Biased agonism of G protein-coupled receptors (GPCRs) offers potential for safer medications. Current efforts have explored the balance between G proteins and β-arrestin; however, other transducers like GPCR kinases (GRKs) remain understudied. GRK2 is essential for β₂ adrenergic receptor (β₂AR)-mediated glucose uptake, but β₂AR agonists are considered poor clinical candidates for glycemic management due to G_s/cyclic AMP (cAMP)-induced cardiac side effects and β-arrestin-dependent desensitization. Using ligand-based virtual screening and chemical evolution, we developed pathway-selective agonists of β₂AR that prefer GRK coupling. These compounds perform well in preclinical models of hyperglycemia and obesity and demonstrate a lower potential for cardiac and muscular side effects compared with standard β₂-receptor agonists and incretin mimetics, respectively. Furthermore, the lead candidate showed favorable pharmacokinetics and was well tolerated in a placebo-controlled clinical trial. GRK-biased β₂AR partial agonists are thus promising oral alternatives to injectable incretin mimetics used in the treatment of type 2 diabetes and obesity.

The primary aim of this thesis was to investigate ways to safely leverage the adrenergic signalling pathway to utilize thermogenic fat and skeletal muscle for treating metabolic disease. To this end, our research first provided key evidence that adrenergically stimulated glucose uptake in brown adipocytes operates independently of the canonical insulin/AKT pathway, highlighting that this pathway provides a mechanism to bypass the core signalling defects present in insulin resistant states (Paper I). We subsequently identified Myo1c as a novel, specific regulator of this process in a BAT specific manner, providing a new molecular target within this pathway (Paper II).

To enable the direct identification of novel modulators of thermogenesis, we established isothermal microcalorimetry as a high-throughput platform capable of quantifying both UCP1 dependent and independent heat production in murine and human adipocytes (Paper III). Additionally, we complimented this work through a detailed pharmacological characterization of the β₃ AR agonist Mirabegron, clarifying that its beneficial effects in our rodent models were indeed β₃ AR and UCP1 dependent (Paper IV). Concurrently, to address the critical issue of translatability, we developed a physiologically humanized mouse model, which demonstrated that rodent classical BAT recapitulates the molecular and morphological signatures of human thermogenic tissue (Paper V).

This mechanistic work provided the framework for the design of next generation therapeutics that could activate adrenergic signalling in a functionally selective manner to avoid cardiovascular side effects and desensitization associated with conventional agonism. We first developed ATR-127, a dual β_2/3 AR agonist that served as an essential proof of concept to show that the separation of metabolic efficacy from cardiovascular effects was indeed possible (Paper VI). This led to the further development of the refined, clinically validated candidate, ATR-258, a GRK2 biased β₂ agonist that demonstrated broad preclinical efficacy, inducing healthy weight loss characterized by a significant reduction of fat mass with the preservation of lean mass. This potent muscle sparing effect was also observed in models of late-stage diabetes and sarcopenia. Furthermore, ATR-258 showed significant utility in combination regimens, providing complementary benefits such as preventing the lean mass loss associated with incretin analogues and producing additive glycemic effects with SGLT2 inhibitors. This robust preclinical profile was ultimately confirmed for its safety and tolerability in a first-in-human clinical trial (Paper VII)

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

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.

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.

The beta(3)-adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at beta-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [H-3]-2-deoxyglucose uptake, cellular glycolysis, and O(2)consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at beta(3)-adrenoceptors as they were largely absent in adipocytes derived from beta(3)-adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the beta(3)-adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the beta(3)-adrenoceptor.

The possibility to use leptin therapeutically for lowering glucose levels in patients with type 1 diabetes has attracted interest. However, earlier animal models of type 1 diabetes are severely catabolic with very low endogenous leptin levels, unlike most patients with diabetes. Here, we aim to test glucose-lowering effects of leptin in novel, more human-like murine models. We examined the glucose-lowering potential of leptin in diabetic models of two types: streptozotocin-treated mice and mice treated with the insulin receptor antagonist S961. To prevent hypoleptinemia, we used combinations of thermoneutral temperature and high-fat feeding. Leptin fully normalized hyperglycemia in standard chow-fed streptozotocin-treated diabetic mice. However, more humanized physiological conditions (high-fat diets or thermoneutral temperatures) that increased adiposity- and thus also leptin level-sin the diabetic mice abrogated the effects of leptin, i.e., the mice developed leptin resistance also in this respect. The glucose-lowering effect of leptin was not dependent on the presence of the uncoupling protein-1 and was not associated with alterations in plasma insulin, insulin-like growth factor 1, food intake or corticosterone but fully correlated with decreased plasma glucagon levels and gluconeogenesis. An important implication of these observations is that the therapeutic potential of leptin as an additional treatment in patients with type 1 diabetes is probably limited. This is because such patients are treated with insulin and do not display low leptin levels. Thus, the potential for a glucose-lowering effect of leptin would already have been attained with standard insulin therapy, and further effects on blood glucose level through additional leptin cannot be anticipated.

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.

Human and rodent brown adipose tissues (BAT) appear morphologically and molecularly different. Here we compare human BAT with both classical brown and brite/beige adipose tissues of 'physiologically humanized' mice: middle-aged mice living under conditions approaching human thermal and nutritional conditions, that is, prolonged exposure to thermoneutral temperature (approximately 30 degrees C) and to an energy-rich (high-fat, high-sugar) diet. We find that the morphological, cellular and molecular characteristics (both marker and adipose-selective gene expression) of classical brown fat, but not of brite/beige fat, of these physiologically humanized mice are notably similar to human BAT. We also demonstrate, both in silico and experimentally, that in physiologically humanized mice only classical BAT possesses a high thermogenic potential. These observations suggest that classical rodent BAT is the tissue of choice for translational studies aimed at recruiting human BAT to counteract the development of obesity and its comorbidities.