metGWAS 1.0: an R workflow for network-driven over-representation analysis between independent metabolomic and meta-genome-wide association studies.

MOTIVATION: The method of genome-wide association studies (GWAS) and metabolomics combined provide an quantitative approach to pinpoint metabolic pathways and genes linked to specific diseases; however, such analyses require both genomics and metabolomics datasets from the same individuals/samples. In most cases, this approach is not feasible due to high costs, lack of technical infrastructure, unavailability of samples, and other factors. Therefore, an unmet need exists for a bioinformatics tool that can identify gene loci-associated polymorphic variants for metabolite alterations seen in disease states using standalone metabolomics. RESULTS: Here, we developed a bioinformatics tool, metGWAS 1.0, that integrates independent GWAS data from the GWAS database and standalone metabolomics data using a network-based systems biology approach to identify novel disease/trait-specific metabolite-gene associations. The tool was evaluated using standalone metabolomics datasets extracted from two metabolomics-GWAS case studies. It discovered both the observed and novel gene loci with known single nucleotide polymorphisms when compared to the original studies. AVAILABILITY AND IMPLEMENTATION: The developed metGWAS 1.0 framework is implemented in an R pipeline and available at: https://github.com/saifurbd28/metGWAS-1.0.

The magnesium transporter NIPAL1 is a pancreatic islet-expressed protein that conditionally impacts insulin secretion.

Type 2 diabetes is a chronic metabolic disease characterized by pancreatic ß-cell dysfunction and peripheral insulin resistance. Among individuals with type 2 diabetes, ∼30% exhibit hypomagnesemia. Hypomagnesemia has been linked to insulin resistance through reduced tyrosine kinase activity of the insulin receptor; however, its impact on pancreatic ß-cell function is unknown. In this study, through analysis of several single-cell RNA-sequencing data sets in tandem with quantitative PCR validation in both murine and human islets, we identified NIPAL1 (NIPA-like domain containing 1), encoding a magnesium influx transporter, as an islet-enriched gene. A series of immunofluorescence experiments confirmed NIPAL1's magnesium-dependent expression and that it specifically localizes to the Golgi in Min6-K8 cells, a pancreatic ß-cell-like cell line (mouse insulinoma 6 clone K8). Under varying magnesium concentrations, NIPAL1 knockdown decreased both basal insulin secretion and total insulin content; in contrast, its overexpression increased total insulin content. Although the expression, distribution, and magnesium responsiveness of NIPAL1 in α-TC6 glucagonoma cells (a pancreatic α-cell line) were similar to the observations in Min6-K8 cells, no effect was observed on glucagon secretion in α-TC6 cells under the conditions studied. Overall, these results suggest that NIPAL1 expression is regulated by extracellular magnesium and that down-regulation of this transporter decreases glucose-stimulated insulin secretion and intracellular insulin content, particularly under conditions of hypomagnesemia.

Intensive lactation among women with recent gestational diabetes significantly alters the early postpartum circulating lipid profile: the SWIFT study.

BACKGROUND: Women with a history of gestational diabetes mellitus (GDM) have a 7-fold higher risk of developing type 2 diabetes (T2D). It is estimated that 20-50% of women with GDM history will progress to T2D within 10 years after delivery. Intensive lactation could be negatively associated with this risk, but the mechanisms behind a protective effect remain unknown. METHODS: In this study, we utilized a prospective GDM cohort of 1010 women without T2D at 6-9 weeks postpartum (study baseline) and tested for T2D onset up to 8 years post-baseline (n=980). Targeted metabolic profiling was performed on fasting plasma samples collected at both baseline and follow-up (1-2 years post-baseline) during research exams in a subset of 350 women (216 intensive breastfeeding, IBF vs. 134 intensive formula feeding or mixed feeding, IFF/Mixed). The relationship between lactation intensity and circulating metabolites at both baseline and follow-up were evaluated to discover underlying metabolic responses of lactation and to explore the link between these metabolites and T2D risk. RESULTS: We observed that lactation intensity was strongly associated with decreased glycerolipids (TAGs/DAGs) and increased phospholipids/sphingolipids at baseline. This lipid profile suggested decreased lipogenesis caused by a shift away from the glycerolipid metabolism pathway towards the phospholipid/sphingolipid metabolism pathway as a component of the mechanism underlying the benefits of lactation. Longitudinal analysis demonstrated that this favorable lipid profile was transient and diminished at 1-2 years postpartum, coinciding with the cessation of lactation. Importantly, when stratifying these 350 women by future T2D status during the follow-up (171 future T2D vs. 179 no T2D), we discovered that lactation induced robust lipid changes only in women who did not develop incident T2D. Subsequently, we identified a cluster of metabolites that strongly associated with future T2D risk from which we developed a predictive metabolic signature with a discriminating power (AUC) of 0.78, superior to common clinical variables (i.e., fasting glucose, AUC 0.56 or 2-h glucose, AUC 0.62). CONCLUSIONS: In this study, we show that intensive lactation significantly alters the circulating lipid profile at early postpartum and that women who do not respond metabolically to lactation are more likely to develop T2D. We also discovered a 10-analyte metabolic signature capable of predicting future onset of T2D in IBF women. Our findings provide novel insight into how lactation affects maternal metabolism and its link to future diabetes onset. TRIAL REGISTRATION: ClinicalTrials.gov NCT01967030 .

Holo-lipocalin-2-derived siderophores increase mitochondrial ROS and impair oxidative phosphorylation in rat cardiomyocytes.

Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by N-acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.

Amino acid and lipid metabolism in post-gestational diabetes and progression to type 2 diabetes: A metabolic profiling study.

BACKGROUND: Women with a history of gestational diabetes mellitus (GDM) have a 7-fold higher risk of developing type 2 diabetes (T2D) during midlife and an elevated risk of developing hypertension and cardiovascular disease. Glucose tolerance reclassification after delivery is recommended, but fewer than 40% of women with GDM are tested. Thus, improved risk stratification methods are needed, as is a deeper understanding of the pathology underlying the transition from GDM to T2D. We hypothesize that metabolites during the early postpartum period accurately distinguish risk of progression from GDM to T2D and that metabolite changes signify underlying pathophysiology for future disease development. METHODS AND FINDINGS: The study utilized fasting plasma samples collected from a well-characterized prospective research study of 1,035 women diagnosed with GDM. The cohort included racially/ethnically diverse pregnant women (aged 20-45 years-33% primiparous, 37% biparous, 30% multiparous) who delivered at Kaiser Permanente Northern California hospitals from 2008 to 2011. Participants attended in-person research visits including 2-hour 75-g oral glucose tolerance tests (OGTTs) at study baseline (6-9 weeks postpartum) and annually thereafter for 2 years, and we retrieved diabetes diagnoses from electronic medical records for 8 years. In a nested case-control study design, we collected fasting plasma samples among women without diabetes at baseline (n = 1,010) to measure metabolites among those who later progressed to incident T2D or did not develop T2D (non-T2D). We studied 173 incident T2D cases and 485 controls (pair-matched on BMI, age, and race/ethnicity) to discover metabolites associated with new onset of T2D. Up to 2 years post-baseline, we analyzed samples from 98 T2D cases with 239 controls to reveal T2D-associated metabolic changes. The longitudinal analysis tracked metabolic changes within individuals from baseline to 2 years of follow-up as the trajectory of T2D progression. By building prediction models, we discovered a distinct metabolic signature in the early postpartum period that predicted future T2D with a median discriminating power area under the receiver operating characteristic curve of 0.883 (95% CI 0.820-0.945, p < 0.001). At baseline, the most striking finding was an overall increase in amino acids (AAs) as well as diacyl-glycerophospholipids and a decrease in sphingolipids and acyl-alkyl-glycerophospholipids among women with incident T2D. Pathway analysis revealed up-regulated AA metabolism, arginine/proline metabolism, and branched-chain AA (BCAA) metabolism at baseline. At follow-up after the onset of T2D, up-regulation of AAs and down-regulation of sphingolipids and acyl-alkyl-glycerophospholipids were sustained or strengthened. Notably, longitudinal analyses revealed only 10 metabolites associated with progression to T2D, implicating AA and phospholipid metabolism. A study limitation is that all of the analyses were performed with the same cohort. It would be ideal to validate our findings in an independent longitudinal cohort of women with GDM who had glucose tolerance tested during the early postpartum period. CONCLUSIONS: In this study, we discovered a metabolic signature predicting the transition from GDM to T2D in the early postpartum period that was superior to clinical parameters (fasting plasma glucose, 2-hour plasma glucose). The findings suggest that metabolic dysregulation, particularly AA dysmetabolism, is present years prior to diabetes onset, and is revealed during the early postpartum period, preceding progression to T2D, among women with GDM. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01967030.

GABA promotes ß-cell proliferation, but does not overcome impaired glucose homeostasis associated with diet-induced obesity.

γ-Aminobutyric acid (GABA) administration has been shown to increase ß-cell mass, leading to a reversal of type 1 diabetes in mice. Whether GABA has any effect on ß cells of healthy and prediabetic/glucose-intolerant obese mice remains unknown. In the present study, we show that oral GABA administration ( ad libitum) to mice indeed increased pancreatic ß-cell mass, which led to a modest enhancement in insulin secretion and glucose tolerance. However, GABA treatment did not further increase insulin-positive islet area in high fat diet-fed mice and was unable to prevent or reverse glucose intolerance and insulin resistance. Mechanistically, whether in vivo or in vitro, GABA treatment increased ß-cell proliferation. In vitro, the effect was shown to be mediated via the GABAA receptor. Single-cell RNA sequencing analysis revealed that GABA preferentially up-regulated pathways linked to ß-cell proliferation and simultaneously down-regulated those networks required for other processes, including insulin biosynthesis and metabolism. Interestingly, single-cell differential expression analysis revealed GABA treatment gave rise to a distinct subpopulation of ß cells with a unique transcriptional signature, including urocortin 3 ( ucn3), wnt4, and hepacam2. Taken together, this study provides new mechanistic insight into the proliferative nature of GABA but suggests that ß-cell compensation associated with prediabetes overlaps with, and negates, its proliferative effects.-Untereiner, A., Abdo, S., Bhattacharjee, A., Gohil, H., Pourasgari, F., Ibeh, N., Lai, M., Batchuluun, B., Wong, A., Khuu, N., Liu, Y., Al Rijjal, D., Winegarden, N., Virtanen, C., Orser, B. A., Cabrera, O., Varga, G., Rocheleau, J., Dai, F. F., Wheeler, M. B. GABA promotes ß-cell proliferation, but does not overcome impaired glucose homeostasis associated with diet-induced obesity.

γ-aminobutyric acid stimulates ß-cell proliferation through the mTORC1/p70S6K pathway, an effect amplified by Ly49, a novel γ-aminobutyric acid type A receptor positive allosteric modulator.

AIM: To examine the mechanism of action of γ-aminobutyric acid (GABA) on ß-cell proliferation and investigate if co-treatment with Ly49, a novel GABA type A receptor positive allosteric modulator (GABAA -R PAM), amplifies this effect. METHODS: Human or mouse islets were co-treated for 4-5 days with GABA and selected receptor or cell signalling pathway modulators. Immunofluorescence was used to determine protein co-localization, cell number or proliferation, and islet size. Osmotic minipumps were surgically implanted in mice to assess Ly49 effects on pancreatic ß-cells. RESULTS: Amplification of GABAA -R signalling enhanced GABA-stimulated ß-cell proliferation in cultured mouse islets. Co-treatment of GABA with an inhibitor specific for PI3K, mTORC1/2, or p70S6K, abolished GABA-stimulated ß-cell proliferation in mouse and human islets. Nuclear p-AktSer473 and p-p70S6KThr421/Ser424 expression in pancreatic ß-cells was increased in GABA-treated mice compared with vehicle-treated mice, an effect augmented with GABA and Ly49 co-treatment. Mice co-treated with GABA and Ly49 exhibited enhanced ß-cell area and proliferation compared with GABA-treated mice. Furthermore, S961 injection (an insulin receptor antagonist) resulted in enhanced plasma insulin in GABA and Ly49 co-treated mice compared with GABA-treated mice. Importantly, GABA co-treated with Ly49 increased ß-cell proliferation in human islets providing a potential application for human subjects. CONCLUSIONS: We show that GABA stimulates ß-cell proliferation via the PI3K/mTORC1/p70S6K pathway in both mouse and human islets. Furthermore, we show that Ly49 enhances the ß-cell regenerative effects of GABA, showing potential in the intervention of diabetes.

The discovery of novel predictive biomarkers and early-stage pathophysiology for the transition from gestational diabetes to type 2 diabetes.

AIMS/HYPOTHESIS: Gestational diabetes mellitus (GDM) affects up to 20% of pregnancies, and almost half of the women affected progress to type 2 diabetes later in life, making GDM the most significant risk factor for the development of future type 2 diabetes. An accurate prediction of future type 2 diabetes risk in the early postpartum period after GDM would allow for timely interventions to prevent or delay type 2 diabetes. In addition, new targets for interventions may be revealed by understanding the underlying pathophysiology of the transition from GDM to type 2 diabetes. The aim of this study is to identify both a predictive signature and early-stage pathophysiology of the transition from GDM to type 2 diabetes. METHODS: We used a well-characterised prospective cohort of women with a history of GDM pregnancy, all of whom were enrolled at 6-9 weeks postpartum (baseline), were confirmed not to have diabetes via 2 h 75 g OGTT and tested anually for type 2 diabetes on an ongoing basis (2 years of follow-up). A large-scale targeted lipidomic study was implemented to analyse ~1100 lipid metabolites in baseline plasma samples using a nested pair-matched case-control design, with 55 incident cases matched to 85 non-case control participants. The relationships between the concentrations of baseline plasma lipids and respective follow-up status (either type 2 diabetes or no type 2 diabetes) were employed to discover both a predictive signature and the underlying pathophysiology of the transition from GDM to type 2 diabetes. In addition, the underlying pathophysiology was examined in vivo and in vitro. RESULTS: Machine learning optimisation in a decision tree format revealed a seven-lipid metabolite type 2 diabetes predictive signature with a discriminating power (AUC) of 0.92 (87% sensitivity, 93% specificity and 91% accuracy). The signature was highly robust as it includes 45-fold cross-validation under a high confidence threshold (1.0) and binary output, which together minimise the chance of data overfitting and bias selection. Concurrent analysis of differentially expressed lipid metabolite pathways uncovered the upregulation of α-linolenic/linoleic acid metabolism (false discovery rate [FDR] 0.002) and fatty acid biosynthesis (FDR 0.005) and the downregulation of sphingolipid metabolism (FDR 0.009) as being strongly associated with the risk of developing future type 2 diabetes. Focusing specifically on sphingolipids, the downregulation of sphingolipid metabolism using the pharmacological inhibitors fumonisin B1 (FB1) and myriocin in mouse islets and Min6 K8 cells (a pancreatic beta-cell like cell line) significantly impaired glucose-stimulated insulin secretion but had no significant impact on whole-body glucose homeostasis or insulin sensitivity. CONCLUSIONS/INTERPRETATION: We reveal a novel predictive signature and associate reduced sphingolipids with the pathophysiology of transition from GDM to type 2 diabetes. Attenuating sphingolipid metabolism in islets impairs glucose-stimulated insulin secretion.

Correction to: The discovery of novel predictive biomarkers and early-stage pathophysiology for the transition from gestational diabetes to type 2 diabetes.

Unfortunately, the graph in Fig. 5f became misaligned during typesetting.

3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) prevents high fat diet-induced insulin resistance via maintenance of hepatic lipid homeostasis.

AIM: Omega-3 fatty acid ethyl ester supplements, available by prescription, are common in the treatment of dyslipidaemia in humans. Recent studies show that 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), a metabolite formed from fish oil supplementation, was able to prevent and reverse high fat diet (HFD)-induced fatty liver in mice. In the present study, we investigated the underlying molecular mechanisms responsible for CMPF's hepatic lipid-lowering effects. MATERIALS AND METHODS: CD1 male mice were i.p. injected with CMPF (dosage, 6 mg/kg) for 7 days, followed by 5 weeks of a 60% HFD to induce a fatty liver phenotype. Metabolic parameters, liver morphology, lipid content, protein expression and microarray analysis were assessed. We also utilized primary hepatocytes, an in vitro model, to further investigate the direct effects of CMPF on hepatic lipid utilization and biosynthesis. RESULTS: CMPF-treated mice display enhanced hepatic lipid clearance while hepatic lipid storage is prevented, thereby protecting against liver lipid accumulation and development of HFD-induced hepatic insulin resistance. Mechanistically, as CMPF enters the liver, it acts as an allosteric acetyl-coA carboxylase (ACC) inhibitor, which directly induces both fatty acid oxidation and hepatic production of fibroblast growth factor 21 (FGF21). A feed-back loop is initiated by CMPF, which exists between ACC inhibition, fatty acid oxidation and production of FGF21. As a consequence, an adaptive decrease in Insig2/SREBP-1c/FAS protein expression results in priming of the liver to prevent a HFD-induced fatty liver phenotype. CONCLUSION: CMPF is a potential driver of hepatic lipid metabolism, preventing diet-induced hepatic lipid deposition and insulin resistance in the long term.

IKKß inhibition prevents fat-induced beta cell dysfunction in vitro and in vivo in rodents.

AIMS/HYPOTHESIS: We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase ß (IKKß), which is activated by oxidative stress, is also implicated. METHODS: Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKß inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKß) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice. RESULTS: 48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet-1 h-1) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet-1 h-1] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKß also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet-1 h-1] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate). CONCLUSIONS/INTERPRETATION: Our results demonstrate a causal role for IKKß in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion.

Characterization of Zinc Influx Transporters (ZIPs) in Pancreatic ß Cells: ROLES IN REGULATING CYTOSOLIC ZINC HOMEOSTASIS AND INSULIN SECRETION.

Zinc plays an essential role in the regulation of pancreatic ß cell function, affecting important processes including insulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in the zinc efflux transport protein ZnT8 have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx transporters (ZIPs). In this study, we examined the ß cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human and mouse islets and MIN6 ß cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic ß cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this dysregulated cytosolic zinc homeostasis led to impaired insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast two-hybrid assay. Knock-down of ZIP6 but not ZIP7 in MIN6 ß cells impaired the protective effects of GLP-1 on fatty acid-induced cell apoptosis, possibly via reduced activation of the p-ERK pathway. Therefore, our data suggest that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in ß cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on ß cell survival.

Progesterone receptor membrane component 1 is a functional part of the glucagon-like peptide-1 (GLP-1) receptor complex in pancreatic ß cells.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic ß cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 ß cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in ß cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor-PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of ß cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.

Deletion of ARNT/HIF1ß in pancreatic beta cells does not impair glucose homeostasis in mice, but is associated with defective glucose sensing ex vivo.

AIMS/HYPOTHESIS: It has been suggested that the transcription factor ARNT/HIF1ß is critical for maintaining in vivo glucose homeostasis and pancreatic beta cell glucose-stimulated insulin secretion (GSIS). Our goal was to gain more insights into the metabolic defects seen after the loss of ARNT/HIF1ß in beta cells. METHODS: The in vivo and in vitro consequences of the loss of ARNT/HIF1ß were investigated in beta cell specific Arnt/Hif1ß knockout mice (ß-Arnt (fl/fl/Cre) mice). RESULTS: The only in vivo defects found in ß-Arnt (fl/fl/Cre) mice were significant increases in the respiratory exchange ratio and in vivo carbohydrate oxidation, and a decrease in lipid oxidation. The mitochondrial oxygen consumption rate was unaltered in mouse ß-Arnt (fl/fl/Cre) islets upon glucose stimulation. ß-Arnt (fl/fl/Cre) islets had an impairment in the glucose-stimulated increase in Ca(2+) signalling and a reduced insulin secretory response to glucose in the presence of KCl and diazoxide. The glucose-stimulated increase in the NADPH/NADP(+) ratio was reduced in ß-Arnt (fl/fl/Cre) islets. The reduced GSIS and NADPH/NADP(+) levels in ß-Arnt (fl/fl/Cre) islets could be rescued by treatment with membrane-permeable tricarboxylic acid intermediates. Small interfering (si)RNA mediated knockdown of ARNT/HIF1ß in human islets also inhibited GSIS. These results suggest that the regulation of GSIS by the KATP channel-dependent and -independent pathways is affected by the loss of ARNT/HIF1ß in islets. CONCLUSIONS/INTERPRETATION: This study provides three new insights into the role of ARNT/HIF1ß in beta cells: (1) ARNT/HIF1ß deletion in mice impairs GSIS ex vivo; (2) ß-Arnt (fl/fl/Cre) mice have an increased respiratory exchange ratio; and (3) ARNT/HIF1ß is required for GSIS in human islets.

LKB1 couples glucose metabolism to insulin secretion in mice.

AIMS/HYPOTHESIS: Precise regulation of insulin secretion by the pancreatic beta cell is essential for the maintenance of glucose homeostasis. Insulin secretory activity is initiated by the stepwise breakdown of ambient glucose to increase cellular ATP via glycolysis and mitochondrial respiration. Knockout of Lkb1, the gene encoding liver kinase B1 (LKB1) from the beta cell in mice enhances insulin secretory activity by an undefined mechanism. Here, we sought to determine the molecular basis for how deletion of Lkb1 promotes insulin secretion. METHODS: To explore the role of LKB1 on individual steps in the insulin secretion pathway, we used mitochondrial functional analyses, electrophysiology and metabolic tracing coupled with by gas chromatography and mass spectrometry. RESULTS: Beta cells lacking LKB1 surprisingly display impaired mitochondrial metabolism and lower ATP levels following glucose stimulation, yet compensate for this by upregulating both uptake and synthesis of glutamine, leading to increased production of citrate. Furthermore, under low glucose conditions, Lkb1(-/-) beta cells fail to inhibit acetyl-CoA carboxylase 1 (ACC1), the rate-limiting enzyme in lipid synthesis, and consequently accumulate NEFA and display increased membrane excitability. CONCLUSIONS/INTERPRETATION: Taken together, our data show that LKB1 plays a critical role in coupling glucose metabolism to insulin secretion, and factors in addition to ATP act as coupling intermediates between feeding cues and secretion. Our data suggest that beta cells lacking LKB1 could be used as a system to identify additional molecular events that connect metabolism to cellular excitation in the insulin secretion pathway.

SDF2L1 interacts with the ER-associated degradation machinery and retards the degradation of mutant proinsulin in pancreatic ß-cells.

Stromal cell-derived factor 2-like 1 (SDF2L1) is an endoplasmic reticulum (ER)-localized protein whose function is undefined. Here we show that SDF2L1 protein levels are increased in response to ER stress-inducing compounds, but not other cell stressors that we tested in insulinoma cell lines. SDF2L1 protein levels were also induced by expression of misfolded proinsulin in insulinoma cells and in islets from diabetic mice. Immunoprecipitation and binding assays demonstrated that SDF2L1 interacts with the ER chaperone GRP78/BiP, the ER-associated degradation (ERAD) machinery and with misfolded proinsulin. Unexpectedly, knockdown of SDF2L1 in INS-1 (insulin 2 C96Y-GFP) cells increased the degradation kinetics of mutant proinsulin, suggesting that SDF2L1 regulates substrate availability for the ERAD system. We suggest that SDF2L1 increases the time that misfolded proteins have to achieve a correctly folded conformation and thus that SDF2L1 can act as a buffer for substrate availability for ERAD in pancreatic ß-cells.

Proinsulin intermolecular interactions during secretory trafficking in pancreatic ß cells.

Classically, exit from the endoplasmic reticulum (ER) is rate-limiting for secretory protein trafficking because protein folding/assembly occurs there. In this study, we have exploited "hPro-CpepSfGFP," a human proinsulin bearing "superfolder" green fluorescent C-peptide expressed in pancreatic ß cells where it is processed to human insulin and CpepSfGFP. Remarkably, steady-state accumulation of hPro-CpepSfGFP and endogenous proinsulin is in the Golgi region, as if final stages of protein folding/assembly were occurring there. The Golgi regional distribution of proinsulin is dynamic, influenced by fasting/refeeding, and increased with ß cell zinc deficiency. However, coexpression of ER-entrapped mutant proinsulin-C(A7)Y shifts the steady-state distribution of wild-type proinsulin to the ER. Endogenous proinsulin coprecipitates with hPro-CpepSfGFP and even more so with hProC(A7)Y-CpepSfGFP. Using Cerulean and Venus-tagged proinsulins, we find that both WT-WT and WT-mutant proinsulin pairs exhibit FRET. The data demonstrate that wild-type proinsulin dimerizes within the ER but accumulates at a poorly recognized slow step within the Golgi region, reflecting either slow kinetics of proinsulin hexamerization, steps in formation of nascent secretory granules, or other unknown molecular events. However, in the presence of ongoing misfolding of a subpopulation of proinsulin in ß cells, the rate-limiting step in transport of the remaining proinsulin shifts to the ER.

Stage-specific signaling through TGFß family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells.

The generation of insulin-producing ß-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFß family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.

Crosstalk between GABAA receptors in astrocytes and neurons triggered by general anesthetic drugs.

General anesthetic drugs cause cognitive deficits that persist after the drugs have been eliminated. Astrocytes may contribute to such cognition-impairing effects through the release of one or more paracrine factors that increase a tonic inhibitory conductance generated by extrasynaptic γ-aminobutyric acid type A (GABAA) receptors in hippocampal neurons. The mechanisms underlying this astrocyte-to-neuron crosstalk remain unknown. Interestingly, astrocytes express anesthetic-sensitive GABAA receptors. Here, we tested the hypothesis that anesthetic drugs activate astrocytic GABAA receptors to initiate crosstalk leading to a persistent increase in extrasynaptic GABAA receptor function in neurons. We also investigated the signaling pathways in neurons and aimed to identify the paracrine factors released from astrocytes. Astrocytes and neurons from mice were grown in primary cell cultures and studied using in vitro electrophysiological and biochemical assays. We discovered that the commonly used anesthetics etomidate (injectable) and sevoflurane (inhaled) stimulated astrocytic GABAA receptors, which in turn promoted the release paracrine factors, that increased the tonic current in neurons via a p38 MAPK-dependent signaling pathway. The increase in tonic current was mimicked by exogenous IL-1ß and abolished by blocking IL-1 receptors; however, unexpectedly, IL-1ß and other cytokines were not detected in astrocyte-conditioned media. In summary, we have identified a novel form of crosstalk between GABAA receptors in astrocytes and neurons that engages a p38 MAPK-dependent pathway. Brief commentary BACKGROUND: Many older patients experience cognitive deficits after surgery. Anesthetic drugs may be a contributing factor as they cause a sustained increase in the function of "memory blocking" extrasynaptic GABAA receptors in neurons. Interestingly, astrocytes are required for this increase; however, the mechanisms underlying the astrocyte-to-neuron crosstalk remain unknown. TRANSLATIONAL SIGNIFICANCE: We discovered that commonly used general anesthetic drugs stimulate GABAA receptors in astrocytes, which in turn release paracrine factors that trigger a persistent increase in extrasynaptic GABAA receptor function in neurons via p38 MAPK. This novel form of crosstalk may contribute to persistent cognitive deficits after general anesthesia and surgery.