thermogenic signaling. This system can offer a convenient method to assay thermogenic activity for both basic and translational research. CONCLUSIONS: The OLTAM system offers a convenient way to measure the activation of thermogenic fat and presents opportunities to discover novel signaling pathways and unknown compounds targeting metabolically active adipocytes to counteract human obesity.

Evidence for kidney rejection after combined bone marrow and renal transplantation despite ongoing whole-blood chimerism in rhesus macaques.

Although there is evidence linking hematopoietic chimerism induction and solid organ transplant tolerance, the mechanistic requirements for chimerism-induced tolerance are not clearly elucidated. To address this, we used an MHC-defined primate model to determine the impact of impermanent, T cell-poor, mixed-chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow and renal transplant ("BMT/renal") and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40-directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole-blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p = 0.46), with histopathology documenting T cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28-/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28-negative Tem and costimulation blockade-resistant rejection. These results suggest that in some settings, transient T cell-poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.

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

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 ß-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.