Department of Medicine, Division of Endocrinology and Diabetes, and Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, USA.
Address correspondence to: Allen M. Spiegel, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Belfer 312, Bronx, New York 10461, USA. Phone: 718.430.2801; Email: email@example.com.
First published January 16, 2018 - More info
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.
Mario Rossi, Lu Zhu, Sara M. McMillin, Sai Prasad Pydi, Shanu Jain, Lei Wang, Yinghong Cui, Regina J. Lee, Amanda H. Cohen, Hideaki Kaneto, Morris J. Birnbaum, Yanling Ma, Yaron Rotman, Jie Liu, Travis J. Cyphert, Toren Finkel, Owen P. McGuinness, Jürgen Wess
Hepatic glucose production (HGP) is a key determinant of glucose homeostasis. Glucagon binding to its cognate seven-transmembrane Gs-coupled receptor in hepatocytes stimulates cAMP production, resulting in increased HGP. In this issue of the JCI, Rossi and colleagues tested the hypothesis that activation of hepatic Gi–coupled receptors, which should inhibit cAMP production, would oppose the cAMP-inducing action of glucagon and thereby decrease HGP. Surprisingly, however, the opposite occurred: activation of Gi signaling increased HGP via a novel mechanism, while inhibition of Gi signaling reduced HGP. These results define a new physiologic role for hepatic Gi signaling and identify a potential therapeutic target for HGP regulation.
Appropriate regulation of hepatic glucose production (HGP) is critical for glucose homeostasis under conditions that range from high glucose demand, such as prolonged fasting, to increased glucose abundance, such as excess dietary carbohydrate intake. HGP is regulated by a complex network of direct and indirect mechanisms (Figure 1) that have been extensively studied in animals and humans (1). Impaired regulation of HGP is an important feature of diabetes and has been attributed to reduced insulin sensitivity and excessive glucagon action. Insulin acts directly on hepatocytes via its tyrosine-kinase receptor to trigger a phosphorylation cascade that inhibits glycogenolysis and gluconeogenesis, thus reducing HGP (Figure 1). Hepatocyte-specific KO of mouse insulin receptors leads to severe glucose intolerance and failure of insulin to suppress HGP (2).
Indirect and direct mechanisms regulate HGP. Free fatty acids (FFAs) and glycerol from adipose cells and amino acids from skeletal muscle provide substrate for liver gluconeogenesis, resulting in increased HGP. HGP is also modulated by adipose-derived cytokines and through neural inputs from the CNS. Additionally, pancreatic islet hormones, insulin, via tyrosine kinase–stimulated phosphorylation of the insulin receptor (IR), and glucagon, via Gs-coupled receptor stimulation of cAMP production, act directly on hepatocytes to decrease and increase HGP, respectively. Hepatocytes also contain other GPCRs that are Gi coupled. In this issue, Rossi and colleagues (7) show, surprisingly, that activation of Gi-coupled receptors increases HGP.
Glucagon activates specific receptors coupled to Gs, the heterotrimeric G protein that stimulates adenylyl cyclase, leading to increased cAMP formation. Increased hepatocyte cAMP activates protein kinase A, initiating a phosphorylation cascade that ultimately leads to increased glycogenolysis, gluconeogenesis, and HGP. Indeed, the roles of cAMP as both a ubiquitous second messenger of hormone action (3) and a downstream effector of Gs (4) were discovered in classic studies on the mechanism of glucagon activation of liver glycogenolysis. Subsequent studies identified the so-called “inhibitory” G protein Gi, which couples to receptors to inhibit adenylyl cyclase. Gi is inactivated by pertussis toxin–catalyzed ADP-ribosylation of a cysteine in the carboxy terminus of its α subunit (5). Three different genes encode subtypes of the Gi-α subunit (6), with Gαi1 and Gαi3 being widely expressed and Gαi2 being ubiquitously expressed. Hepatocytes contain abundant amounts of Gi, but the role of this G protein in HGP regulation has not been well defined.
In this issue, Rossi and colleagues (7) use in vitro and in vivo murine studies and apply a number of genetic and pharmacologic tools to probe the role of Gi in HGP regulation. On the basis of the classic, cAMP production–inhibiting definition of Gi, the authors hypothesized that activation of Gi in hepatocytes should oppose glucagon action and decrease HGP. Surprisingly, this was not the case.
Rossi and colleagues used viral vectors and a liver-specific promoter to express a designer Gi–coupled receptor (designer receptor exclusively activated by a designer drug [DREADD]) that is only activated by a specific compound devoid of pharmacologic effects elsewhere to study the liver-specific effects of Gi activation and avoid indirect effects on other organs involved in regulating HGP (Figure 1). DREADD activation did indeed inhibit glucagon-stimulated cAMP production and did not alter intracellular Ca++ (a Gαq-mediated effect), consistent with the classic functional definition of Gi. Nonetheless, DREADD activation of Gi in the livers of mice impaired glucose tolerance, activated both glycogenolysis and gluconeogenesis, and potentiated, rather than inhibited, the hyperglycemic effect of glucagon. Interestingly, DREADD activation did not cause insulin resistance.
Pertussis toxin was first termed islet-activating protein because of its stimulatory effect on islets, which was shown to be due to loss of catecholamine inhibition of insulin secretion via Gi-coupled α-adrenergic receptors in β cells (5). Rossi and colleagues were able to study the effect of liver-specific Gi KO by selectively expressing the catalytic subunit of pertussis toxin (S1-PTX) in hepatocytes. The effectiveness of S1-PTX expression was confirmed, as S1-PTX expression abolished the hyperglycemic effect of DREADD activation. Furthermore, Gi KO in the liver improved glucose tolerance in normal chow-fed mice, as well as in mice with high-fat diet–induced insulin resistance.
Overall, the results by Rossi et al. suggest possible physiologic roles for Gi stimulation and Gi inhibition in the liver by increasing and decreasing HGP, respectively. However, these results beg the question of which Gi signaling pathway accounts for these effects? Rossi and colleagues addressed this issue by testing the effect of DREADD activation of hepatocyte Gi on various signaling pathways, including MAPK and PI3K pathways. The authors found evidence of JNK activation due to an increased oxygen consumption rate and generation of ROS.
While the methods used by Rossi and colleagues allowed them to define a role for liver Gi in the regulation of HGP, liver-specific expression of DREADD or S1-PTX obviously does not reflect normal physiologic conditions. Endogenous expression of Gi-coupled receptors, including the cannabinoid CB1 receptor and the α2A-adrenergic receptor, in mouse hepatocytes allowed the authors to assess the role of liver Gi under physiologic conditions. The hyperglycemic action of a CB1 receptor agonist was blocked in mice with liver-specific KO of Gi and in animals treated with ROS-scavenging agents or selective JNK inhibitors, supporting the notion of liver Gi signaling in HGP regulation, as elucidated by hepatocyte DREAAD expression. Rossi et al. provided further suggestive evidence of a physiologic role of hepatic Gi signaling in HGP regulation by showing that transcription of Gαi1- and α2A-adrenergic receptor–encoding genes is increased in livers from mice that have been fasted for 16 hours.
Are the results obtained in mouse liver relevant to human hepatocytes? Rossi and colleagues showed that expression of constitutively activated Gαi in human hepatocytes increases HGP via the ROS/JNK signaling pathway identified in mouse hepatocytes. Moreover, examination of gene expression in livers from control subjects compared with expression in patients with nonalcoholic steatohepatitis (NASH) with clear signs of insulin resistance revealed increased transcription of the α2A-adrenergic receptor and the CB1 receptor, both of which are Gi coupled, and markedly decreased transcription of the genes encoding Gαi1 and Gαi3 in livers from patients with NASH. Rossi et al. attribute the reduction in RNA levels of Gαi1 and Gαi3 to counterregulatory mechanisms caused by enhanced Gi signaling; however, it is unclear why this result is opposite of the increase in Gαi1 RNA observed in the livers of fasted mice.
The findings of Rossi and colleagues raise a number of important issues that need to be further addressed. Their observation that stimulation of mouse hepatocyte Gi signaling via JNK activation increases HGP without reducing insulin sensitivity is puzzling, as JNK activation has been recognized as a major factor causing insulin resistance (8). Is this discrepancy a function of cell-specific differences in JNK action? Perhaps, but the lack of effect of Gi stimulation on insulin sensitivity observed by Rossi et al. differs from results obtained in studies of mice with hepatocyte-specific KO of the CB1 receptor (9). The effects of hepatocyte-specific KO or overexpression of the CB1 receptor on overall glucose homeostasis are consistent with those seen by Rossi and colleagues; however, these studies also provide evidence of CB1-induced insulin resistance. The reasons for this discrepancy are unclear and deserve further study.
Another question concerns the identity of the signaling pathways downstream of Gi that lead to increased HGP. JNK activation is not the sole mechanism, as hepatic expression of a dominant-negative form of JNK only partially inhibited the increased HGP caused by Gi activation. Comparison of RNA profiles from the livers of mice with and without DREADD stimulation showed differential expression of more than 1,000 genes, including many associated with the unfolded protein stress response. Additionally, genes involved in several other pathways also showed altered expression, suggesting that pathways other than JNK activation should be investigated in future studies.
The specific form(s) and subunits of heterotrimeric Gi (Gαi1, Gαi2, and Gαi3 individually or in combination; the β/γ subunits) involved in the direct stimulation of HGP and the nature of the effector with which Gi interacts to mediate its distal effect remain unclear. Various forms of Gαi, as well as β/γ subunits, have been shown to regulate effectors beyond adenylyl cyclase, including K+ and Ca2+ channels and PI3K (5, 10). No phenotypic changes were reported in mice with germline KO of genes encoding either Gαi1 or Gαi3, and germline KO of the Gαi2-encoding gene led to an ulcerative colitis–like disease ascribed to abnormal T cell function (6). Failure to observe overt defects in glucose homeostasis may reflect functional redundancy of Gαi genes and/or opposing roles for Gi in the liver and other organs. It is also possible that defects in glucose homeostasis in Gαi-KO mice remain to be discovered.
The enormity of the worldwide diabetes epidemic demands new, more effective forms of therapy. Of the drugs that are currently available to treat diabetes (aside from insulin itself), metformin is the only one that inhibits HGP (1). Other agents (11) act by distinct mechanisms, including insulin sensitization (thiazolidinediones), incretin effects (GLP-1 agonists and DPP-IV inhibitors), and increased renal glucose excretion (SGLT-2 inhibitors). The limited efficacy and side-effect profiles of these drugs have spurred the search for novel therapeutic targets. Recent studies, for example, have identified selective modulators of FOXO1 (12), a key downstream target of insulin action in the liver, and inhibitors of GPR21, an orphan receptor coupled to the Gq phospholipase C pathway (13), as potential therapeutic options. Given their finding that liver Gi KO improves glucose tolerance by reducing HGP, Rossi and colleagues suggest that inhibition of Gi signaling in the liver could represent a novel target for the treatment of diabetes. While theoretically true, developing agents that can selectively inhibit Gi only in liver will represent a formidable challenge, but agents that block hepatic Gi–coupled receptors, e.g., CB1 receptor antagonists, may hold more promise.
The CB1 receptor antagonist rimonabant was approved in Europe for the treatment of obesity on the basis of its ability to suppress appetite. Unfortunately, serious psychiatric side effects led to its withdrawal (14) and the development of peripherally restricted CB1 receptor antagonists (15). While the effects of such antagonists in the liver should improve glucose homeostasis (based on the present work), the ultimate role of CB1 receptor–targeting drugs in treating diabetes will depend on their integrated effect on adipose tissue, skeletal muscle, and the pancreas, where CB1 receptors are also expressed (15).
I am grateful to Jeffrey Pessin and Jonathan Backer (Albert Einstein College of Medicine) for their review of this Commentary.
Conflict of interest: A.M. Spiegel holds US-awarded patents on polyclonal antibodies against G proteins that have been licensed nonexclusively by the NIH to several companies.
Reference information: J Clin Invest. 2018;128(2):567–569. https://doi.org/10.1172/JCI99037.
See the related article at Hepatic Gi signaling regulates whole-body glucose homeostasis.