Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells.

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with ß2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective ß2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of ß-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle ß2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic ß2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating ß2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

Metabolic effects of skeletal muscle-specific deletion of beta-arrestin-1 and -2 in mice.

Type 2 diabetes (T2D) has become a major health problem worldwide. Skeletal muscle (SKM) is the key tissue for whole-body glucose disposal and utilization. New drugs aimed at improving insulin sensitivity of SKM would greatly expand available therapeutic options. ß-arrestin-1 and -2 (Barr1 and Barr2, respectively) are two intracellular proteins best known for their ability to mediate the desensitization and internalization of G protein-coupled receptors (GPCRs). Recent studies suggest that Barr1 and Barr2 regulate several important metabolic functions including insulin release and hepatic glucose production. Since SKM expresses many GPCRs, including the metabolically important ß2-adrenergic receptor, the goal of this study was to examine the potential roles of Barr1 and Barr2 in regulating SKM and whole-body glucose metabolism. Using SKM-specific knockout (KO) mouse lines, we showed that the loss of SKM Barr2, but not of SKM Barr1, resulted in mild improvements in glucose tolerance in diet-induced obese mice. SKM-specific Barr1- and Barr2-KO mice did not show any significant differences in exercise performance. However, lack of SKM Barr2 led to increased glycogen breakdown following a treadmill exercise challenge. Interestingly, mice that lacked both Barr1 and Barr2 in SKM showed no significant metabolic phenotypes. Thus, somewhat surprisingly, our data indicate that SKM ß-arrestins play only rather subtle roles (SKM Barr2) in regulating whole-body glucose homeostasis and SKM insulin sensitivity.

Hepatic Gi signaling regulates whole-body glucose homeostasis.

An increase in hepatic glucose production (HGP) is a key feature of type 2 diabetes. Excessive signaling through hepatic Gs-linked glucagon receptors critically contributes to pathologically elevated HGP. Here, we tested the hypothesis that this metabolic impairment can be counteracted by enhancing hepatic Gi signaling. Specifically, we used a chemogenetic approach to selectively activate Gi-type G proteins in mouse hepatocytes in vivo. Unexpectedly, activation of hepatic Gi signaling triggered a pronounced increase in HGP and severely impaired glucose homeostasis. Moreover, increased Gi signaling stimulated glucose release in human hepatocytes. A lack of functional Gi-type G proteins in hepatocytes reduced blood glucose levels and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Additionally, we delineated a signaling cascade that links hepatic Gi signaling to ROS production, JNK activation, and a subsequent increase in HGP. Taken together, our data support the concept that drugs able to block hepatic Gi-coupled GPCRs may prove beneficial as antidiabetic drugs.

Hepatic ß-arrestin 2 is essential for maintaining euglycemia.

An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein ß-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific ß-arrestin 2 deficiency did not affect hepatic insulin sensitivity or ß-adrenergic signaling. Adult mice lacking ß-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of ß-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic ß-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes.

Functional role of the three conserved cysteines in the N domain of visual arrestin-1.

Arrestins specifically bind active and phosphorylated forms of their cognate G protein-coupled receptors, blocking G protein coupling and often redirecting the signaling to alternative pathways. High-affinity receptor binding is accompanied by two major structural changes in arrestin: release of the C-tail and rotation of the two domains relative to each other. The first requires detachment of the arrestin C-tail from the body of the molecule, whereas the second requires disruption of the network of charge-charge interactions at the interdomain interface, termed the polar core. These events can be facilitated by mutations destabilizing the polar core or the anchoring of the C-tail that yield "preactivated" arrestins that bind phosphorylated and unphosphorylated receptors with high affinity. Here we explored the functional role in arrestin activation of the three native cysteines in the N domain, which are conserved in all arrestin subtypes. Using visual arrestin-1 and rhodopsin as a model, we found that substitution of these cysteines with serine, alanine, or valine virtually eliminates the effects of the activating polar core mutations on the binding to unphosphorylated rhodopsin while only slightly reducing the effects of the C-tail mutations. Thus, these three conserved cysteines play a role in the domain rotation but not in the C-tail release.

ß-arrestin-2 is an essential regulator of pancreatic ß-cell function under physiological and pathophysiological conditions.

ß-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the ß-arrestin-2 gene, barr2, in ß-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in ß-arrestin-2 (barr2) deficient ß-cells. In human ß-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in ß-cells. Importantly, overexpression of barr2 in ß-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of ß-cell function, which may serve as a new target to improve ß-cell function.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.