Inhibiting arachidonic acid generation mitigates aging-induced hyperinsulinemia and insulin resistance in mice.

BACKGROUND: Aging-related type 2 diabetes (T2DM) is characterized by hyperinsulinemia, insulin resistance, and ß-cell dysfunction. However, the underlying molecular mechanisms remain to be unclear. METHODS: We conducted non-targeted metabolomics to compare human serum samples from young adults (YA), elderly adults (EA), and elderly adults with diabetes (EA + DM) of Chinese population. Adult mice and aged mice were intragastrically administered with varespladib every day for two weeks and metabolic characteristics were monitored. Serum levels of arachidonic acid, insulin, and C-peptide, as well as serum activity of secretory phospholipase A2 (sPLA2) were detected in mice. Mouse islet perfusion assays were used to assess insulin secretion ability. Phosphorylated AKT levels were measured to evaluate insulin sensitivities of peripheral tissues in mice. RESULTS: Non-targeted metabolomics analysis of human serum samples revealed differential metabolic signatures among the YA, EA, and EA + DM groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed significant enhancement of arachidonic acid metabolism and glycerophospholipid metabolism in the EA group compared with the YA group. Further analysis identified two metabolic fluxes that favored the accumulation of arachidonic acid in the elderly. Increased levels of arachidonic acid were also confirmed in aged mice with hyperinsulinemia and insulin resistance, together with subsequent glucose intolerance. Conversely, inhibiting the generation of arachidonic acid with varespladib, an inhibitor of sPLA2, reduced aging-associated diabetes by improving hyperinsulinemia and hepatic insulin resistance in aged mice but not in adult mice. Islet perfusion assays also showed that varespladib treatment suppressed the enhanced insulin secretion observed in aged islets. CONCLUSIONS: Collectively, our findings uncover that arachidonic acid serves as a metabolic hub in Chinese elderly population. Our results also suggest that arachidonic acid plays a fundamental role in regulating ß-cell function during aging and point to a novel therapy for aging-associated diabetes.

Islet-resident macrophage-derived miR-155 promotes ß cell decompensation via targeting PDX1.

Chronic inflammation is critical for the initiation and progression of type 2 diabetes mellitus via causing both insulin resistance and pancreatic ß cell dysfunction. miR-155, highly expressed in macrophages, is a master regulator of chronic inflammation. Here we show that blocking a macrophage-derived exosomal miR-155 (MDE-miR-155) mitigates the insulin resistances and glucose intolerances in high-fat-diet (HFD) feeding and type-2 diabetic db/db mice. Lentivirus-based miR-155 sponge decreases the level of miR-155 in the pancreas and improves glucose-stimulated insulin secretion (GSIS) ability of ß cells, thus leading to improvements of insulin sensitivities in the liver and adipose tissues. Mechanistically, miR-155 increases its expression in HFD and db/db islets and is released as exosomes by islet-resident macrophages under metabolic stressed conditions. MDE-miR-155 enters ß cells and causes defects in GSIS function and insulin biosynthesis via the miR-155-PDX1 axis. Our findings offer a treatment strategy for inflammation-associated diabetes via targeting miR-155.

M2 macrophages independently promote beige adipogenesis via blocking adipocyte Ets1.

Adipose tissue macrophages can promote beige adipose thermogenesis by altering local sympathetic activity. Here, we perform sympathectomy in mice and further eradicate subcutaneous adipose macrophages and discover that these macrophages have a direct beige-promoting function that is independent of sympathetic system. We further identify adipocyte Ets1 as a vital mediator in this process. The anti-inflammatory M2 macrophages suppress Ets1 expression in adipocytes, transcriptionally activate mitochondrial biogenesis, as well as suppress mitochondrial clearance, thereby increasing the mitochondrial numbers and promoting the beiging process. Male adipocyte Ets1 knock-in mice are completely cold intolerant, whereas male mice lacking Ets1 in adipocytes show enhanced energy expenditure and are resistant to metabolic disorders caused by high-fat-diet. Our findings elucidate a direct communication between M2 macrophages and adipocytes, and uncover a function for Ets1 in responding to macrophages and negatively governing mitochondrial content and beige adipocyte formation.

ß-Cell miRNA-503-5p Induced by Hypomethylation and Inflammation Promotes Insulin Resistance and ß-Cell Decompensation.

Chronic inflammation promotes pancreatic ß-cell decompensation to insulin resistance because of local accumulation of supraphysiologic interleukin 1ß (IL-1ß) levels. However, the underlying molecular mechanisms remain elusive. We show that miR-503-5p is exclusively upregulated in islets from humans with type 2 diabetes and diabetic rodents because of its promoter hypomethylation and increased local IL-1ß levels. ß-Cell-specific miR-503 transgenic mice display mild or severe diabetes in a time- and expression-dependent manner. By contrast, deletion of the miR-503 cluster protects mice from high-fat diet-induced insulin resistance and glucose intolerance. Mechanistically, miR-503-5p represses c-Jun N-terminal kinase-interacting protein 2 (JIP2) translation to activate mitogen-activated protein kinase signaling cascades, thus inhibiting glucose-stimulated insulin secretion (GSIS) and compensatory ß-cell proliferation. In addition, ß-cell miR-503-5p is packaged in nanovesicles to dampen insulin signaling transduction in liver and adipose tissues by targeting insulin receptors. Notably, specifically blocking the miR-503 cluster in ß-cells effectively remits aging-associated diabetes through recovery of GSIS capacity and insulin sensitivity. Our findings demonstrate that ß-cell miR-503-5p is required for the development of insulin resistance and ß-cell decompensation, providing a potential therapeutic target against diabetes. ARTICLE HIGHLIGHTS: Promoter hypomethylation during natural aging permits miR-503-5p overexpression in islets under inflammation conditions, conserving from rodents to humans. Impaired ß-cells release nanovesicular miR-503-5p to accumulate in liver and adipose tissue, leading to their insulin resistance via the miR-503-5p/insulin receptor/phosphorylated AKT axis. Accumulated miR-503-5p in ß-cells impairs glucose-stimulated insulin secretion via the JIP2-coordinated mitogen-activated protein kinase signaling cascades. Specific blockage of ß-cell miR-503-5p improves ß-cell function and glucose tolerance in aging mice.

BRSK2 in pancreatic ß cells promotes hyperinsulinemia-coupled insulin resistance and its genetic variants are associated with human type 2 diabetes.

Brain-specific serine/threonine-protein kinase 2 (BRSK2) plays critical roles in insulin secretion and ß-cell biology. However, whether BRSK2 is associated with human type 2 diabetes mellitus (T2DM) has not been determined. Here, we report that BRSK2 genetic variants are closely related to worsening glucose metabolism due to hyperinsulinemia and insulin resistance in the Chinese population. BRSK2 protein levels are significantly elevated in ß cells from T2DM patients and high-fat diet (HFD)-fed mice due to enhanced protein stability. Mice with inducible ß-cell-specific Brsk2 knockout (ßKO) exhibit normal metabolism with a high potential for insulin secretion under chow-diet conditions. Moreover, ßKO mice are protected from HFD-induced hyperinsulinemia, obesity, insulin resistance, and glucose intolerance. Conversely, gain-of-function BRSK2 in mature ß cells reversibly triggers hyperglycemia due to ß-cell hypersecretion-coupled insulin resistance. Mechanistically, BRSK2 senses lipid signals and induces basal insulin secretion in a kinase-dependent manner. The enhanced basal insulin secretion drives insulin resistance and ß-cell exhaustion and thus the onset of T2DM in mice fed an HFD or with gain-of-function BRSK2 in ß cells. These findings reveal that BRSK2 links hyperinsulinemia to systematic insulin resistance via interplay between ß cells and insulin-sensitive tissues in the populations carrying human genetic variants or under nutrient-overload conditions.

NR3C1/Glucocorticoid receptor activation promotes pancreatic ß-cell autophagy overload in response to glucolipotoxicity.

Diabetes is a complex and heterogeneous disorder characterized by chronic hyperglycemia. Its core cause is progressively impaired insulin secretion by pancreatic ß-cell failures, usually upon a background of preexisting insulin resistance. Recent studies demonstrate that macroautophagy/autophagy is essential to maintain architecture and function of ß-cells, whereas excessive autophagy is also involved in ß-cell dysfunction and death. It has been poorly understood whether autophagy plays a protective or harmful role in ß-cells, while we report here that it is dependent on NR3C1/glucocorticoid receptor activation. We proved that deleterious hyperactive autophagy happened only upon NR3C1 activation in ß-cells under glucolipotoxic conditions, which eventually promoted diabetes. The transcriptome and the N6-methyladenosine (m6A) methylome revealed that NR3C1-enhancement upregulated the RNA demethylase FTO (fat mass and obesity associated) protein in ß-cells, which caused diminished m6A modifications on mRNAs of four core Atg (autophagy related) genes (Atg12, Atg5, Atg16l2, Atg9a) and, hence, hyperactive autophagy and defective insulin output; by contrast, FTO inhibition, achieved by the specific FTO inhibitor Dac51, prevented NR3C1-instigated excessive autophagy activation. Importantly, Dac51 effectively alleviated impaired insulin secretion and glucose intolerance in hyperglycemic ß-cell specific NR3C1 overexpression mice. Our results determine that the NR3C1-FTO-m6A modifications-Atg genes axis acts as a key mediator of balanced autophagic flux in pancreatic ß-cells, which offers a novel therapeutic target for the treatment of diabetes.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; Ac: acetylation; Ad: adenovirus; AL: autolysosome; ATG: autophagy related; AUC: area under curve; Baf A1: bafilomycin A1; ßNR3C1 mice: pancreatic ß-cell-specific NR3C1 overexpression mice; cFBS: charcoal-stripped FBS; Ctrl: control; ER: endoplasmic reticulum; FTO: fat mass and obesity associated; GC: glucocorticoid; GRE: glucocorticoid response element; GSIS: glucose-stimulated insulin secretion assay; HFD: high-fat diet; HG: high glucose; HsND: non-diabetic human; HsT2D: type 2 diabetic human; i.p.: intraperitoneal injected; KSIS: potassium-stimulated insulin secretion assay; m6A: N6-methyladenosine; MeRIP-seq: methylated RNA immunoprecipitation sequencing; NR3C1/GR: nuclear receptor subfamily 3, group C, member 1; NR3C1-Enhc.: NR3C1-enhancement; NC: negative control; Palm.: palmitate; RNA-seq: RNA sequencing; T2D: type 2 diabetes; TEM: transmission electron microscopy; UTR: untranslated region; WT: wild-type.

Lentinan confers protection against type 1 diabetes by inducing regulatory T cell in spontaneous non-obese diabetic mice.

BACKGROUND: Lentinan (LNT) is a complex fungal component that possesses effective antitumor and immunostimulating properties. However, there is a paucity of studies regarding the effects and mechanisms of LNT on type 1 diabetes. OBJECTIVE: In the current study, we investigated whether an intraperitoneal injection of LNT can diminish the risk of developing type 1 diabetes (T1D) in non-obese diabetic (NOD) mice and further examined possible mechanisms of LNT's effects. METHODS: Pre-diabetic female NOD mice 8 weeks of age, NOD mice with 140-160 mg/dL, 200-230 mg/dL or 350-450 mg/dL blood glucose levels were randomly divided into two groups and intraperitoneally injected with 5 mg/kg LNT or PBS every other day. Then, blood sugar levels, pancreas slices, spleen, PnLN and pancreas cells from treatment mice were examined. RESULTS: Our results demonstrated that low-dosage injections (5 mg/kg) of LNT significantly suppressed immunopathology in mice with autoimmune diabetes but increased the Foxp3+ regulatory T cells (Treg cells) proportion in mice. LNT treatment induced the production of Tregs in the spleen and PnLN cells of NOD mice in vitro. Furthermore, the adoptive transfer of Treg cells extracted from LNT-treated NOD mice confirmed that LNT induced Treg function in vivo and revealed an enhanced suppressive capacity as compared to the Tregs isolated from the control group. CONCLUSION: LNT was capable of stimulating the production of Treg cells from naive CD4 + T cells, which implies that LNT exhibits therapeutic values as a tolerogenic adjuvant and may be used to reverse hyperglycaemia in the early and late stages of T1D.

The heparan sulfate mimetic Muparfostat aggravates steatohepatitis in obese mice due to its binding affinity to lipoprotein lipase.

BACKGROUND AND PURPOSE: Heparanase is the only confirmed endoglycosidase that cleaves heparan sulfate (HS), a ubiquitous glycosaminoglycan with various essential roles in multiple pathological processes. Thus, the development of heparanase inhibitors has become an attractive strategy for drug discovery, especially in tumour therapy, in which HS mimetics are the most promising compounds. The various biological effects of heparanase also suggest a role for HS mimetics in many non-cancer indications, such as type 1 diabetes. However, the potential benefits of HS mimetics in obesity-related type 2 diabetes have not been elucidated. EXPERIMENTAL APPROACH: In this study, we investigated muparfostat (PI-88), a developed HS mimetic currently enrolled in Phase III clinical trials, in obese mouse models and in vitro cultured murine hepatocytes. KEY RESULTS: Daily administration of muparfostat for 4 weeks caused hyperlipidaemia and aggravated hepatic steatosis in obese mice models, but not in lean animals. In cultured hepatocytes, muparfostat did not alter lipid accumulation. Acute tests suggested that muparfostat binds to lipoprotein lipase in competition with HS on vascular endothelial cell surfaces, thereby reducing the degradation of circulating triglycerides by lipoprotein lipase and subsequent uptake of fatty acids into vascular endothelial cells and causing hyperlipidaemia. This hyperlipidaemia aggravates hepatic steatosis and causes liver injury in muparfostat-treated obese mice. CONCLUSIONS AND IMPLICATIONS: The binding activity of HS mimetics to lipoprotein lipase should be investigated as an additional pharmacological effect during heparanase inhibitor drug discovery. This study also provides novel evidence for an increased risk of drug-induced liver injury in obese individuals.

Recurrent hypoglycemia increases hepatic gluconeogenesis without affecting glycogen metabolism or systemic lipolysis in rat.

INTRODUCTION: Recurrent hypoglycemia (RH) impairs secretion of counterregulatory hormones. Whether and how RH affects responses within metabolically important peripheral organs to counterregulatory hormones are poorly understood. OBJECTIVE: To study the effects of RH on metabolic pathways associated with glucose counterregulation within liver, white adipose tissue and skeletal muscle. METHODS: Using a widely adopted rodent model of 3-day recurrent hypoglycemia, we first checked expression of counterregulatory hormone G-protein coupled receptors (GPCRs), their inhibitory regulators and downstream enzymes catalyzing glycogen metabolism, gluconeogenesis and lipolysis by qPCR and western blot. Then, we examined epinephrine-induced phosphorylation of PKA substrates to validate adrenergic sensitivity in each organ. Next, we measured hepatic and skeletal glycogen content, degree of breakdown by epinephrine and abundance of phosphorylated glycogen phosphorylase under hypoglycemia and that of phosphorylated glycogen synthase during recovery to evaluate glycogen turnover. Further, we performed pyruvate and lactate tolerance tests to assess gluconeogenesis. Additionally, we measured circulating FFA and glycerol to check lipolysis. The abovementioned studies were repeated in streptozotocin-induced diabetic rat model. Finally, we conducted epinephrine tolerance test to investigate systemic glycemic excursions to counterregulatory hormones. Saline-injected rats served as controls. RESULTS: RH increased counterregulatory hormone GPCR signaling in liver and epidydimal white adipose tissue (eWAT), but not in skeletal muscle. For glycogen metabolism, RH did not affect total content or epinephrine-stimulated breakdown in liver and skeletal muscle. Although RH decreased expression of phosphorylated glycogen synthase 2, it did not affect hepatic glycogen biosynthesis during recovery from hypoglycemia or after fasting-refeeding. For gluconeogenesis, RH upregulated fructose 1,6-bisphosphatase 1 and monocarboxylic acid transporter 1 that imports lactate as precursor, resulting in a lower blood lactate profile during hypoglycemia. In agreement, RH elevated fasting blood glucose and caused higher glycemic excursions during pyruvate tolerance test. For lipolysis, RH did not affect circulating levels of FFA and glycerol after overnight fasting or upon epinephrine stimulation. Interestingly, RH upregulated the trophic fatty acid transporter FATP1 and glucose transporter GLUT4 to increase lipogenesis in eWAT. These aforementioned changes of gluconeogenesis, lipolysis and lipogenesis were validated in streptozotocin-diabetic rats. Finally, RH increased insulin sensitivity to accelerate glucose disposal, which was attributable to upregulated visceral adipose GLUT4. CONCLUSIONS: RH caused metabolic adaptations related to counterregulation within peripheral organs. Specifically, adrenergic signaling was enhanced in liver and visceral fat, but not in skeletal muscle. Glycogen metabolism remained unchanged. Hepatic gluconeogenesis was augmented. Systemic lipolysis was unaffected, but visceral lipogenesis was enhanced. Insulin sensitivity was increased. These findings provided insights into mechanisms underlying clinical problems associated with intensive insulin therapy, such as high gluconeogenic flux and body weight gain.

Perfluorooctanoic acid promotes pancreatic ß cell dysfunction and apoptosis through ER stress and the ATF4/CHOP/TRIB3 pathway.

Perfluorooctanoic acid (PFOA), a widely used chemical substance, causes an increased risk of human type 2 diabetes (T2D), but its underlying mechanism is not well elucidated. The aim of the present study was to investigate whether PFOA regulates the functions of pancreatic ß cells, which are specialized for the biosynthesis and secretion of insulin. The treatment of the mouse pancreatic ß cell line (MIN6 cells) with PFOA caused a time- and dose-dependent inhibition of cell viability in CCK-8 assays. Annexin V/PI and TUNEL staining results confirmed that exposure to a high PFOA dose (500 µM) promoted apoptosis of ß cells, while a low dose (300 µM) had no effects on ß cell survival. PFOA treatment, even at a low dose, diminished glucose-stimulated insulin secretion (GSIS) in both primary islet perfusion and MIN6 cell experiments. RNA-sequencing data showed significantly increased expression of endoplasmic reticulum (ER) stress-associated genes, with tribbles homolog 3 (Trib3) ranking first among the altered genes. The activation of ER stress pathways was verified by qRT-PCR assays, and the ATF4/CHOP/TRIB3 pathway contributed to PFOA-induced ß cell damage. The inhibition of TRIB3 expression significantly protected MIN6 cells from PFOA-induced GSIS defects and apoptosis by ameliorating ER stress. These findings reveal a link between ER stress and PFOA-induced ß cell defects, opening up a new set of questions about the pathogenesis of T2D due to environmental chemicals.

Rab31, a receptor of advanced glycation end products (RAGE) interacting protein, inhibits AGE induced pancreatic ß-cell apoptosis through the pAKT/BCL2 pathway.

Receptor of advanced glycation end products (RAGE) mediates diverse signal transduction following ligand stimulation and plays an important role in diabetes complications and aging associated disease. We have previously verified that advanced glycation end products (AGE) bind to RAGE to cause pancreatic ß-cell apoptosis through the mitochondrial pathway. However, the direct interacting protein(s) of RAGE in ß cells has never been appreciated. In the present study, we utilized GST pull-down assay combined with mass spectrometry to identify the interacting proteins of the RAGE intracellular domain (C-terminal 43 amino acid of RAGE). Overall four RAGE interacting proteins, including Rab31, were identified with scores over 160. Rab31 was detected in three ß-cell lines and confirmed to have interacted with RAGE via co-immunoprecipitation and immunostaining assays. This interaction was further enhanced by glycation-serum (GS) stimulation due to membrane distribution of Rab31 following treatment with GS. We further confirmed that Rab31 promoted RAGE endocytosis and inhibited GS-induced ß-cell apoptosis through the pAKT/BCL2 pathway. These findings reveal a new RAGE interaction protein Rab31 that prevents AGE/RAGE-induced pancreatic ß-cell apoptosis. Rab31 is therefore a promising therapeutic target for preserving functional ß cells under diabetes conditions.

miR-25 and miR-92b regulate insulin biosynthesis and pancreatic ß-cell apoptosis.

PURPOSE: Pancreatic ß-cell failure is a central hallmark of the pathogenesis of diabetes mellitus; however, the molecular basis underlying chronic inflammation-caused ß-cell failure remains unclear. This study reported here specifically assessed the association between miR-25/miR-92b family and ß-cell failure in diabetes. METHODS: IL-1ß and two additional ER stress activators, palmitate and tunicamycin were applied to evaluate the expression level miR-25 by Taqman® RT-PCR. Glucose- and potassium-stimulated insulin secretion assays were performed to assess ß-cell function. Dual-luciferase activity, and western blotting assays were utilized for miR-25 target gene verification. CCK-8 and TUNEL staining were used to evaluate ß-cell viability and apoptosis. RESULTS: miRNA ChIP identified the increased level of miR-25 in INS-1 cells by IL-1ß treatment. Expression levels of miR-25 were significantly upregulated with the treatment of IL-1ß, palmitate or tunicamycin in both INS-1 cells and human islets. Ectopic elevation of miR-25 recapitulated most featured ß-cell defects caused by IL-1ß, including inhibition of insulin biosynthesis and increased ß-cell apoptosis. These detrimental effects of miR-25 relied on its seed sequence recognition and repressed expression of its target genes Neurod1 and Mcl1. The miR-25/NEUROD1 axis reduced insulin biosynthesis via transcriptional regulation of ß-cell specific genes. The miR-25/MCL1 axis caused ß-cell apoptosis in a CASPASE-3/PARP1-dependent manner. Comparable impairments were generated by miR-92b and miR-25, emphasizing the redundant biological roles of miRNA family members with the same seed sequence. CONCLUSION: MiR-25/miR-92b family plays a major role in ß-cell failure occurring under inflammation and diabetes states.

Protocol for in vivo and ex vivo assessments of glucose-stimulated insulin secretion in mouse islet ß cells.

Pancreatic islet ß cells secrete insulin in a biphasic manner when sensing high blood glucose level. This protocol describes the evaluation of different phases of insulin secretion, as well as basal, glucose-stimulated and total insulin secretion abilities, thereby enabling precise assessment of ß cell function both in vivo and ex vivo. The in vivo assay consists of intravenous tube imbedding surgery and hyperglycemic clamp. The ex vivo assay consists of islet isolation, dynamic perfusion and static immersion. For complete details on the use and execution of this protocol, please refer to Sun et al. (2021).

M1 macrophage-derived exosomes impair beta cell insulin secretion via miR-212-5p by targeting SIRT2 and inhibiting Akt/GSK-3ß/ß-catenin pathway in mice.

AIMS/HYPOTHESIS: Macrophage levels are elevated in pancreatic islets, and the resulting inflammatory response is a major contributor to beta cell failure during obesity and type 2 diabetes mellitus. Previous studies by us and others have reported that exosomes released by macrophages play important roles in mediating cell-to-cell communication, and represent a class of inflammatory factors involved in the inflammatory process associated with type 2 diabetes mellitus. However, to date, no reports have demonstrated the effect of macrophage-derived exosomes on beta cells, and little is known regarding their underlying mechanisms in beta cell injury. Thus, we aimed to study the impact of macrophage-derived exosomes on islet beta cell injury in vitro and in vivo. METHODS: The phenotypic profiles of islet-resident macrophages were analysed in C57BL/6J mice fed a high-fat diet (HFD). Exosomes were collected from the medium of cultured bone marrow-derived macrophages (BMDMs) and from isolated islet-resident macrophages of HFD-fed mice (HFD-Exos). The role of exosomes secreted by inflammatory M1 phenotype BMDMs (M1-Exos) and HFD-Exos on beta cell function was assessed. An miRNA microarray and quantitative real-time PCR (qPCR) were conducted to test the level of M1-Exos-derived miR-212-5p in beta cells. Then, miR-212-5p was overexpressed or inhibited in M1-Exos or beta cells to determine its molecular and functional impact. RESULTS: M1-polarised macrophages were enriched in the islets of obese mice. M1 macrophages and islet-resident macrophages of HFD-fed mice impaired beta cell insulin secretion in an exosome-dependent manner. miR-212-5p was notably upregulated in M1-Exos and HFD-Exos. Enhancing the expression of miR-212-5p impaired beta cell insulin secretion. Blocking miR-212-5p elicited a significant improvement in M1-Exos-mediated beta cell insulin secretion during injury. Mechanistically, M1-Exos mediated an intercellular transfer of the miR-212-5p, targeting the sirtuin 2 gene and regulating the Akt/GSK-3ß/ß-catenin pathway in recipient beta cells to restrict insulin secretion. CONCLUSIONS/INTERPRETATION: A novel exosome-modulated mechanism was delineated for macrophage-beta cell crosstalk that drove beta cell dysfunction and should be explored for its therapeutic utility.

Exploiting D2 receptor ß-arrestin2-biased signalling to suppress tumour growth of pituitary adenomas.

BACKGROUND AND PURPOSE: Dopamine agonists targeting D2 receptor have been used for decades in treating pituitary adenomas. There has been little clear evidence implicating the canonical G protein signalling as the mechanism by which D2 receptor suppresses the growth of pituitary tumours. We hypothesize that ß-arrestin2-dependent signalling is the molecular mechanism dictating D2 receptor inhibitory effects on pituitary tumour growth. EXPERIMENTAL APPROACH: The involvement of G protein and ß-arrestin2 in bromocriptine-mediated growth suppression in rat MMQ and GH3 tumour cells was assessed. The anti-growth effect of a ß-arrestin2-biased agonist, UNC9994, was tested in cultured cells, tumour-bearing nude mice and primary cultured human pituitary adenomas. The effect of G protein signalling on tumour growth was also analysed by using a G protein-biased agonist, MLS1547, and a Gßγ inhibitor, gallein, in vitro. KEY RESULTS: ß-arrestin2 signalling but not G protein pathways mediated the suppressive effect of bromocriptine on pituitary tumour growth. UNC9994 inhibited pituitary tumour cell growth in vitro and in vivo. The suppressive function of UNC9994 was obtained by inducing intracellular reactive oxygen species generation through downregulating mitochondrial complex I subunit NDUFA1. The effects of Gαi/o signalling and Gßγ signalling via D2 receptor on pituitary tumour growth were cell-type-dependent. CONCLUSION AND IMPLICATIONS: Given the very low expression of Gαi/o proteins in pituitary tumours and the complexity of the responses of pituitary tumours to G protein signalling pathways, our study reveals D2 receptor ß-arrestin2-biased ligand may be a more promising choice to treat pituitary tumours with improved therapeutic selectivity.

Expression of miRNA-29 in Pancreatic ß Cells Promotes Inflammation and Diabetes via TRAF3.

Type 2 diabetes mellitus (T2DM) is recognized as a chronic, low-grade inflammatory disease characterized by insulin resistance and pancreatic ß cell dysfunction; however, the underlying molecular mechanism remains unclear. Here, we report a key ß cell-macrophage crosstalk pathway mediated by the miRNA-29-TNF-receptor-associated factor 3 (TRAF3) axis. ß cell-specific transgenic miR-29a/b/c mice are predisposed to develop glucose intolerance and insulin resistance when fed a high-fat diet (HFD). The metabolic effect of ß cell miR-29 is largely mediated through macrophages because either depletion of macrophages or reconstitution with miR-29-signaling defective bone marrow improves metabolic parameters in the transgenic mice. Mechanistically, our data show that miR-29 promotes the recruitment and activation of circulating monocytes and macrophages and, hence, inflammation, via miR-29 exosomes in a TRAF3-dependent manner. Our results demonstrate the ability of ß cells to modulate the systemic inflammatory tone and glucose homeostasis via miR-29 in response to nutrient overload.

Radiotherapy Enhancement for Human Pancreatic Carcinoma Using a Peptide-Gold Nanoparticle Hybrid.

Pancreatic ductal adenocarcinoma (PDAC) is radioresistant. Due to their strong X-ray absorption capacity, gold nanoparticles (AuNPs) have been used as radiosensitizers for cancer therapeutics. Herein, we describe a novel conjugate complex consisting of a peptide for targeting plectin-1 (PTP) specifically expressed on the PDAC cell membrane and AuNPs, termed AuNP-PTP, to be used for PDAC radiotherapy in vitro and in vivo. Previous studies revealed that compared with unmodified AuNPs, AuNP-PTP along with relevant low-energy X-ray irradiation of 6 MV at a dose of 2 Gy (RF) increased the targeting efficiency and induced apoptosis in treated PANC-1 cells and tumours. Importantly, extensive histopathological examination did not reveal evidence of acute or chronic injury in mice due to AuNPs or AuNP-PTP for up to six weeks despite the presence of X-ray exposure. The delicate AuNP-PTP hybrid provides a novel strategy to enhance radiotherapy efficiency in PDAC treatment.

Ets-1 deficiency alleviates nonalcoholic steatohepatitis via weakening TGF-ß1 signaling-mediated hepatocyte apoptosis.

Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis (NASH) and contributes to liver injury, fibrosis, and inflammation. However, the molecular mechanisms underlying excessive hepatocyte apoptosis in NASH remain largely unknown. This study aimed to explore whether and how the v-ets avian erythroblastosis virus E26 oncogene homolog 1 (Ets-1) is involved in diet-induced hepatocyte apoptosis in mice. The study found that the expression level of hepatic Ets-1 was elevated in a NASH mouse model as a result of the activation of transforming growth factor beta1 (TGF-ß1) signaling. In the presence of TGF-ß1, phosphorylated mothers against decapentaplegic homolog 2/3 (p-Smad2/3) translocated to the binding sites of the Ets-1 promoter to upregulate the expression of Ets-1 in primary hepatocytes. In addition, Ets-1 bound directly to phosphorylated Smad3 (p-Smad3), thereby preventing the ubiquitination and proteasomal degradation of p-Smad3 and enhancing the activity of TGF-ß1/Smad3 signaling. Consequently, elevated Ets-1 stimulated TGF-ß1-induced hepatocyte apoptosis. However, Ets-1 knockdown alleviated diet-induced hepatocyte apoptosis and NASH with reduced liver injury, inflammation, and fibrosis. Taken together, Ets-1 had an adverse impact on hepatocyte survival under TGF-ß1 treatment and accelerated the development of NASH in mice.

Ets1-Mediated Acetylation of FoxO1 Is Critical for Gluconeogenesis Regulation during Feed-Fast Cycles.

The homeostatic balance of hepatic glucose uptake and production is exquisitely controlled by hormonal signals during feed-fast cycles. FoxO1, a transcription factor that functions in the regulation of glucose homeostasis, undergoes posttranslational modifications, such as acetylation, in response to hormonal signals, yet the mechanism remains poorly elucidated. Through expression profiling of 324 co-factors of CBP, a well-known acetyl-transferase of FoxO1, we identify Ets1 as a modulator of FoxO1 acetylation that is highly associated with feed-fast cycles. Mechanistic assays suggest that Ets1 enhances FoxO1 acetylation through the formation of a complex with CBP, which further promotes FoxO1 nuclear exclusion and inhibits its binding to gluconeogenic promoters. Functional studies further reveal that Ets1 inhibits gluconeogenesis under physiological and diabetes statuses, while the hyperinsulinemic-euglycemic clamp assay suggests hepatocyte Ets1 knockout mice have enhanced hepatic glucose production. Our study identifies Ets1 as an enhancer of FoxO1 acetylation and a repressor of hepatic gluconeogenesis in response to hormonal signals.

Drosophila Histone Demethylase KDM5 Regulates Social Behavior through Immune Control and Gut Microbiota Maintenance.

Loss-of-function mutations in the histone demethylases KDM5A, KDM5B, or KDM5C are found in intellectual disability (ID) and autism spectrum disorders (ASD) patients. Here, we use the model organism Drosophila melanogaster to delineate how KDM5 contributes to ID and ASD. We show that reducing KDM5 causes intestinal barrier dysfunction and changes in social behavior that correlates with compositional changes in the gut microbiota. Therapeutic alteration of the dysbiotic microbiota through antibiotic administration or feeding with a probiotic Lactobacillus strain partially rescues the behavioral, lifespan, and cellular phenotypes observed in kdm5-deficient flies. Mechanistically, KDM5 was found to transcriptionally regulate component genes of the immune deficiency (IMD) signaling pathway and subsequent maintenance of host-commensal bacteria homeostasis in a demethylase-dependent manner. Together, our study uses a genetic approach to dissect the role of KDM5 in the gut-microbiome-brain axis and suggests that modifying the gut microbiome may provide therapeutic benefits for ID and ASD patients.