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.

Objective: The potential of brown adipose tissue (BAT) to influence energy homeostasis in animals and humans is encouraging as this tissue can increase fatty acid and glucose utilization to produce heat through uncoupling protein 1 (UCP1), but the actual mechanism of how the cell regulates glucose uptake is not fully understood. Myosin 1c (Myo1c) is an unconventional motor protein involved in several cellular processes, including insulin-mediated glucose uptake via GLUT4 vesicle fusion in white adipocytes, but its role in glucose uptake in BAT has not previously been investigated.

Methods: Using the specific inhibitor pentachloropseudilin (PClP), a neutralizing antibody assay, and siRNA, we examined the role of Myo1c in mechanisms leading to glucose uptake both in vitro in isolated mouse primary adipocytes and in vivo in mice.

Results: Our results show that inhibition of Myo1c removes insulin-stimulated glucose uptake in white adipocytes, while inducing glucose uptake in brown adipocytes, independent of GLUT4, by increasing the expression, translation, and translocation of GLUT1 to the plasma membrane. Inhibition of Myo1c leads to the activation of PKA and downstream substrates p38 and ATF-2, which are known to be involved in the expression of β-adrenergic genes.

Conclusions: Myo1c is a PKA repressor and regulates glucose uptake into BAT.

Metabolic diseases like type II diabetes (T2D) and obesity largely stems from an unbalanced energy homeostasis with the fails of the insulin pathway to the point in which the glucose homeostasis is severely disturbed leading to hyperglycemia. We have investigated if the β-adrenergic signaling pathways, in both brown adipose tissue (BAT) and skeletal muscle, could be used as a strategy to alleviate metabolic disease.

As an important protein regulating energy metabolism, Akt has been an interesting target for study also in BAT, but its role in glucose uptake downstream the β-adrenergic receptors (β-ARs) have had conflicting outcomes. We have therefore made efforts to separate Akt and insulin from the β-adrenergic pathway and shown that Akt is not involved in β-adrenergic glucose uptake or thermogenesis in the brown adipocyte and norepinephrine (NE)-driven glucose clearance in vivo (Paper I). We have also shown that a β2-adrenergic agonist, clenbuterol, at low dose, can be used to induce glucose uptake to skeletal muscle, glucose clearance and increase insulin sensitivity in diet-induced obese mice, independently of insulin (Paper II). Administrating an agonist that binds to either the β-ARs on BAT or on skeletal muscle, at low dose to minimize cardiovascular adverse effects, would initiate processes independent of Akt and insulin, and could therefore be administered to patients with T2D to target idle assets.

Further, we have identified Myosin 1c (Myo1c) as a major regulator of basal protein kinase A (PKA) activity and basal glucose uptakein the brown adipocyte (Paper III) and also as a cofactor in chromatin remodeling for uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1α (Pgc1α) transcription in the thermogenic adipocyte (Paper IV). These novel functions of Myo1c will open up the possibilities for future explorations concerning motor proteins in thermogenic adipocytes.

This thesis has expanded current understanding about the independence of Akt and insulin in β-adrenergic metabolism as well as identifying Myo1c as a key component of the β-adrenergic pathway in the thermogenic adipocyte. The work presented herein will hopefully contribute to further exploration into adrenergic signaling in BAT and skeletal muscle.

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.

Objective: β-adrenoceptor mediated activation of brown adipose tissue (BAT) has been associated with improvements in metabolic health in models of type 2 diabetes and obesity due to its unique ability to increase whole body energy expenditure, and rate of glucose and free fatty acid disposal. While the thermogenic arm of this phenomenon has been studied in great detail, the underlying mechanisms involved in β-adrenoceptor mediated glucose uptake in BAT are relatively understudied. As β-adrenoceptor agonist administration results in increased hepatic gluconeogenesis that can consequently result in secondary pancreatic insulin release, there is uncertainty regarding the importance of insulin and the subsequent activation of its downstream effectors in mediating β-adrenoceptor stimulated glucose uptake in BAT. Therefore, in this study, we made an effort to discriminate between the two pathways and address whether the insulin signaling pathway is dispensable for the effects of β-adrenoceptor activation on glucose uptake in BAT.

Methods: Using a specific inhibitor of phosphoinositide 3-kinase α (PI3Kα), which effectively inhibits the insulin signaling pathway, we examined the effects of various β-adrenoceptor agonists, including the physiological endogenous agonist norepinephrine on glucose uptake and respiration in mouse brown adipocytes in vitro and on glucose clearance in mice in vivo.

Results: PI3Kα inhibition in mouse primary brown adipocytes in vitro, did not inhibit β-adrenoceptor stimulated glucose uptake, GLUT1 synthesis, GLUT1 translocation or respiration. Furthermore, β-adrenoceptor mediated glucose clearance in vivo did not require insulin or Akt activation but was attenuated upon administration of a β3-adrenoceptor antagonist.

Conclusions: We conclude that the β-adrenergic pathway is still functionally intact upon the inhibition of PI3Kα, showing that the activation of downstream insulin effectors is not required for the acute effects of β-adrenoceptor agonists on glucose homeostasis or thermogenesis.