Thrombospondin 2 is a key determinant of fibrogenesis in non-alcoholic fatty liver disease.

OBJECTIVE: Hepatic overexpression of the thrombospondin 2 gene (THBS2) and elevated levels of circulating thrombospondin 2 (TSP2) have been observed in patients with chronic liver disease. This study aimed to identify the specific cells expressing THBS2/TSP2 in non-alcoholic fatty liver disease (NAFLD) and investigate the underlying mechanism behind THBS2/TSP2 upregulation. DESIGN: Comprehensive NAFLD liver gene datasets, including single-cell RNA sequencing (scRNA-seq), in-house NAFLD liver tissue, and LX-2 cells derived from human hepatic stellate cells (HSCs), were analysed using a combination of computational biology, genetic, immunological, and pharmacological approaches. RESULTS: Analysis of the genetic dataset revealed the presence of 1433 variable genes in patients with advanced fibrosis NAFLD, with THBS2 ranked among the top 2 genes. Quantitative polymerase chain reaction (qPCR) examination of NAFLD livers showed a significant correlation between THBS2 expression and fibrosis stage (r = .349, p < .001). In support of this, scRNA-seq data and in situ hybridization demonstrated that the THBS2 gene was highly expressed in HSCs of NAFLD patients with advanced fibrosis. Pathway analysis of the gene dataset revealed THBS2 expression to be associated with the transforming growth factor beta (TGFß) pathway and collagen gene activation. Moreover, the activation of LX-2 cells with TGFß increased THBS2/TSP2 and collagen expression independently of the TGFß-SMAD2/3 pathway. THBS2 gene knockdown significantly decreased collagen expression in LX-2 cells. CONCLUSIONS: THBS2/TSP2 is highly expressed in HSCs and plays a role in regulating fibrogenesis in NAFLD patients. THBS2/TSP2 may therefore represent a potential target for anti-fibrotic therapy in NAFLD.

Circulating thrombospondin 2 levels reflect fibrosis severity and disease activity in HCV-infected patients.

Among several secreted glycoproteins belonging to the thrombospondin family, thrombospondin 2 (TSP2) is involved in various functions, including collagen/fibrin formation. Liver/serum TSP2 levels have been correlated to liver fibrosis stage and disease activity in nonalcoholic fatty liver disease. This study investigated whether serum TSP2 was associated with clinicopathological features in hepatitis C virus (HCV)-infected patients as well. A total of 350 patients with HCV who had undergone liver biopsy were retrospectively enrolled and divided into a discovery cohort (n = 270) and a validation cohort (n = 80). In the discovery cohort, serum TSP2 levels were moderately correlated with both liver fibrosis stage (r = 0.426, P < 0.0001) and activity grade (r = 0.435, P < 0.0001). The area under the receiver operating characteristic curve of TSP2 for predicting severe fibrosis (≥ F3) was 0.78 and comparable to or better than those of autotaxin (0.78), FIB-4 index (0.78), and APRI (0.76). The discovery cohort findings were closely replicated in the validation cohort. Moreover, comprehensive liver genetic analysis of HCV-infected patients confirmed that the expression of the THBS2 gene encoding TSP2 was significantly higher in severely fibrotic F4 than in F1 patients. Circulating TSP2 levels may reflect the severity of hepatic fibrosis/inflammation in HCV-infected patients.

Adipocyte Gq signaling is a regulator of glucose and lipid homeostasis in mice.

Obesity is the major driver of the global epidemic in type 2 diabetes (T2D). In individuals with obesity, impaired insulin action leads to increased lipolysis in adipocytes, resulting in elevated plasma free fatty acid (FFA) levels that promote peripheral insulin resistance, a hallmark of T2D. Here we show, by using a combined genetic/biochemical/pharmacologic approach, that increased adipocyte lipolysis can be prevented by selective activation of adipocyte Gq signaling in vitro and in vivo (in mice). Activation of this pathway by a Gq-coupled designer receptor or by an agonist acting on an endogenous adipocyte Gq-coupled receptor (CysLT2 receptor) greatly improved glucose and lipid homeostasis in obese mice or in mice with adipocyte insulin receptor deficiency. Our findings identify adipocyte Gq signaling as an essential regulator of whole-body glucose and lipid homeostasis and should inform the development of novel classes of GPCR-based antidiabetic drugs.

2-Step PLT16-AST44 method: Simplified liver fibrosis detection system in patients with non-alcoholic fatty liver disease.

AIM: Accurate detection of the hepatic fibrosis stage is essential to estimate the outcome of patients with non-alcoholic fatty liver disease (NAFLD). Many formulas, biomarkers, and imaging tests are being developed to predict advanced liver fibrosis without performing a liver biopsy. However, these tests do not have high efficiency in detecting early-stage hepatic fibrosis. Therefore, we aimed to detect the presence of hepatic fibrosis (≥F1) merely by using only standard clinical markers. METHODS: A total of 436 patients with NAFLD who underwent liver biopsy were retrospectively enrolled as the discovery cohort (316 patients) and the validation cohort (120 patients). Liver biopsy and laboratory data were matched to extract simple parameters for identifying ≥F1. RESULTS: We developed a novel simplified ≥F1 detecting system, designated as 2-Step PLT16-AST44 method, where (1) PLT of 16 × 104 /µl or less, or (2) PLT greater than 16 × 104 /µl and AST greater than 44 U/L is determined as having ≥F1 fibrosis. The 2-Step PLT16-AST44 method had a sensitivity of 68%, a specificity of 90%, a positive predictive value (PPV) of 97%, a negative predictive value (NPV) of 40%, and an accuracy of 72% to detect ≥F1 fibrosis in the discovery cohort. Validation studies further supported these results. Despite its simplicity, the 2-Step PLT16-AST44 method's power to detect ≥F1 fibrosis in total NAFLD patients was comparable to hyaluronic acid, type 4 collagen 7S, FIB-4, and APRI. CONCLUSIONS: We propose the 2-Step PLT16-AST44 method as a simple and beneficial early-stage hepatic fibrosis detection system.

ß-Arrestins as Important Regulators of Glucose and Energy Homeostasis.

ß-Arrestin-1 and -2 (also known as arrestin-2 and -3, respectively) are ubiquitously expressed cytoplasmic proteins that dampen signaling through G protein-coupled receptors. However, ß-arrestins can also act as signaling molecules in their own right. To investigate the potential metabolic roles of the two ß-arrestins in modulating glucose and energy homeostasis, recent studies analyzed mutant mice that lacked or overexpressed ß-arrestin-1 and/or -2 in distinct, metabolically important cell types. Metabolic analysis of these mutant mice clearly demonstrated that both ß-arrestins play key roles in regulating the function of most of these cell types, resulting in striking changes in whole-body glucose and/or energy homeostasis. These studies also revealed that ß-arrestin-1 and -2, though structurally closely related, clearly differ in their metabolic roles under physiological and pathophysiological conditions. These new findings should guide the development of novel drugs for the treatment of various metabolic disorders, including type 2 diabetes and obesity.

ß-Arrestin-1 is required for adaptive ß-cell mass expansion during obesity.

Obesity is the key driver of peripheral insulin resistance, one of the key features of type 2 diabetes (T2D). In insulin-resistant individuals, the expansion of beta-cell mass is able to delay or even prevent the onset of overt T2D. Here, we report that beta-arrestin-1 (barr1), an intracellular protein known to regulate signaling through G protein-coupled receptors, is essential for beta-cell replication and function in insulin-resistant mice maintained on an obesogenic diet. Specifically, insulin-resistant beta-cell-specific barr1 knockout mice display marked reductions in beta-cell mass and the rate of beta-cell proliferation, associated with pronounced impairments in glucose homeostasis. Mechanistic studies suggest that the observed metabolic deficits are due to reduced Pdx1 expression levels caused by beta-cell barr1 deficiency. These findings indicate that strategies aimed at enhancing barr1 activity and/or expression in beta-cells may prove useful to restore proper glucose homeostasis in T2D.

Adipocyte P2Y14 receptors play a key role in regulating whole-body glucose and lipid homeostasis.

Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated Gi-type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y14R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y14R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y14R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y14R antagonist. In conclusion, we demonstrate that adipose P2Y14Rs were critical regulators of whole-body glucose and lipid homeostasis, suggesting that P2Y14R antagonists might be beneficial for the therapy of obesity and T2D.

Metabolic roles of G protein-coupled receptor signaling in obesity and type 2 diabetes.

The incidence of obesity and type 2 diabetes (T2D) has been increasing steadily worldwide. It is estimated that by 2045 more than 800 million people will be suffering from diabetes. Despite the advancements in modern medicine, more effective therapies for treating obesity and T2D are needed. G protein-coupled receptors (GPCRs) have emerged as important drug targets for various chronic diseases, including obesity, T2D, and liver diseases. During the past two decades, many laboratories worldwide focused on understanding the role of GPCR signaling in regulating glucose metabolism and energy homeostasis. The information gained from these studies can guide the development of novel therapeutic agents. In this review, we summarize recent studies providing insights into the role of GPCR signaling in peripheral, metabolically important tissues such as pancreas, liver, skeletal muscle, and adipose tissue, focusing primarily on the use of mutant animal models and human data.

Chemogenetic approaches to identify metabolically important GPCR signaling pathways: Therapeutic implications.

DREADDs (Designer Receptors Exclusively Activated by a Designer Drug) are designer G protein-coupled receptors (GPCRs) that are widely used in the neuroscience field to modulate neuronal activity. In this review, we will focus on DREADD studies carried out with genetically engineered mice aimed at elucidating signaling pathways important for maintaining proper glucose and energy homeostasis. The availability of muscarinic receptor-based DREADDs endowed with selectivity for one of the four major classes of heterotrimeric G proteins (Gs , Gi , Gq , and G12 ) has been instrumental in dissecting the physiological and pathophysiological roles of distinct G protein signaling pathways in metabolically important cell types. The novel insights gained from this work should inform the development of novel classes of drugs useful for the treatment of several metabolic disorders including type 2 diabetes and obesity.

Serum thrombospondin 2 is a novel predictor for the severity in the patients with NAFLD.

AIM: Thrombospondins are a family of multidomain and secretory glycoproteins. Among them, thrombospondin 2 (TSP2) encoded by TSP2 gene has been reported to be involved in various functions such as collagen/fibrin formation, maintenance of normal blood vessel density and cell adhesion properties. Microarray analyses ranked TSP2 as one of the most highly up-regulated genes in the fibrotic liver in patients with non-alcoholic fatty liver disease (NAFLD). Since TSP2 possesses unique properties as a secretory protein, we hypothesized that hepatic TSP2 gene expression levels would be reflected in serum TSP2 levels. In this study, we examined the relationship between serum TSP2 concentrations and clinicopathological findings in NAFLD patients. METHODS: One hundred and thirty NAFLD patients who had undergone liver biopsy between 2009 and 2015 were retrospectively enrolled. Serum samples were collected at the time of biopsy, and TSP2 was measured by enzyme immunoassays. RESULTS: Serum TSP2 levels moderately correlated with ballooning (r = 0.56, P < .001) and fibrosis stage (r = 0.53, P < .001). The AUC values of TSP2 for predicting mild fibrosis (≧F1), moderate fibrosis (≧F2) and severe fibrosis (≧F3) were 0.73, 0.76 and 0.82 respectively. Additionally, NAFLD activity score (NAS) correlated best with TSP2 (r = 0.52, P < .001) compared to conventional NAFLD-related biomarkers, such as cytokeratin 18 M30, hyaluronic acid, type IV collagen 7S, APRI and FIB-4 index. CONCLUSION: Serum TSP2 levels reflected hepatocyte ballooning, fibrosis and NAS in NAFLD patients. For clinical application of serum TSP2 as a predictor of NAFLD histological activity, additional validation and mechanistic investigations are required.

Key Metabolic Functions of ß-Arrestins: Studies with Novel Mouse Models.

ß-Arrestin-1 and -2 are intracellular proteins that are able to inhibit signaling via G protein-coupled receptors (GPCRs). However, both proteins can also modulate cellular functions in a G protein-independent fashion. During the past few years, studies with mutant mice selectivity lacking ß-arrestin-1 and/or -2 in metabolically important cell types have led to novel insights into the mechanisms through which ß-arrestins regulate key metabolic processes in vivo, including whole-body glucose and energy homeostasis. The novel information gained from these studies should inform the development of novel drugs, including ß-arrestin- or G protein-biased GPCR ligands, that could prove useful for the therapy of several important pathophysiological conditions, including type 2 diabetes and obesity.

Use of DREADD Technology to Identify Novel Targets for Antidiabetic Drugs.

G protein-coupled receptors (GPCRs) form a superfamily of plasma membrane receptors that couple to four major families of heterotrimeric G proteins, Gs, Gi, Gq, and G12. GPCRs represent excellent targets for drug therapy. Since the individual GPCRs are expressed by many different cell types, the in vivo metabolic roles of a specific GPCR expressed by a distinct cell type are not well understood. The development of designer GPCRs known as DREADDs (designer receptors exclusively activated by a designer drug) that selectively couple to distinct classes of heterotrimeric G proteins has greatly facilitated studies in this area. This review focuses on the use of DREADD technology to explore the physiological and pathophysiological roles of distinct GPCR/G protein cascades in several metabolically important cell types. The novel insights gained from these studies should stimulate the development of GPCR-based treatments for major metabolic diseases such as type 2 diabetes and obesity.

Lack of adipocyte purinergic P2Y6 receptor greatly improves whole body glucose homeostasis.

Uridine diphosphate (UDP)-activated purinergic receptor P2Y6 (P2Y6R) plays a crucial role in controlling energy balance through central mechanisms. However, P2Y6R's roles in peripheral tissues regulating energy and glucose homeostasis remain unexplored. Here, we report the surprising finding that adipocyte-specific deletion of P2Y6R protects mice from diet-induced obesity, improving glucose tolerance and insulin sensitivity with reduced systemic inflammation. These changes were associated with reduced JNK signaling and enhanced expression and activity of PPARα affecting downstream PGC1α levels leading to beiging of white fat. In contrast, P2Y6R deletion in skeletal muscle reduced glucose uptake, resulting in impaired glucose homeostasis. Interestingly, whole body P2Y6R knockout mice showed metabolic improvements similar to those observed with mice lacking P2Y6R only in adipocytes. Our findings provide compelling evidence that P2Y6R antagonists may prove useful for the treatment of obesity and type 2 diabetes.

Metabolic Functions of G Protein-Coupled Receptors in Hepatocytes-Potential Applications for Diabetes and NAFLD.

G protein-coupled receptors (GPCRs) are cell surface receptors that mediate the function of extracellular ligands. Understanding how GPCRs work at the molecular level has important therapeutic implications, as 30-40% of the drugs currently in clinical use mediate therapeutic effects by acting on GPCRs. Like many other cell types, liver function is regulated by GPCRs. More than 50 different GPCRs are predicted to be expressed in the mouse liver. However, knowledge of how GPCRs regulate liver metabolism is limited. A better understanding of the metabolic role of GPCRs in hepatocytes, the dominant constituent cells of the liver, could lead to the development of novel drugs that are clinically useful for the treatment of various metabolic diseases, including type 2 diabetes, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). In this review, we describe the functions of multiple GPCRs expressed in hepatocytes and their role in metabolic processes.

Beneficial metabolic role of ß-arrestin-1 expressed by AgRP neurons.

ß-Arrestin-1 and ß-arrestin-2 have emerged as important signaling molecules that modulate glucose fluxes in several peripheral tissues. The potential roles of neuronally expressed ß-arrestins in regulating glucose homeostasis remain unknown. We here report that mice lacking ß-arrestin-1 (barr1) selectively in AgRP neurons displayed impaired glucose tolerance and insulin sensitivity when consuming an obesogenic diet, while mice overexpressing barr1 selectively in AgRP neurons were protected against obesity-associated metabolic impairments. Additional physiological, biochemical, and electrophysiological data indicated that the presence of barr1 is essential for insulin-mediated hyperpolarization of AgRP neurons. As a result, barr1 expressed by AgRP neurons regulates efferent neuronal pathways that suppress hepatic glucose production and promote lipolysis in adipose tissue. Mice lacking ß-arrestin-2 (barr2) selectively in AgRP neurons showed no substantial metabolic phenotypes. Our data suggest that agents able to enhance the activity of barr1 in AgRP neurons may prove beneficial as antidiabetic drugs.

ß-arrestin-1 suppresses myogenic reprogramming of brown fat to maintain euglycemia.

A better understanding of the signaling pathways regulating adipocyte function is required for the development of new classes of antidiabetic/obesity drugs. We here report that mice lacking ß-arrestin-1 (barr1), a cytoplasmic and nuclear signaling protein, selectively in adipocytes showed greatly impaired glucose tolerance and insulin sensitivity when consuming an obesogenic diet. In contrast, transgenic mice overexpressing barr1 in adipocytes were protected against the metabolic deficits caused by a high-calorie diet. Barr1 deficiency led to a myogenic reprogramming of brown adipose tissue (BAT), causing elevated plasma myostatin (Mstn) levels, which in turn led to impaired insulin signaling in multiple peripheral tissues. Additional in vivo studies indicated that barr1-mediated suppression of Mstn expression by BAT is required for maintaining euglycemia. These findings convincingly identify barr1 as a critical regulator of BAT function. Strategies aimed at enhancing barr1 activity in BAT may prove beneficial for the treatment of type 2 diabetes.

Adipocyte Gi signaling is essential for maintaining whole-body glucose homeostasis and insulin sensitivity.

Adipocyte dysfunction links obesity to insulin resistance and type 2 diabetes. Adipocyte function is regulated by receptor-mediated activation of heterotrimeric G proteins. Little is known about the potential in vivo metabolic roles of Gi-type G proteins expressed by adipocytes, primarily due to the lack of suitable animal models. To address this question, we generated mice lacking functional Gi proteins selectively in adipocytes. Here we report that these mutant mice displayed significantly impaired glucose tolerance and reduced insulin sensitivity when maintained on an obesogenic diet. In contrast, using a chemogenetic strategy, we demonstrated that activation of Gi signaling selectively in adipocytes greatly improved glucose homeostasis and insulin signaling. We also elucidated the cellular mechanisms underlying the observed metabolic phenotypes. Our data support the concept that adipocyte Gi signaling is essential for maintaining euglycemia. Drug-mediated activation of adipocyte Gi signaling may prove beneficial for restoring proper glucose homeostasis in type 2 diabetes.

Selective activation of Gs signaling in adipocytes causes striking metabolic improvements in mice.

OBJECTIVE: Given the worldwide epidemics of obesity and type 2 diabetes, novel antidiabetic and appetite-suppressing drugs are urgently needed. Adipocytes play a central role in the regulation of whole-body glucose and energy homeostasis. The goal of this study was to examine the metabolic effects of acute and chronic activation of Gs signaling selectively in adipocytes (activated Gs stimulates cAMP production), both in lean and obese mice. METHODS: To address this question, we generated a novel mutant mouse strain (adipo-GsD mice) that expressed a Gs-coupled designer G protein-coupled receptor (Gs DREADD or short GsD) selectively in adipocytes. Importantly, the GsD receptor can only be activated by administration of an exogenous agent (CNO) that is otherwise pharmacologically inert. The adipo-GsD mice were maintained on either regular chow or a high-fat diet and then subjected to a comprehensive series of metabolic tests. RESULTS: Pharmacological (CNO) activation of the GsD receptor in adipocytes of adipo-GsD mice caused profound improvements in glucose homeostasis and protected mice against the metabolic deficits associated with the consumption of a calorie-rich diet. Moreover, chronic activation of Gs signaling in adipocytes led to a striking increase in energy expenditure and reduced food intake, resulting in a decrease in body weight and fat mass when mice consumed a calorie-rich diet. CONCLUSION: Systematic studies with a newly developed mouse model enabled us to assess the metabolic consequences caused by acute or chronic activation of Gs signaling selectively in adipocytes. Most strikingly, chronic activation of this pathway led to reduced body fat mass and restored normal glucose homeostasis in obese mice. These findings are of considerable relevance for the development of novel antidiabetic and anti-obesity drugs.

Adipocyte ß-arrestin-2 is essential for maintaining whole body glucose and energy homeostasis.

ß-Arrestins are major regulators of G protein-coupled receptor-mediated signaling processes. Their potential roles in regulating adipocyte function in vivo remain unexplored. Here we report the novel finding that mice lacking ß-arrestin-2 (barr2) selectively in adipocytes show significantly reduced adiposity and striking metabolic improvements when consuming excess calories. We demonstrate that these beneficial metabolic effects are due to enhanced signaling through adipocyte ß3-adrenergic receptors (ß3-ARs), indicating that barr2 represents a potent negative regulator of adipocyte ß3-AR activity in vivo. Interestingly, essentially all beneficial metabolic effects caused by adipocyte barr2 deficiency are absent in adipocyte barr2-PRDM16 double KO mice, indicating that the metabolic improvements caused by the lack of barr2 in adipocytes are mediated by the browning/beiging of white adipose tissue. Our data support the novel concept that 'G protein-biased' ß3-AR agonists that do not promote ß3-AR/barr2 interactions may prove useful for the treatment of obesity and related metabolic disorders.