Primary Human Islets Research Articles

Improved Beta-Cell Differentiation and Function in Isolated Islets Established in Dynamic Culture to Reduce Hypoxia

Background: Pancreatic islet transplantation effectively prevents severe hypoglycaemia in type 1 diabetes. Restoration of normal β-cell functional mass remains elusive and we have recently demonstrated loss of β-cell end-differentiation in clinical islet recipients. We hypothesised that reducing islet hypoxia in culture would restore β-cell differentiation and function. Methods: MIN6 pseudo-islets (PI) and primary human islets (HI) were cultured in either static (SC) or horizontal rotation (RC) culture for 7 days. Hypoxia was detected by pimonidazole (Pim) adduct formation and hypoxia-induced gene expression (qPCR). β-cell end-differentiation was assessed by UCN3 expression and IF staining (urocortin-3+ insulin+ cells/total insulin+ cells) with function assayed by glucose-stimulated insulin secretion (GSIS). Results: Hypoxia was confirmed in established PI (70±1.0% Pim+). This was reduced in rotation culture (Pim+: 15±1.5% RC vs. 80±2.5% SC; p<0.05) with reduced hypoxia-induced gene expression compared with adherent MIN6 (LDHA: 6.0±2.1 RC vs. 86.0±7.8 fold SC; MCT4: RC 2.9±0.4 vs. 54.6±0.6 fold SC; p<0.05). RC enhanced UCN3 gene expression (4.2±0.5 RC vs. 2.2±0.2 fold SC; p<0.05) and increased UCN3/insulin co-staining (80±1.2% RC vs. 30±2.4% SC; p<0.0001) with enhanced GSIS stimulation index (4.1±0.1 RC vs. 2.8±0.1 SC; p<0.0001). Hypoxia-induced expression was reduced in HI RC compared with freshly isolated islets (LDHA: 0.3±0.1 RC vs. 1.8±0.04 fold SC; MCT4: 0.08±0.01 RC vs. 1.1±0.2 fold SC; p<0.05) with increased UCN3 expression (5.9±1.6 RC vs. 3.3±0.9 fold SC; p<0.05) and enhanced GSIS stimulation index (4.1±0.9 RC vs. 3.0±1.2 SC; p<0.05). Conclusion: Hypoxia in established PI and isolated HI was reduced over 7 days in dynamic culture. This was associated with enhanced β-cell differentiation and function providing a potential approach to maximise optimal functional mass in islet transplantation. Disclosure N.A. Al-Jahdhami: None. S.J. Anderson: None. A. Aldibbiat: None. J.A. Shaw: Advisory Panel; Self; Novo Nordisk A/S.

MiR-204 Antagomir Enhances GLP-1 Receptor Function and Glucose Control

Glucagon-like peptide 1 receptor (GLP-1R) is a G protein-coupled receptor that is highly expressed on pancreatic beta-cells and activated by endogenous incretins or antidiabetic GLP-1R agonist drugs. GLP-1R function is important in maintaining glucose homeostasis and involves stimulation of glucose-induced beta-cell insulin secretion via cAMP generation. While GLP-1R expression has been shown to be downregulated in diabetes, the molecular mechanisms have not been fully elucidated. In this study, we have discovered that miR-204, a highly beta-cell-enriched microRNA that is upregulated in diabetes, directly targets the 3’ UTR of GLP-1R and decreases its expression in INS-1 cells as well as primary mouse and human islets. Moreover, genetic knockout of miR-204 in vivo increased islet GLP-1R expression and enhanced GLP-1R agonist-induced cAMP production and insulin secretion, resulting in improved glucose tolerance as well as protection against diabetes. In addition, miR-204 antagomir administration was able to mimic the effects of genetic miR-204 deletion and resulted again in GLP-1R agonist-induced insulin secretion as well as improved glucose tolerance. Thus, these results indicate for the first time that GLP-1R is under the control of a micro RNA and suggest that antagomir-mediated inhibition of miR-204 represents a potential novel therapeutic approach to promote the actions of GLP-1R agonist drugs in a beta-cell-specific manner and thereby help treat diabetes. Disclosure S. Jo: None. J. Chen: None. G. Xu: None. T. Grayson: None. L. Thielen: None. A. Shalev: None.

Mir-128 Targets Thioredoxin-Interacting Protein 3’UTR and Downregulates Its Expression in Pancreatic Beta Cells

Thioredoxin-interacting protein (TXNIP) expression is induced by glucose, promotes beta cell apoptosis and is essential for glucotoxicity-induced beta cell death, whereas TXNIP deficiency protects against diabetes. Carbohydrate-response element-binding protein (ChREBP/MondoA) is the major transcription factor conferring glucose-induced TXNIP expression. However, we recently discovered that glucose was still able to induce TXNIP in ChREBP knockout mouse islets suggesting the existence of other factors involved in glucose-induced TXNIP regulation. Analyses of the TXNIP 3’UTR revealed several potential miRNA binding sites including miR-93, miR-106b and miR-128. However, using INS-1 beta cells, primary mouse islets and T2D human islets, we found that only miR-128 was downregulated by glucose or diabetes. This suggested that glucose might decrease miR-128 and thereby promote TXNIP expression. In fact, we found overexpression of miR-128 significantly reduced TXNIP mRNA and protein expression whereas inhibition of miR-128 increased beta cell TXNIP expression. Furthermore, luciferase assays using reporter constructs with the wild type TXNIP 3’UTR or a mutated TXNIP 3’UTR confirmed that TXNIP is indeed a direct target of miR-128. Taken together, the results of these studies have identified a novel microRNA directly targeting TXNIP and revealed an alternative, post-transcriptional pathway by which glucose induces TXNIP expression. Disclosure G. Jing: None. J. Chen: None. G. Xu: None. A. Shalev: None.

Novel Small Molecule TXNIP Inhibitor Protects Against Diabetes

Loss of functional beta cell mass represents a major factor in the pathogenesis of diabetes. Currently, there are no therapies that halt this process; however, thioredoxin-interacting protein (TXNIP) has recently emerged as a promising therapeutic target. TXNIP was found to be the top glucose-induced gene in a human pancreatic islet microarray, is increased in diabetes, and TXNIP overexpression results in beta cell apoptosis. TXNIP reduction has shown favorable effects in vivo, where whole body TXNIP-deficient and beta cell-specific TXNIP knockout mice have decreased beta cell apoptosis, increased beta cell mass, elevated insulin levels, and are protected from diabetes. We have also shown that pharmacologic TXNIP inhibition, via the anti-hypertensive medication verapamil, was able to mimic the protective effects of genetic TXNIP deletion and reversed overt diabetes in mice. In addition, it has been established that TXNIP downregulation is beneficial in multiple tissues, making it unnecessary and even undesirable to have a beta cell-specific TXNIP inhibitor and suggesting that detrimental off target effects are unlikely. We have since undertaken a high-throughput screen of 300,000 small molecules that has yielded a novel compound, TI-37330, which is more effective than verapamil in inhibiting TXNIP expression. Moreover, TI-37330 protected primary human islets and INS-1 beta cells against glucose- and cytokine-induced TXNIP and beta cell apoptosis. TI-37330 has also shown in vivo potential as it was found to have good pharmacokinetic properties and was well tolerated in healthy mice. In the context of streptozotocin-induced diabetes, oral administration of TI-37330 promoted beta cell survival, prevented diabetes, and even significantly improved overt diabetes. Collectively, these data establish small molecule TXNIP inhibitors, especially TI-37330, as robust candidates for further development. Disclosure L. Thielen: None. J. Chen: None. G. Xu: None. G. Jing: None. T. Grayson: None. S. Jo: None. A. Shalev: None.

Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-βH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.

Coordination of GPR40 and Ketogenesis Signaling by Medium Chain Fatty Acids Regulates Beta Cell Function.

Diabetes prevalence increases with age, and β-cell dysfunction contributes to the incidence of the disease. Dietary lipids have been recognized as contributory factors in the development and progression of the disease. Unlike long chain triglycerides, medium chain triglycerides (MCT) increase fat burning in animal and human subjects as well as serum C-peptide in type 2 diabetes patients. We evaluated the beneficial effects of MCT on β-cells in vivo and in vitro. MCT improved glycemia in aged rats via β-cell function assessed by measuring insulin secretion and content. In β-cells, medium chain fatty acid (MCFA)-C10 activated fatty acid receptor 1 FFAR1/GPR40, while MCFA-C8 induced mitochondrial ketogenesis and the C8:C10 mixture improved β cell function. We showed that GPR40 signaling positively impacts ketone body production in β-cells, and chronic treatment with β-hydroxybutyrate (BHB) improves β-cell function. We also showed that BHB and MCFA help β-cells recover from lipotoxic stress by improving mitochondrial function and increasing the expression of genes involved in β-cell function and insulin biogenesis, such as Glut2, MafA, and NeuroD1 in primary human islets. MCFA offers a therapeutic advantage in the preservation of β-cell function as part of a preventative strategy against diabetes in at risk populations.

Human EndoC-βH1 β-cells form pseudoislets with improved glucose sensitivity and enhanced GLP-1 signaling in the presence of islet-derived endothelial cells.

Three-dimensional (3D) pseudoislets (PIs) can be used for the study of insulin-producing β-cells in free-floating islet-like structures similar to that of primary islets. Previously, we demonstrated the ability of islet-derived endothelial cells (iECs) to induce PIs using murine insulinomas, where PI formation enhanced insulin production and glucose responsiveness. In this report, we examined the ability of iECs to spontaneously induce the formation of free-floating 3D PIs using the EndoC-βH1 human β-cell line murine MS1 iEC. Within 14 days, the coculturing of both cell types produced fully humanized EndoC-βH1 PIs with little to no contaminating murine iECs. The size and shape of these PIs were similar to primary human islets. iEC-induced PIs demonstrated reduced dysregulated insulin release under low glucose levels and higher insulin secretion in response to high glucose and exendin-4 [a glucagon-like peptide-1 (GLP-1) analog] compared with monolayer cells cultured alone. Interestingly, iEC-PIs were also better at glucose sensing in the presence of extendin-4 compared with PIs generated on a low-adhesion surface plate in the absence of iECs and showed an overall improvement in cell viability. iEC-induced PIs exhibited increased expression of key genes involved in glucose transport, glucose sensing, β-cell differentiation, and insulin processing, with a concomitant decrease in glucagon mRNA expression. The enhanced responsiveness to exendin-4 was associated with increased protein expression of GLP-1 receptor and phosphokinase A. This rapid coculture system provides an unlimited number of human PIs with improved insulin secretion and GLP-1 responsiveness for the study of β-cell biology.

CORRIGENDUM FOR "Coxsackievirus-Induced Proteomic Alterations in Primary Human Islets Provide Insights for the Etiology of Diabetes".

[This corrects the article DOI: 10.1210/js.2017-00278.].

MiR-204 Controls Glucagon-Like Peptide 1 Receptor Expression and Agonist Function.

Glucagon-like peptide 1 receptor (GLP1R) agonists are widely used to treat diabetes. However, their function is dependent on adequate GLP1R expression, which is downregulated in diabetes. GLP1R is highly expressed on pancreatic β-cells, and activation by endogenous incretin or GLP1R agonists increases cAMP generation, which stimulates glucose-induced β-cell insulin secretion and helps maintain glucose homeostasis. We now have discovered that the highly β-cell–enriched microRNA, miR-204, directly targets the 3′ UTR of GLP1R and thereby downregulates its expression in the β-cell–derived rat INS-1 cell line and primary mouse and human islets. Furthermore, in vivo deletion of miR-204 promoted islet GLP1R expression and enhanced responsiveness to GLP1R agonists, resulting in improved glucose tolerance, cAMP production, and insulin secretion as well as protection against diabetes. Since we recently identified thioredoxin-interacting protein (TXNIP) as an upstream regulator of miR-204, we also assessed whether in vivo deletion of TXNIP could mimic that of miR-204. Indeed, it also enhanced islet GLP1R expression and GLP1R agonist–induced insulin secretion and glucose tolerance. Thus, the present studies show for the first time that GLP1R is under the control of a microRNA, miR-204, and uncover a previously unappreciated link between TXNIP and incretin action.

Coxsackievirus-Induced Proteomic Alterations in Primary Human Islets Provide Insights for the Etiology of Diabetes.

Enteroviral infections have been associated with the development of type 1 diabetes (T1D), a chronic inflammatory disease characterized by autoimmune destruction of insulin-producing pancreatic beta cells. Cultured human islets, including the insulin-producing beta cells, can be infected with coxsackievirus B4 (CVB4) and thus are useful for understanding cellular responses to infection. We performed quantitative mass spectrometry analysis on cultured primary human islets infected with CVB4 to identify molecules and pathways altered upon infection. Corresponding uninfected controls were included in the study for comparative protein expression analyses. Proteins were significantly and differentially regulated in human islets challenged with virus compared with their uninfected counterparts. Complementary analyses of gene transcripts in CVB4-infected primary islets over a time course validated the induction of RNA transcripts for many of the proteins that were increased in the proteomics studies. Notably, infection with CVB4 results in a considerable decrease in insulin. Genes/proteins modulated during CVB4 infection also include those involved in activation of immune responses, including type I interferon pathways linked to T1D pathogenesis and with antiviral, cell repair, and inflammatory properties. Our study applies proteomics analyses to cultured human islets challenged with virus and identifies target proteins that could be useful in T1D interventions.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

The dual DPP4 inhibitor and GPR119 agonist HBK001 regulates glycemic control and beta cell function ex and in vivo

Glucagon like peptide-1 (GLP-1) plays a vital role in glucose homeostasis and sustaining β-cell function. Currently there are two major methods to enhance endogenous GLP-1 activity; inhibiting dipeptidyl peptidase-4 (DPP4) or activating G protein-coupled receptor 119 (GPR119). Here we describe and validate a novel dual-target compound, HBK001, which can both inhibit DPP4 and activate GPR119 ex and in vivo. We show that HBK001 can promote glucose-stimulated insulin secretion in mouse and human primary islets. A single administration of HBK001 in ICR mice can increase plasma incretins levels much more efficiently than linagliptin, a classic DPP4 inhibitor. Long-term treatment of HBK001 in KKAy mice can ameliorate hyperglycemia as well as improve glucose tolerance, while linagliptin fails to achieve such glucose-lowing effects despite inhibiting 95% of serum DPP4 activity. Moreover, HBK001 can increase first-phase insulin secretion in KKAy mice, suggesting a direct effect on islet β-cells via GPR119 activation. Furthermore, HBK001 can improve islet morphology, increase β-cell proliferation and up-regulate genes involved in improved β-cell function. Thus, we have identified, designed and synthesized a novel dual-target compound, HBK001, which represents a promising therapeutic candidate for type 2 diabetes, especially for patients who are insensitive to current DPP4 inhibitors.

Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells

Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed, respiration, mitonuclear protein imbalance or down-stream signaling may be altered.

High-throughput screening and bioinformatic analysis to ascertain compounds that prevent saturated fatty acid-induced β-cell apoptosis

Pancreatic β-cell lipotoxicity is a central feature of the pathogenesis of type 2 diabetes. To study the mechanism by which fatty acids cause β-cell death and develop novel approaches to prevent it, a high-throughput screen on the β-cell line INS1 was carried out. The cells were exposed to palmitate to induce cell death and compounds that reversed palmitate-induced cytotoxicity were ascertained. Hits from the screen were analyzed by an increasingly more stringent testing funnel, ending with studies on primary human islets treated with palmitate. MAP4K4 inhibitors, which were not part of the screening libraries but were ascertained by a bioinformatics analysis, and the endocannabinoid anandamide were effective at inhibiting palmitate-induced apoptosis in INS1 cells as well as primary rat and human islets. These targets could serve as the starting point for the development of therapeutics for type 2 diabetes.

Abstract 393: Pancreatic Beta Cell Export of miR-375 to High-Density Lipoproteins is Inversely Associated With Insulin Secretion

microRNAs (miRNAs) are critical regulators of glucose metabolism and contribute to the pathogenesis of Type 2 Diabetes (T2D). Recently, we reported that high-density lipoproteins (HDL) transport and deliver functional miRNAs to recipient cells. Here, we report that miR-375 is decreased on HDL in two models of chronic hyperglycemia -- T2D human subjects and Zucker Diabetic Fatty (ZDF) rats. Since miR-375 expression in the islets is 10X greater than in other organs, we tested whether pancreatic beta cells have the ability to export miR-375 to HDL through in vitro export assays, incubating HDL with INS1 beta cells or primary human islets. Indeed, we found miR-375 to be readily exported to HDL from INS1 cells and primary islets in vitro . To determine if cholesterol transporters contribute to HDL-miR-375 export from beta cells, Abca1, Abcg1 and Scarb1 (SR-BI) were inhibited using siRNAs; however, we found that knockdown of each of these transporters failed to affect the beta cell’s ability to export miR-375 to HDL. Nonetheless, enhancing insulin secretion with tolbutamide resulted in the suppression of HDL-miR-375 export, suggesting that miRNA export and insulin secretion are inversely regulated. To determine the roles of Argonaute (Ago) family proteins in HDL-miRNA export, INS1 cells were transfected with siRNAs against Eif2c1-4 to knockdown Ago1-4. We found HDL-miR-375 export to be suppressed when Ago1, but not Ago2-4, were inhibited, suggesting that miRNA export is downstream of miRNA processing by Ago1. We are currently investigating the relationship between HDL-miR-375 export, insulin secretion, and miRNA processing in pancreatic beta cells to elucidate the mechanism(s) controlling HDL-miR-375 export. Collectively, results suggest that a large fraction of HDL-miRNAs originate from pancreatic beta cells and HDL-miRNAs are exported independent of cholesterol transporters.

Preparation of monolayer cultures of primary rat and human islet cells on glass for super-resolution and live cell imaging

Preparation of monolayer cultures of primary rat and human islet cells on glass for super-resolution and live cell imaging

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.

Impact of gain-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) on glucose and lipid homeostasis.

Wnt signaling through LRP5 represents a newly appreciated metabolic pathway, which potentially represents a target for drug discovery in type 2 diabetes and hyperlipidemia. Studies in animal models suggest a physiologic link between LRP5 and glucose and lipid homeostasis; however, whether it plays a similar role in humans is unclear. As current literature links loss-of-function LRP5 to impaired glucose and lipid metabolism, we hypothesized that individuals with an HBM-causing mutation in LRP5 would exhibit improved glucose and lipid homeostasis. Since studies in animal models have suggested that Wnt signaling augments insulin secretion, we also examined the effect of Wnt signaling on glucose-stimulated insulin secretion on human pancreatic islets. This was a matched case-control study. We used several methods to assess glucose and lipid metabolism in 11 individuals with HBM-causing mutations in LRP5. Affected study participants were recruited from previously identified kindreds with HBM-causing LRP5 mutations and included 9 males and 2 females. Two subjects that were being treated with insulin for type 2 diabetes were excluded from our analysis, as this would have obscured our ability to determine the impact of gain-of-function LRP5 mutations on glucose metabolism. The mean age of the evaluated study subjects was 55±7 with a mean BMI of 27.2±2.0. Control subjects were matched and recruited from the general community at an equivalent ratio, with 18 males and 4 females (mean age 56±4; mean BMI 27.2±1.0). Study testing was conducted at an academic medical center. There were no statistically significant differences between affected and matched control populations for HbA1c (p=0.06), eAG (p=0.06), insulin (p=0.82), HOMA-B (p=0.34), or HOMA-IR (p=0.66). The mean Insulin Sensitivity Index (ISI) was also similar between control and affected individuals. Total cholesterol (p=0.43), triglycerides (TG) (p=0.56), and HDL (p=0.32) were not different between the same two groups. In a small subset of studied subjects, intramyocellular and hepatic lipid content were similar in the affected individuals and controls when quantified by proton magnetic resonance spectroscopy (MRS). However, the mean value for serum LDL was significantly lower (p=0.04) in affected individuals. In primary human islets, there were no differences between control and Wnt treatment groups for insulin secretion measured as area under the curve (AUC) for first phase (p=0.17) or second phase (p=0.33) insulin secretion. Although our sample size was small, our data do not support the hypothesis that HBM-causing LRP5 mutations, associated with increased Wnt signaling, improve glucose metabolism in humans. However, it does appear that LRP5 variants may affect LDL metabolism, a major risk factor for coronary artery disease. The molecular mechanisms underpinning this effect warrant further study.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

SummaryType 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells.

The relationship between iron and β-cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in β-cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human β-cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZRT/IRT-like protein 14 and 8 (ZIP14 and ZIP8) and divalent metal-ion transporter-1 (DMT1) to iron uptake by human β-cells. Iron was provided to the cells as nontransferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by βlox5 cells, a human β-cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in βlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to β-cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1β. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by β-cells and suggest differential regulation of ZIP14 in primary human islets compared with other cell types such as hepatocytes.