Functional screening and rational design of compounds targeting GPR132 to treat diabetes.

Chronic inflammation due to islet-residing macrophages plays key roles in the development of type 2 diabetes mellitus. By systematically profiling intra-islet lipid-transmembrane receptor signalling in islet-resident macrophages, we identified endogenous 9(S)-hydroxy-10,12-octadecadienoic acid-G-protein-coupled receptor 132 (GPR132)-Gi signalling as a significant contributor to islet macrophage reprogramming and found that GPR132 deficiency in macrophages reversed metabolic disorders in mice fed a high-fat diet. The cryo-electron microscopy structures of GPR132 bound with two endogenous agonists, N-palmitoylglycine and 9(S)-hydroxy-10,12-octadecadienoic acid, enabled us to rationally design both GPR132 agonists and antagonists with high potency and selectivity through stepwise translational approaches. We ultimately identified a selective GPR132 antagonist, NOX-6-18, that modulates macrophage reprogramming within pancreatic islets, decreases weight gain and enhances glucose metabolism in mice fed a high-fat diet. Our study not only illustrates that intra-islet lipid signalling contributes to islet macrophage reprogramming but also provides a broadly applicable strategy for the identification of important G-protein-coupled receptor targets in pathophysiological processes, followed by the rational design of therapeutic leads for refractory diseases such as diabetes.

Endogenous Lipid-GPR120 Signaling Modulates Pancreatic Islet Homeostasis to Different Extents.

Long-chain fatty acids (LCFAs) are not only energy sources but also serve as signaling molecules. GPR120, an LCFA receptor, plays key roles in maintaining metabolic homeostasis. However, whether endogenous ligand-GPR120 circuits exist and how such circuits function in pancreatic islets are unclear. Here, we found that endogenous GPR120 activity in pancreatic δ-cells modulated islet functions. At least two unsaturated LCFAs, oleic acid (OA) and linoleic acid (LA), were identified as GPR120 agonists within pancreatic islets. These two LCFAs promoted insulin secretion by inhibiting somatostatin secretion and showed bias activation of GPR120 in a model system. Compared with OA, LA exerted higher potency in promoting insulin secretion, which is dependent on ß-arrestin2 function. Moreover, GPR120 signaling was impaired in the diabetic db/db model, and replenishing OA and LA improved islet function in both the db/db and streptozotocin-treated diabetic models. Consistently, the administration of LA improved glucose metabolism in db/db mice. Collectively, our results reveal that endogenous LCFA-GPR120 circuits exist and modulate homeostasis in pancreatic islets. The contributions of phenotype differences caused by different LCFA-GPR120 circuits within islets highlight the roles of fine-tuned ligand-receptor signaling networks in maintaining islet homeostasis.

In vitro expansion of pancreatic islet clusters facilitated by hormones and chemicals.

Tissue regeneration, such as pancreatic islet tissue propagation in vitro, could serve as a promising strategy for diabetes therapy and personalised drug testing. However, such a strategy has not been realised yet. Propagation could be divided into two steps, in vitro expansion and repeated passaging. Even the first step of the in vitro islet expansion has not been achieved to date. Here, we describe a method that enables the expansion of islet clusters isolated from pregnant mice or wild-type rats by employing a combination of specific regeneration factors and chemical compounds in vitro. The expanded islet clusters expressed insulin, glucagon and somatostatin, which are markers corresponding to pancreatic ß cells, α cells and δ cells, respectively. These different types of cells grouped together, were spatially organised and functioned similarly to primary islets. Further mechanistic analysis revealed that forskolin in our recipe contributed to renewal and regeneration, whereas exendin-4 was essential for preserving islet cell identity. Our results provide a novel method for the in vitro expansion of islet clusters, which is an important step forward in developing future protocols and media used for islet tissue propagation in vitro. Such method is important for future regenerative diabetes therapies and personalised medicines using large amounts of pancreatic islets derived from the same person.

Fatty acid-induced endoplasmic reticulum stress promoted lipid accumulation in calf hepatocytes, and endoplasmic reticulum stress existed in the liver of severe fatty liver cows.

Disruption of endoplasmic reticulum (ER) homeostasis, often termed ER stress, is intrinsically linked with perturbation of lipid metabolism in humans and mice. Whether ER homeostasis is affected in cows experiencing fatty liver is unknown. The aim of this study was to investigate the potential role of ER stress in hepatic lipid accumulation in calf hepatocytes and ER stress status in dairy cows with severe fatty liver. In vitro experiments were conducted in which hepatocytes were isolated from calves and treated with different concentrations of fatty acids, tauroursodeoxycholic acid (TUDCA; a canonical inhibitor of ER stress), or both. The increase in phosphorylation level of protein kinase RNA-like ER kinase (PERK) and inositol requiring protein-1α (IRE1α) proteins, and the cleavage of activating transcription factor-6 (ATF6) protein in response to increasing doses of fatty acids (which were reversed by TUDCA treatment) in primary hepatocytes underscored a mechanistic link between fatty acids and ER stress. In addition, fatty acid treatment increased the abundance of sterol regulatory element-binding protein 1c, acetyl-CoA carboxylase-α, fatty acid synthase, and diacylglycerol acyltransferase 1, and lipid accumulation in calf primary hepatocytes, whereas inhibition of ER stress by incubating with TUDCA significantly weakened these effects. Overall, results in vitro indicate that inhibition of ER stress in calf hepatocytes alleviates fatty acid-induced lipid accumulation by downregulating the expression of lipogenic genes. In vivo experiments, liver and blood samples were collected from cows diagnosed as healthy (n = 15) or with severe fatty liver (n = 15). The phosphorylation level of PERK and IRE1α, the cleavage of ATF6 protein, and the abundance of several unfolded protein response genes (78 kDa glucose-regulated protein, AMP-dependent transcription factor 4, and spliced X-box binding protein 1) were greater in liver of cows with severe fatty liver. The present in vivo study confirms the occurrence of ER stress in dairy cows with severe fatty liver. Considering the causative role of fatty acid-induced ER stress in hepatic lipid accumulation in calf hepatocytes, the existence of ER stress in the liver of severe fatty liver cows may presage its participation in fatty liver progression in dairy cows. However, the mechanistic relationship between ER stress and fatty liver in dairy cows remain to be determined.

Expression patterns of hepatic genes involved in lipid metabolism in cows with subclinical or clinical ketosis.

Dairy cows with ketosis are characterized by high blood concentrations of ketone bodies and hepatic lipid metabolism disorder. The discrepancies in the abundance of mRNA encoding a variety of hepatic candidate genes in varying degrees of ketotic cows represent specific responses of the liver to the challenge of fatty acids and ketone bodies. Importantly, the expression disorder of hepatic genes involved in lipid metabolism plays a promoting role in the onset and progression of ketosis. Thus, the aim of this study was to investigate the expression patterns of genes involved in the hepatic fatty acids uptake, transport, activation, ß-oxidation, synthesis, and esterification in the cows with subclinical ketosis (SCK) or clinical ketosis (CK). Twenty-four cows were selected into control [n = 8, ß-hydroxybutyrate (BHB) ≤0.6 mM], SCK (n = 8, 3.0 > BHB ≥ 1.2 mM), and CK (n = 8, BHB ≥3.0 mM) groups according to the blood BHB concentration and clinical symptoms. The accumulation of hepatic lipid, as indicated by triglycerides (TG) contents and Oil Red O and hematoxylin and eosin staining, was pronouncedly exacerbated in the tCK group compared with the control and SCK groups. The hepatic mRNA expression of fatty acids transport and activation genes, liver fatty acid-binding protein (FABP1) and long-chain acyl-CoA synthetase 1 (ACSL1), were both significantly higher in the SCK and CK groups than in the control group. The expression levels of peroxisome proliferator-activated receptor α (PPARA) and its target genes, carnitine palmitoyltransferase 1A (CPT1A) and carnitine palmitoyltransferase 2 (CPT2), were significantly elevated in the SCK group but reduced in the CK group compared with control group. Furthermore, the gene expression level of sterol regulatory element-binding protein 1 (SREBP1) and the protein expression level of sterol regulatory element-binding protein 1c and its target genes acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD1) and TG synthesis genes diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) were significantly higher in the CK group relative to the control group. In short, the present data indicated that hepatic fatty acids uptake, transport, and activation are significantly increased in cows with SCK and CK, hepatic fatty acids ß-oxidation is significantly increased in SCK cows but markedly decreased in CK cows, and hepatic fatty acids and TG synthesis are significantly increased in CK cows, thereby inducing hepatic steatosis in CK cows.

High concentrations of fatty acids and ß-hydroxybutyrate impair the growth hormone-mediated hepatic JAK2-STAT5 pathway in clinically ketotic cows.

The hepatic growth hormone (GH)-insulin-like growth factor (IGF)-I axis is essential for regulating intrahepatic lipid metabolism. Ketotic cows are characterized by high blood concentrations of fatty acids and ß-hydroxybutyrate (BHB), which display lipotoxicity. The aim of this study was to investigate changes in the hepatic GH-IGF-I axis in ketotic cows and to determine the effects of fatty acids and BHB on the GH-IGF-I axis in calf hepatocytes. Liver and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) cows. Hepatocytes were isolated from calves and treated with various concentrations of GH, fatty acids, and BHB. The results showed that clinically ketotic cows displayed a high blood concentration of GH, a low blood concentration of IGF-I, and decreased hepatic GHR1A expression as well as impaired hepatic Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling. In vitro, GH treatment induced activation of the JAK2-STAT5 pathway to increase the mRNA expression and secretion of IGF-I in calf hepatocytes. More importantly, treatment with fatty acids or BHB significantly inhibited GHR1A mRNA and JAK2 protein expression, as well as the STAT5 phosphorylation level and phospho-STAT5 nuclear translocation; these effects markedly reduced IGF1 mRNA expression and secretion in calf hepatocytes. In summary, these results indicate that high blood concentrations of fatty acids or BHB can impair the intrahepatic GH-mediated JAK2-STAT5 pathway and downregulate IGF-I expression and secretion in ketotic cows.