Role In Beta-cell Function Research Articles

Sox9 regulates alternative splicing and pancreatic beta cell function

Despite significant research, mechanisms underlying the failure of islet beta cells that result in type 2 diabetes (T2D) are still under investigation. Here, we report that Sox9, a transcriptional regulator of pancreas development, also functions in mature beta cells. Our results show that Sox9-depleted rodent beta cells have defective insulin secretion, and aging animals develop glucose intolerance, mimicking the progressive degeneration observed in T2D. Using genome editing in human stem cells, we show that beta cells lacking SOX9 have stunted first-phase insulin secretion. In human and rodent cells, loss of Sox9 disrupts alternative splicing and triggers accumulation of non-functional isoforms of genes with key roles in beta cell function. Sox9 depletion reduces expression of protein-coding splice variants of the serine-rich splicing factor arginine SRSF5, a major splicing enhancer that regulates alternative splicing. Our data highlight the role of SOX9 as a regulator of alternative splicing in mature beta cell function.

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic β cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair β cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on β cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during β cell glucose toxicity that impairs expression of proteins with critical roles in β cell function.

Role of Beta Cell Function and Insulin Resistance in the Development of Gestational Diabetes Mellitus.

Background: Gestational diabetes mellitus (GDM) is a pregnancy complication characterized by second trimester hyperglycemia. Untreated, GDM is related to an increased risk for adverse pregnancy outcomes. Both beta cell dysfunction and insulin resistance underlie impaired glucose tolerance. Understanding the dominant mechanism predisposing to GDM may be important to provide effective treatment in order to improve perinatal outcomes. We hypothesize that insulin resistance rather that beta cell dysfunction predisposes to GDM. Methods: A 75g oral glucose tolerance test (OGTT) was performed on 2112 second-trimester pregnant women to determine the relationship between insulin resistance (HOMA-IR), beta cell function (HOMA-β), and the prevalence of abnormal glucose handling. Results: High insulin resistance raised the risk of GDM (relative risk (RR) 6.1, 95% confidence interval (CI) (4.4–8.5)), as did beta cell dysfunction (RR 3.8, 95% CI (2.7–5.4)). High insulin resistance, but not beta cell function, enhances the necessity for additional glucose lowering medication on top of a low carbohydrate diet in women diagnosed with GDM. Conclusions: Both high insulin resistance and beta cell dysfunction increase the risk of GDM. As increased insulin resistance, rather than beta cell function, is related to an insufficient response to a low carbohydrate diet, we speculate that insulin sensitizers rather than insulin therapy may be the most targeted therapeutic modality in diet-insensitive GDM.

Beta Cell Function as a Baseline Predictor of Weight Loss After Bariatric Surgery.

BackgroundObesity is a multifactorial disease, which is strongly associated to other metabolic disorders. Bariatric surgery is the most effective treatment of morbid obesity. The role of beta cell function in weight loss after bariatric surgery is uncertain.AimTo evaluate the association between beta cell function and percentage of total body weight loss (TBWL%) 1, 2, 3, and 4 years after bariatric surgery in patients with morbid obesity.MethodsRetrospective longitudinal study in patients with morbid obesity followed in our center between January 2010 and July 2018. Patients were excluded if they had diabetes at baseline or missing data on the needed parameters. We evaluated baseline Homeostatic Model Assessment of IR, Homeostatic Model Assessment of β-cell function (HOMA-beta), Quantitative Insulin Sensitivity Check Index, and Matsuda and DeFronzo index, and TBWL% at years 1 to 4. Linear regression models were used to evaluate the association of indexes of insulin resistance with TBWL% (unadjusted and adjusted for age, sex, BMI, and type of surgery).ResultsThere were 1,561 patients included in this analysis. HOMA-beta was negatively associated with TBWL% at second, third, and fourth years post-surgery (β = −1.04 [−1.82 to −0.26], p<0.01; β = −1.16 [−2.13 to −0.19], p=0.02; β = −1.29 [−2.64 to 0.06], p=0.061, respectively). This was not observed in the first year post-surgery nor for the other indexes. Glycemia at baseline was positively associated to EWL% at second and third years post-surgery.Conclusionβ-cell function at baseline seems to be associated to long-term weight loss, explicitly after the first year post bariatric surgery. This might be a helpful predictor of weight loss in clinical practice.

Smad3 deficiency promotes beta cell proliferation and function in db/db mice via restoring Pax6 expression.

Rationale: Transforming Growth Factor-beta (TGF-β) /Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases, but its role in beta cell function and type 2 diabetes is unknown.Methods: The role of Smad3 in beta cell function under type 2 diabetes condition was investigated by genetically deleting Smad3 from db/db mice. Phenotypic changes of pancreatic islets and beta cell function were compared between Smad3 knockout db/db (Smad3KO-db/db) mice and Smad3 wild-type db/db (Smad3WT-db/db) mice, and other littermate controls. Islet-specific RNA-sequencing was performed to identify Smad3-dependent differentially expressed genes associated with type 2 diabetes. In vitro beta cell proliferation assay and insulin secretion assay were carried out to validate the mechanism by which Smad3 regulates beta cell proliferation and function.Results: The results showed that Smad3 deficiency completely protected against diabetes-associated beta cell loss and dysfunction in db/db mice. By islet-specific RNA-sequencing, we identified 8160 Smad3-dependent differentially expressed genes associated with type 2 diabetes, where Smad3 deficiency markedly prevented the down-regulation of those genes. Mechanistically, Smad3 deficiency preserved the expression of beta cell development mediator Pax6 in islet, thereby enhancing beta cell proliferation and function in db/db mice in vivo and in Min6 cells in vitro.Conclusions: Taken together, we discovered a pathogenic role of Smad3 in beta cell loss and dysfunction via targeting the protective Pax6. Thus, Smad3 may represent as a novel therapeutic target for type 2 diabetes prevention and treatment.

Endothelial Cell‐Specific Deletion of Small GTPase ARF6 Results in Blunted Insulin‐Stimulated Vasodilation and Systemic Insulin Resistance via Arf6/Akt/eNOS Signaling Pathway

Endothelial Cell (EC) dysfunction has classically been viewed as a consequence of systemic metabolic dysfunction. However, recent evidence suggests that EC dysfunction may precede and contribute to systemic metabolic dysfunction, although the underlying mechanisms remain unclear. Adenosine diphosphate (ADP) ribosylation factor 6 (Arf6), a small GTPase of the Ras superfamily that regulates cellular proliferation and motility, has been found to be dysregulated in a host of human vascular pathologies. We sought to test the hypothesis that endothelial Arf6 plays a critical role in vascular and systemic metabolic function. To do so, we used a model of constitutive EC specific Arf6 deletion, tie2‐cre (tie2Arf6 KO). To achieve efficient EC deletion of Arf6 in this model, the EC KO was created on a whole body Arf6 heterozygous (HET) background. Using ex vivo pressure myography, we found insulin‐mediated vasodilation to be blunted (p&lt;0.05) in adipose tissue arteries, via an NO dependent mechanism (Fig 1A). Importantly, this was not observed in response to either acetylcholine or sodium nitroprusside, suggesting that Arf6 influences EC insulin signaling specifically. To explore this, we assessed insulin‐stimulated phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in HUVECs with and without siRNA‐mediated Arf6 knockdown or NAV‐2729‐induced inhibition of Arf6. We found insulin‐mediated phosphorylation of both Akt (p&lt;0.05) and eNOS (p&lt;0.05) to be lower compared to scramble or vehicle control treated cells (Fig 1B). Concomitantly, insulin sensitivity (1U/kg, ip, p&lt;0.01), but not glucose tolerance (2g/kg, ip), was impaired in tie2Arf6 KO compared to whole body HET mice. Because heterozygosity for Arf6 in the whole body may impact systemic metabolism and activation of tie2‐cre may induce changes in other cell types including macrophages, we developed a superior EC specific, tamoxifen inducible Arf6 knockout (ECArf6KO) mouse using a VECAD‐cre. In agreement with our previous model, insulin sensitivity, but not glucose tolerance, was impaired in ECArf6KO compared to controls (CON) (Fig 2A). Furthermore, both HOMA‐IR and ‐B% were ~40% higher (both p&lt;0.01) in ECArf6KO mice compared to CON mice. These results support a role for EC Arf6 in the development of systemic insulin resistance as well as suggest a role in beta cell function. Thus, we assessed glucose stimulated insulin secretion and found that both fasting (4–5 hr) and glucose‐stimulated (2g/kg, ip, 5 min) insulin secretion were higher in ECArf6KO mice compared to CON mice (Fig 2B). Taken together, we demonstrate a novel role of EC Arf6 signaling as both a modulator of vascular function and as a critical regulator of systemic metabolic function. The findings provide insight into a therapeutic target to improve insulin sensitivity.Support or Funding InformationNIA R01 AG048366, R01 AG050238 and K02 AG045339, R01 AG060395 and US Department of Veterans Affairs I01 BX002151, I01 BX004492.Endothelial cell (EC) Arf6 deletion leads to blunted insulin‐stimulated vasodilation.(A) Vasodilatory responses to insulin in the presence or absence of nitric oxide synthase inhibitor, L‐NAME, in adipose tissue arteries from constitutive ECArf6KO (tie2Arf6KO) and wildtype (WT) mice, N=6–8/group. Data are Mean ± SEM. Differences were assessed by one way ANOVA with LSD post hoc. * denotes differences between WT and tie2Arf6KO, † denotes difference after L‐NAME. (B) Representative western blot images of multiple replicates for p‐Akt, Akt, Arf6, Actin, and p‐eNOS from Arf6 siRNA and scrambled treated human umbilical vein ECs in presence and absence of insulin.Figure 1EC Arf6 deletion results in systemic insulin resistance.(A) Blood glucose during insulin tolerance test in EC specific, tamoxifen inducible VECAD‐cre Arf6 KO (ECArf6KO) and littermate cre− control mice treated with tamoxifen (CON), N=24–25/group. (B) Plasma insulin after 4–5 hr fast and 5 min after glucose stimulation (1U/kg, ip), N=14–15/group. Data are Mean ± SEM. *P&lt;0.05 vs control, †P&lt;0.05 vs fasting, repeated measure ANOVA was performed to assess group differences and independent Student’s t test was performed to assess differences at individual time points.Figure 2

Serum Free Zinc as A Predictor for Excessive Function of Pancreatic Beta-Cells in Central-Obese Men

BACKGROUND: Central obesity is known as a risk factor for type 2 diabetes mellitus (T2DM). Its development is influenced by many factors such as a progressive failure of pancreatic beta cell function. The beta cells increase their function to secret insulin along T2DM development to compensate before it becomes exhausted. Zinc (Zn) plays a crucial role in beta cell function and insulin secretion. The majority of Zn in serum are bound to protein which is not readily available interact with cells. The free Zn in serum has been suggested as being more representative than total Zn in beta cell function. This research aimed to investigate the correlation between serum free Zn and homeostasis model assessment for beta cell function (HOMA-B) and to predict the pancreatic beta cell function in the development of T2DM.METHODS: This study was designed as an observational with a cross-sectional approach. The subjects were centrally obese men aged 30-50 years and who had met the inclusion and exclusion criteria from the screening tests. Control subjects were lean men without T2DM. Serum free Zn and serum total Zn were measured by using inductively coupled plasma-mass spectrometry (ICP-MS).RESULTS: There was positive correlation between serum free Zn and HOMA-B (R=0.361, p-value&lt;0.001) but there was no correlation between serum total Zn and HOMA-B (R=-0.062, p-value=0.563). This study found that if the concentration of serum free Zn &gt;1.7 ug/dL in central obese men was suggested as an excessive function of pancreatic beta cell and as an early warning before its exhausted.CONCLUSION: This study suggested that serum free Zn had a correlation with beta cell function and had a predictive ability for beta cell excessive function before its exhausted.KEYWORDS: Type 2 diabetes mellitus, HOMA-B, serum free zinc, central obesity

Regional variation of human pancreatic islets dimension and its impact on beta cells in Indian population

ABSTRACTBackground & objectives: Islet of Langerhans, the endocrine pancreas plays a significant role in glucose metabolism. Obesity and insulin resistance are the major factors responsible for beta cell dysfunction. Asian Indian population has increased susceptibility to diabetes in spite of having lower BMI. The morphology of islets plays a significant role in beta cell function. The present study was designed for better understanding the morphology, composition and distribution of islets in different parts of the pancreas and its impact on beta cell proportion. Methods: We observed islet morphology and beta cell area proportion by Large-scale computer-assisted analysis in 20 adult human pancreases in non-diabetic Indian population. Immunohistochemical staining with anti-synaptophysin and anti-insulin antibody was used to detect islet and beta cells respectively. Whole slide images were analyzed using ImageJ software. Results: Endocrine proportion were heterogeneously increasing from head to tail with maximum islet and beta cell distribution in the tail region. Larger islets were predominately confined to the tail region. The islets in Indian population were relatively smaller in size, but they have more beta cells (20%) when compared to American population. Interpretation & conclusions: The beta cells of larger islets are functionally more active than the smaller islets via paracrine effect. Thus, reduction in the number of larger islets may be one of the probable reasons for increased susceptibility of Indians to diabetes even at lower BMI. Knowledge about the regional distribution of islets will help the surgeons to preserve the islet rich regions during surgery.

HCV-Induced Beta Cell Dysfunction Contributes to HCV-Associated Type-2 Diabetes

Chronic hepatitis C virus (HCV) infection has a close association with type 2 diabetes mellitus (T2DM). Although the mechanisms of insulin resistant in hepatitis C (CHC) patients have been extensively studied, little attention has been given to the role of beta cell function in HCV-associated diabetes. Here, we found HCV could infected the beta cell directly and impair pancreatic beta cell function in CHC and mouse model. Both first-phase and second-phase insulin secretion was impaired, at least partially due to the reduction of exocytosis of secretory insulin-containing granules following HCV infection. Upregulated p38δ in HCV-infected beta cells resulted in inactivation of protein kinase D (PKD), which was responsible for impaired insulin secretory capacity of beta cells. These findings provided a new perspective into mechanism of HCV induced type 2 diabetes and a new inspiration for the important prognostic and therapeutic implications in the management of CHC patients with glucose intolerance. Funding Statement: This work was supported by grants from the National Natural Sciences Foundation of China [grant number 81770859, 81271828, 81471079, 31271268], the National Basic Research Program of China [grant number 2015CB554304], the Youth Innovation Promotion Association, Open Research Fund Program of the State Key Laboratory of Virology of China (grant number 2018IOV003) Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: Human studies were approved by the Ethics Committee of the First Hospital of Jilin University and the First Affiliated Hospital of Xinjiang Medical University. The written informed consents were received from participants prior to inclusion in the study. All animal experiments were approved by the Institutional Animal Ethical Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (WIV, CAS).

Normoglycemia in Mice Lacking Beta-Cell Prohormone Convertase 1/3

Introduction: In islet beta cells, the endoprotease activity of prohormone convertases (PC) PC1/3 and PC2 are believed necessary for efficient processing of proinsulin to mature insulin. PC1/3 expression is a marker of beta-cell maturity and function, and polymorphisms in PC1/3 are associated with obesity, impaired glucose tolerance and elevated circulating proinsulin:insulin. Given PC1/3's key role in beta-cell function, we hypothesized that beta-cell PC1/3 deficiency would lead to beta-cell dysfunction and glucose intolerance.

Oxytocin is present in islets and plays a role in beta-cell function and survival

Oxytocin is associated mainly with modulating reproductive function. However, studies suggest that oxytocin also plays a role in endocrine pancreatic function. In the present study, islet expression of oxytocin and its related receptor was confirmed in mouse islets as well as cultured rodent and human beta-cells. Oxytocin significantly stimulated glucose-induced insulin secretion from isolated mouse islets. Similar insulinotropic actions were also observed in rodent BRIN BD11 and human 1.1B4 beta-cells. Positive effects of oxytocin on insulin secretion were almost fully annulled by the oxytocin receptor antagonist, atosiban. In terms of mechanism of insulin secretory action, oxytocin had no effect on beta-cell membrane potential or cAMP generation, but did augment intracellular calcium concentrations. In vivo administration of oxytocin to mice significantly reduced overall blood glucose levels and increased plasma insulin concentrations in response to a glucose challenge. Oxytocin also had a modest, but significant, appetite suppressive effect. As expected, streptozotocin diabetic mice had marked loss of beta-cell area accompanied by increases in alpha-cell area, whilst hydrocortisone treatment increased beta-cell and overall islet areas. Both mouse models of diabetes presented with dramatically decreased percentage islet oxytocin co-localisation with insulin and increased co-localisation with glucagon. More detailed studies in cultured beta-cell lines revealed direct positive effects of oxytocin on beta-cell proliferation and protection against apoptosis. Together, these data highlight a potentially important role of islet-derived oxytocin and related receptor signalling pathways on the modulation of beta-cell function and survival.

Confluence does not affect the expression of miR-375 and its direct targets in rat and human insulin-secreting cell lines.

MicroRNAs are small non-coding RNAs, which negatively regulate the expression of target genes. They have emerged as important modulators in beta cell compensation upon increased metabolic demand, failure of which leads to reduced insulin secretion and type 2 diabetes. To elucidate the function of miRNAs in beta cells, insulin-secreting cell lines, such as the rat insulinoma INS-1 832/13 and the human EndoC-βH1, are widely used. Previous studies in the cancer field have suggested that miRNA expression is influenced by confluency of adherent cells. We therefore aimed to investigate whether one of the most enriched miRNAs in the pancreatic endocrine cells, miR-375, and two of its validated targets in mouse, Cav1 and Aifm1, were differentially-expressed in cell cultures with different confluences. Additionally, we measured the expression of other miRNAs, such as miR-152, miR-130a, miR-132, miR-212 and miR-200a, with known roles in beta cell function. We did not see any significant expression changes of miR-375 nor any of the two targets, in both the rat and human beta cell lines at different confluences. Interestingly, among the other miRNAs measured, the expression of miR-132 and miR-212 positively correlated with confluence, but only in the INS-1 832/13 cells. Our results show that the expression of miR-375 and other miRNAs with known roles in beta cell function is independent of, or at least minimally influenced by the density of proliferating adherent cells, especially within the confluence range optimal for functional assays to elucidate miRNA-dependent regulatory mechanisms in the beta cell.

Group X Secretory Phospholipase A2 Regulates Insulin Secretion through a Cyclooxygenase-2-dependent Mechanism

Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA2 is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA2-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA2 overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA2 (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA2 on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.

Glucose modulates Pax6 expression through the JNK/p38 MAP kinase pathway in pancreatic beta-cells

AimThe paired and homeodomain-containing transcription factor, paired box 6 (Pax6), has shown to play pivotal roles in beta-cell function, including cell survival, insulin biosynthesis and secretion. The present study investigates the signaling events that regulate the modulation of Pax6 expression by glucose and the role of this modulation in cell survival in rat insulinoma-1E (INS-1E) cells. Main methodsINS-1E cells were incubated on 1mM (low) or 25mM (high) glucose overnight. To elucidate the signaling pathways that regulate Pax6 expression, we utilized specific inhibitors. The siRNA transfection of Pax6 into INS-1E cells was performed by electroporation. The mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. Key findingsWe found that the mRNA and protein levels of Pax6 were reduced by approximately 4-fold in high, compared to low, glucose-treated cells. Staurosporine, the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 significantly increased Pax6 levels in high glucose-treated INS-1E cells compared to their respective controls. However, neither calcium ionophore nor the extracellular signal-regulated kinase (ERK) inhibitor U0126 resulted in any alteration in Pax6 protein expression. Further, a siRNA-mediated knockdown of Pax6 significantly decreased the expression of tumor-suppressor phosphatase with tensin homology (PTEN) while increasing cell viability in low glucose-treated INS-1E cells. SignificanceThis study addresses the signaling events that regulate the glucose-dependent expression of Pax6 and the role of these events in cell survival in pancreatic beta cells.

Expression, phosphorylation and function of the Rab-GTPase activating protein TBC1D1 in pancreatic beta-cells

The Rab-GTPase activating protein TBC1D1 is a paralog of AS160/TBC1D4. AS160/TBC1D4, a downstream effector of Akt, has been shown to play a central role in beta-cell function and survival. The two proteins have overlapping function in insulin signalling in muscle cells. However, the expression and the potential role of TBC1D1 in beta-cells remain unknown. Therefore, the aim of this study is to investigate whether TBC1D1 is expressed in beta-cells and whether it plays, as AS160/TBC1D4, a role in beta-cell function and survival. Using human and rat beta-cells, this study shows for the first time that TBC1D1 is expressed and phosphorylated in response to glucose in these cells. Knockdown of TBC1D1 in beta-cells resulted in increased basal and glucose-stimulated insulin release, decreased proliferation but no change in apoptosis.

A rare SNP in pre-miR-34a is associated with increased levels of miR-34a in pancreatic beta cells

Changes in the levels of specific microRNAs (miRNAs) can reduce glucose-stimulated insulin secretion and increase beta-cell apoptosis, two causes of islet dysfunction and progression to type 2 diabetes. Studies have shown that single nucleotide polymorphisms (SNPs) within miRNA genes can affect their expression. We sought to determine whether miRNAs, with a known role in beta-cell function, possess SNPs within the pre-miRNA structure which can affect their expression. Using published literature and dbSNP, we aimed to identify miRNAs with a role in beta-cell function that also possess SNPs within the region encoding its pre-miRNA. Following transfection of plasmids, encoding the pre-miRNA and each allele of the SNP, miRNA expression was measured. Two rare SNPs located within the pre-miRNA structure of two miRNA genes important to beta-cell function (miR-34a and miR-96) were identified. Transfection of INS-1 and MIN6 cells with plasmids encoding pre-miR-34a and the minor allele of rs72631823 resulted in significantly (p < 0.05) higher miR-34a expression, compared to cells transfected with plasmids encoding the corresponding major allele. Similarly, higher levels were also observed upon transfection of HeLa cells. Transfection of MIN6 cells with plasmids encoding pre-miR-96 and each allele of rs41274239 resulted in no significant differences in miR-96 expression. A rare SNP in pre-miR-34a is associated with increased levels of mature miR-34a. Given that small changes in miR-34a levels have been shown to cause increased levels of beta-cell apoptosis this finding may be of interest to studies looking at determining the effect of rare variants on type 2 diabetes susceptibility.

The Dynamic Roles of Visfatin and Obestatin Serum Concentration in Pancreatic Beta Cells Dysfunction (HOMA-beta) and Insulin Resistance (HOMA-IR) in Centrally Obese Men

BACKGROUND: Obesity is a major health problem in the world today. Obesity is closely associated with insulin resistance and type 2 diabetes. Epidemiological studies have shown that obese persons are in a state of insulin resistance, however, most of them do not progress to type 2 diabetes. This occurs because the beta cell function is still good enough for maintaining normal glucose level. Obestatin and visfatin are cytokines that are known to have a role in beta cell function. The aim of this study was to assess the relationship between visfatin and obestatin and Homeostasis Model Assessment of beta cell function (HOMA-β) and Homeostasis Model Assessment of insulin resistance (HOMA-IR).METHODS: This was a cross-sectional study involving 80 central obesity men with waist circumference &gt;90 cm, age 30-65 years old. Visfatin and obestatin were measured by ELISA method. Beta pancreas cell dysfunction and insulin resistance were calculated by HOMA model.RESULTS: Our study showed a correlation between visfatin and HOMA-β (r=0.244 and p = 0.029) and visfatin with HOMA-IR (r=0.287 and p=0.001) and no correlation was found between obestatin with HOMA-β (r=0.010 and p=0.990) and obestatin with HOMA-IR (r=0.080 and p=0.480). We also found visfatin and obestatin concentrations were fluctuative depending on the measurements of the waist circumferences.CONCLUSIONS: High visfatin and low obestatin concentration were independently associated with increased beta pancreas cell dysfunction and insulin resistance.KEYWORDS: obesity. visfatin, obestatin, beta cell dysfunction (HOMA-β), insulin resistance (HOMA-IR)

An siRNA Screen in Pancreatic Beta Cells Reveals a Role for Gpr27 in Insulin Production

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.

The CDK4–pRB–E2F1 pathway controls insulin secretion

CDK4-pRB-E2F1 cell-cycle regulators are robustly expressed in non-proliferating beta cells, suggesting that besides the control of beta-cell number the CDK4-pRB-E2F1 pathway has a role in beta-cell function. We show here that E2F1 directly regulates expression of Kir6.2, which is a key component of the K(ATP) channel involved in the regulation of glucose-induced insulin secretion. We demonstrate, through chromatin immunoprecipitation analysis from tissues, that Kir6.2 expression is regulated at the promoter level by the CDK4-pRB-E2F1 pathway. Consistently, inhibition of CDK4, or genetic inactivation of E2F1, results in decreased expression of Kir6.2, impaired insulin secretion and glucose intolerance in mice. Furthermore we show that rescue of Kir6.2 expression restores insulin secretion in E2f1(-/-) beta cells. Finally, we demonstrate that CDK4 is activated by glucose through the insulin pathway, ultimately resulting in E2F1 activation and, consequently, increased expression of Kir6.2. In summary we provide evidence that the CDK4-pRB-E2F1 regulatory pathway is involved in glucose homeostasis, defining a new link between cell proliferation and metabolism.

β‐cell function assessment from modelling of oral tests: an effective approach

The quantitative study of beta-cell function has relied on mathematical models for almost 40 years. During the last decades, the initial elaborate models have been simplified with the aim of obtaining mathematical methods suitable for the assessment of beta-cell function in an individual from an intravenous test. In more recent times, modelling methods have been proposed for analysing oral glucose tolerance tests or meal tests. In this review, we describe these methods with particular emphasis on an approach we have developed. We discuss the relevance of potentiation of insulin secretion in an oral glucose test, which our model has reintroduced after the historical models. We provide evidence on the validity of the method and illustrate significant applications. We emphasize the importance of quantifying beta-cell function through multiple indices and highlight how our approach has provided new insight on the relationships between beta-cell function and insulin sensitivity and on the role of beta-cell function in glucose intolerance and diabetes.