Induction of thermogenesis in brown and brite adipocytes has recently emerged as a therapeutic target for novel anti obesogenic therapies necessitating the development of methods that can accurately measure heat production in these cells. Modern isothermal microcalorimetric techniques allow for the high throughput quantitative measurement of cellular heat production with limited sample material. Here, we describe the application of this technique for the measurement of thermogenesis in both floating and adherent adipocytes from various murine depots and human cell lines.

The activation of thermogenesis in adipose tissue has emerged as an important target for the development of novel anti-obesity therapies. Using multi-well isothermal microcalorimetry, we have demonstrated that mature murine brown and brite adipocytes produce quantifiable heat upon β₃-AR stimulation, independently of any anaerobic mechanisms. Additionally, in brite adipocytes lacking UCP1 protein, β₃-AR stimulation still induces heat production, albeit to a much lower extent than in their wildtype counterparts, suggesting that UCP1 is an essential component of adrenergic induced thermogenesis in murine brite adipocytes exvivo. Similarly, we could observe an increase in heat production in human-derived adipocytes (hMADS) upon β-AR stimulation. Collectively, these results establish the use of isothermal microcalorimetry as a sensitive and accurate technique for measuring thermogenic responses in intact mature brite adipocytes from murine and human origin.

Aims/hypothesis Chronic stimulation of beta(2)-adrenoceptors, opposite to acute treatment, was reported to reduce blood glucose levels, as well as to improve glucose and insulin tolerance in rodent models of diabetes by essentially unknown mechanisms. We recently described a novel pathway that mediates glucose uptake in skeletal muscle cells via stimulation of beta(2)-adrenoceptors. In the current study we further explored the potential therapeutic relevance of beta(2)-adrenoceptor stimulation to improve glucose homeostasis and the mechanisms responsible for the effect.

Methods C57Bl/6N mice with diet-induced obesity were treated both acutely and for up to 42 days with a wide range of clenbuterol dosages and treatment durations. Glucose homeostasis was assessed by glucose tolerance test. We also measured in vivo glucose uptake in skeletal muscle, insulin sensitivity by insulin tolerance test, plasma insulin levels, hepatic lipids and glycogen.

Results Consistent with previous findings, acute clenbuterol administration increased blood glucose and insulin levels. However, already after 4 days of treatment, beneficial effects of clenbuterol were manifested in glucose homeostasis (32% improvement of glucose tolerance after 4 days of treatment,p < 0.01) and these effects persisted up to 42 days of treatment. These favourable metabolic effects could be achieved with doses as low as 0.025 mg kg(-1) day(-1)(40 times lower than previously studied). Mechanistically, these effects were not due to increased insulin levels, but clenbuterol enhanced glucose uptake in skeletal muscle in vivo both acutely in lean mice (by 64%,p < 0.001) as well as during chronic treatment in diet-induced obese mice (by 74%,p < 0.001). Notably, prolonged treatment with low-dose clenbuterol improved whole-body insulin sensitivity (glucose disposal rate after insulin injection increased up to 1.38 +/- 0.31%/min in comparison with 0.15 +/- 0.36%/min in control mice,p < 0.05) and drastically reduced hepatic steatosis (by 40%,p < 0.01) and glycogen (by 23%,p < 0.05).

Conclusions/interpretation Clenbuterol improved glucose tolerance after 4 days of treatment and these effects were maintained for up to 42 days. Effects were achieved with doses in a clinically relevant microgram range. Mechanistically, prolonged treatment with a low dose of clenbuterol improved glucose homeostasis in insulin resistant mice, most likely by stimulating glucose uptake in skeletal muscle and improving whole-body insulin sensitivity as well as by reducing hepatic lipids and glycogen. We conclude that selective beta(2)-adrenergic agonists might be an attractive potential treatment for type 2 diabetes. This remains to be confirmed in humans.