The nucleotide exchange factor SIL1 is required for glucose-stimulated insulin secretion from mouse pancreatic beta cells in vivo

Aims/hypothesis

Regulation of insulin secretion along the secretory pathway is incompletely understood. We addressed the expression of SIL1, a nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 kD (GRP78), in pancreatic beta cells and investigated whether or not SIL1 is involved in beta cell function.

Methods

SIL1 expression was analysed by immunoblotting and immunofluorescence. Metabolic and islet variables, including glucose tolerance, beta cell mass, insulin secretion, islet ultrastructure, insulin content and levels of ER stress marker proteins, were addressed in Sil1 knockout (Sil1 ^?/?) mice. Insulin, proinsulin and C-peptide release was addressed in Sil1 ^?/? islets, and SIL1 overexpression or knockdown was explored in MIN6 cells in vitro. Models of type 1 diabetes and insulin resistance were induced in Sil1 ^?/? mice by administration of streptozotocin (STZ) and a high-fat diet (HFD), respectively.

Results

We show that SIL1 is expressed in pancreatic beta cells and is required for islet insulin content, islet sizing, glucose tolerance and glucose-stimulated insulin secretion in vivo. Levels of pancreatic ER stress markers are increased in Sil1 ^?/? mice, and Sil1 ^?/? beta cell ER is ultrastructurally compromised. Isolated Sil1 ^?/? islets show lower proinsulin and insulin content and impaired glucose-stimulated insulin secretion. Modulation of SIL1 protein levels in MIN6 cells correlates with changes in insulin content and secreted insulin. Furthermore, Sil1 ^?/? mice are more susceptible to STZ-induced type 1 diabetes with increased apoptosis. Upon HFD feeding, Sil1 ^?/? mice show markedly lower insulin secretion and exacerbated glucose intolerance compared with control mice. Surprisingly, however, HFD-fed Sil1 ^?/? mice display pronounced islet hyperplasia with low amounts of insulin in total pancreas.

Conclusions/interpretation

These results reveal a novel role for the nucleotide exchange factor SIL1 in pancreatic beta cell function under physiological and disease conditions such as diabetes and the metabolic syndrome.     

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model

Aims/hypothesis

Cardiotrophin 1 (CT-1) is a recently described cytokine originally isolated from the heart where it has been shown to play an important role in apoptotic protection of cardiomyocytes and heart hypertrophy. Its beneficial properties have also been described in other organs such as liver and neuromuscular tissue. In the present study, we investigated whether CT-1 can confer protection against pro-apoptotic stimuli in pancreatic beta cells, and its role in insulin secretion and diabetes development.

Methods

The effects of CT-1 on apoptosis and function were studied using MIN6B1 cells and freshly isolated murine pancreatic islets. The impact on the development of diabetes was evaluated in Ct1-null (Ct1 ^?/?) mice (the gene Ct1 is also known as Ctf1) using two streptozotocin (STZ)-induced models of diabetes.

Results

CT-1 has a protective effect in MIN6B1 cells and murine islets under the pro-apoptotic stimulus of serum deprivation, which correlates with the expression of B cell lymphoma-extra large, or following exposure to a mixture of cytokines. In addition, CT-1 enhances glucose-stimulated insulin secretion in MIN6B1 cells and this was repressed by inhibitors of phospholipase C. Furthermore, Ct1 ^?/? mice were more prone to develop diabetes, and their glucose tolerance test showed impaired plasma glucose clearance which correlated with decreased pancreatic insulin secretion.

Conclusions/interpretation

The results obtained from both in vitro and in vivo experiments show that CT-1 improves beta cell function and survival, and protects mice against STZ-induced diabetes.     

Deficiency of APPL1 in mice impairs glucose-stimulated insulin secretion through inhibition of pancreatic beta cell mitochondrial function

Aims/hypothesis

Adaptor protein, phosphotyrosine interaction, pleckstrin homology domain and leucine zipper containing 1 (APPL1) is an adapter protein that positively mediates adiponectin signalling. Deficiency of APPL1 in the target tissues of insulin induces insulin resistance. We therefore aimed, in the present study, to determine its role in regulating pancreatic beta cell function.

Methods

A hyperglycaemic clamp test was performed to determine insulin secretion in APPL1 knockout (KO) mice. Glucose- and adiponectin-induced insulin release was measured in islets from APPL1 KO mice or INS-1(832/13) cells with either APPL1 knockdown or overproduction. RT-PCR and western blotting were conducted to analyse gene expression and protein abundance. Oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential were assayed to evaluate mitochondrial function.

Results

APPL1 is highly expressed in pancreatic islets, but its levels are decreased in mice fed a high-fat diet and db/db mice compared with controls. Deletion of the Appl1 gene leads to impairment of both the first and second phases of insulin secretion during hyperglycaemic clamp tests. In addition, glucose-stimulated insulin secretion (GSIS) is significantly decreased in islets from APPL1 KO mice. Conversely, overproduction of APPL1 leads to an increase in GSIS in beta cells. In addition, expression levels of several genes involved in insulin production, mitochondrial biogenesis and mitochondrial OCR, ATP production and mitochondrial membrane potential are reduced significantly in APPL1-knockdown beta cells. Moreover, suppression or overexproduction of APPL1 inhibits or stimulates adiponectin-potentiated GSIS in beta cells, respectively.

Conclusions/interpretation

Our study demonstrates the roles of APPL1 in regulating GSIS and mitochondrial function in pancreatic beta cells, which implicates APPL1 as a therapeutic target in the treatment of type 2 diabetes.     

Aldosterone deficiency prevents high-fat-feeding-induced hyperglycaemia and adipocyte dysfunction in mice

Aims/hypothesis

Obesity is associated with aldosterone excess, hypertension and the metabolic syndrome, but the relative contribution of aldosterone to obesity-related complications is debated. We previously demonstrated that aldosterone impairs insulin secretion, and that genetic aldosterone deficiency increases glucose-stimulated insulin secretion in vivo. We hypothesised that elimination of endogenous aldosterone would prevent obesity-induced insulin resistance and hyperglycaemia.

Methods

Wild-type and aldosterone synthase-deficient (As ^?/?) mice were fed a high-fat (HF) or normal chow diet for 12 weeks. We assessed insulin sensitivity and insulin secretion using clamp methodology and circulating plasma adipokines, and examined adipose tissue via histology.

Results

HF diet induced weight gain similarly in the two groups, but As ^?/? mice were protected from blood glucose elevation. HF diet impaired insulin sensitivity similarly in As ^?/? and wild-type mice, assessed by hyperinsulinaemic–euglycaemic clamps. Fasting and glucose-stimulated insulin were higher in HF-fed As ^?/? mice than in wild-type controls. Although there was no difference in insulin sensitivity during HF feeding in As ^?/? mice compared with wild-type controls, fat mass, adipocyte size and adiponectin increased, while adipose macrophage infiltration decreased. HF feeding significantly increased hepatic steatosis and triacylglycerol content in wild-type mice, which was attenuated in aldosterone-deficient mice.

Conclusions/interpretation

These studies demonstrate that obesity induces insulin resistance independently of aldosterone and adipose tissue inflammation, and suggest a novel role for aldosterone in promoting obesity-induced beta cell dysfunction, hepatic steatosis and adipose tissue inflammation.     

The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice

Aims/hypothesis

Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function.

Methods

Rkip1 (also known as Pebp1) knockout (Rkip1 ^?/?) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 ^?/? mice after streptozotocin treatment.

Results

Rkip1 ^?/? mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 ^?/? mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice.

Conclusions/interpretation

These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.     

Hydrogen sulfide-induced inhibition of L-type Ca2+ channels and insulin secretion in mouse pancreatic beta cells

Aims/hypothesis

L-type voltage-dependent Ca²⁺ channels (VDCCs) in pancreatic beta cells play a critical role in regulating insulin secretion. The gasotransmitter H₂S is mostly generated from l-cysteine in pancreatic beta cells by cystathionine γ-lyase (CSE) and has been reported to inhibit insulin release by opening ATP-sensitive K⁺ channels. However, whether and how H₂S affects VDCCs in beta cells is unknown.

Methods

The whole-cell patch-clamp technique was used to record VDCCs in beta cells from Cse (also known as Cth)-knockout (KO) and wild-type (WT) mice. Insulin secretion from pancreatic islets and endogenous H₂S production in pancreas were measured.

Results

The H₂S donor NaHS reversibly decreased L-type VDCC current density in a concentration-dependent fashion in WT pancreatic beta cells, and the current density was further inhibited by nifedipine. Furthermore, NaHS inhibited the channel recovery from depolarisation-induced inactivation, but did not shift the current–voltage (I–V) relationship. ACS67, another H₂S donor, also inhibited L-type VDCCs in beta cells. Inhibiting CSE activity with dl-propargylglycine increased the basal L-channel activity of beta cells from WT mice, but not that of beta cells from Cse-KO mice. Beta cells from Cse-KO mice displayed higher L-type VDCC density than those from WT mice. Insulin secretion from pancreatic islets was elevated in Cse-KO mice compared with WT mice. NaHS dose-dependently inhibited glucose-stimulated insulin secretion, which was further inhibited by nifedipine. Bay K-8644 increased glucose-stimulated insulin secretion, but this was counteracted by NaHS and nifedipine.

Conclusions/interpretation

Exogenous and endogenous H₂S inhibit L-type VDCC activity and pancreatic insulin secretion, constituting a novel mechanism for the regulation of insulin secretion by the CSE/H₂S system.     

miRNA-30a-5p-mediated silencing of Beta2/NeuroD expression is an important initial event of glucotoxicity-induced beta cell dysfunction in rodent models

Aims/hypothesis

The loss of beta cell function is a critical factor in the development of type 2 diabetes. Glucotoxicity plays a major role in the progressive deterioration of beta cell function and development of type 2 diabetes mellitus. Here we demonstrate that microRNA (miR)-30a-5p is a key player in early-stage glucotoxicity-induced beta cell dysfunction.

Methods

We performed northern blots, RT-PCR and western blots in glucotoxicity-exposed primary rat islets and INS-1 cells. We also measured glucose-stimulated insulin secretion and insulin content. In vivo approaches were used to evaluate the role of miR-30a-5p in beta cell dysfunction.

Results

miR-30a-5p expression was increased in beta cells after exposure to glucotoxic conditions, and exogenous miR-30a-5p overexpression also induced beta cell dysfunction in vitro. miR-30a-5p directly suppressed expression of Beta2/NeuroD (also known as Neurod1) by binding to a specific binding site in its 3′-untranslated region. After restoration of Beta2/NeuroD expression by knockdown miR-30a-5p or transfection of the Beta2/NeuroD gene, beta cell dysfunction, including decreased insulin content, gene expression and glucose-stimulated insulin secretion, recovered. Glucose tolerance and beta cell dysfunction improved on direct injection of Ad-si30a-5p into the pancreas of diabetic mice.

Conclusions/interpretation

Our data demonstrate that miR-30a-5p-mediated direct suppression of Beta2/NeuroD gene expression is an important initiation step of glucotoxicity-induced beta cell dysfunction.     

Toll-like receptor 2-deficient mice are protected from insulin resistance and beta cell dysfunction induced by a high-fat diet

Aims/hypothesis

Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice.

Methods

Male and female littermate Tlr2 ^+/+ and Tlr2 ^?/? mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production.

Results

While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2 ^?/? mice were protected from the adverse effects of HFD compared with Tlr2 ^+/+ littermate controls. Female Tlr2 ^?/? mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2 ^?/? mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2 ^?/? mice did not increase IL-1β expression in response to a NEFA mixture, whereas Tlr2 ^+/+ control tissues did.

Conclusion/interpretation

These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.     

Glucose metabolism-related protein 1 (GMRP1) regulates pancreatic beta cell proliferation and apoptosis via activation of Akt signalling pathway in rats and mice

Aims/hypothesis

We attempted to elucidate the impacts on and possible mechanisms by which glucose metabolism-related protein 1 (GMRP1) affects beta cell survival.

Methods

Adenovirus-mediated GMRP1 overproduction and siRNA-mediated knockdown were performed in INS-1E cells and rat islets, after which cell proliferation or apoptosis were determined, and phosphorylation of Akt and BCL2-associated agonist of cell death (BAD) investigated. INS-1E cells and rat islets were cultured at 5.6 (low) or 25?mmol/l (high) glucose for 24 or 48?h, and cell proliferation or apoptosis and GMRP1 levels were investigated. INS-1E cells were treated for 24?h with 0, 10, 50 and 100?nmol/l insulin, and GMRP1 levels were determined. After INS-1E cells were transfected with siRNA for 72?h, high glucose-induced cell proliferation and insulin-stimulated Akt phosphorylation were investigated. Glucose-infused rat models were established and beta cell proliferation and mass were evaluated. Levels of GMRP1, and phosphorylation of Akt and BAD were determined in glucose-infused islets. The GMRP1-mediated Akt pathway was also investigated in db/db mice.

Results

Overproduction of GMRP1 promoted beta cell proliferation via increased phosphorylation of Akt. Knockdown of Gmrp1 (also known as Btbd10) reduced phosphorylation of Akt with enhanced beta cell apoptosis. High glucose increased GMRP1 levels and cell proliferation in INS-1E cells and islet cells. Knockdown of Gmrp1 decreased high glucose-induced cell proliferation and insulin-stimulated Akt phosphorylation. Increased GMRP1 levels were involved in the enhancement of beta cell proliferation and mass in glucose-infused islets. Decreased GMRP1 levels may participate in beta cell apoptosis of db/db mice.

Conclusions/interpretation

GMRP1 regulates pancreatic beta cell proliferation and apoptosis via activation of Akt signalling pathway.     

Ablation of AMP-activated protein kinase α1 and α2 from mouse pancreatic beta cells and RIP2.Cre neurons suppresses insulin release in vivo

Aims/hypothesis

AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain.

Methods

The catalytic α1 and α2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkα1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkα2 [also known as Prkaa2] alleles (Ampkα1 ^?/? ,α2 ^fl/fl ,), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca²⁺ were measured with standard techniques.

Results

Trigenic Ampkα1 ^?/? ,α2 ^fl/fl expressing Cre recombinase and lacking both AMPKα subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca²⁺, while granule docking and insulin secretion were enhanced. Conversely, βAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion.

Conclusions/interpretation

Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.     

Per-arnt-sim (PAS) domain-containing protein kinase is downregulated in human islets in type 2 diabetes and regulates glucagon secretion

Aims/hypothesis

We assessed whether per-arnt-sim (PAS) domain-containing protein kinase (PASK) is involved in the regulation of glucagon secretion.

Methods

mRNA levels were measured in islets by quantitative PCR and in pancreatic beta cells obtained by laser capture microdissection. Glucose tolerance, plasma hormone levels and islet hormone secretion were analysed in C57BL/6 Pask homozygote knockout mice (Pask ^?/?) and control littermates. Alpha-TC1-9 cells, human islets or cultured E13.5 rat pancreatic epithelia were transduced with anti-Pask or control small interfering RNAs, or with adenoviruses encoding enhanced green fluorescent protein or PASK.

Results

PASK expression was significantly lower in islets from human type 2 diabetic than control participants. PASK mRNA was present in alpha and beta cells from mouse islets. In Pask ^?/? mice, fasted blood glucose and plasma glucagon levels were 25?±?5% and 50?±?8% (mean ± SE) higher, respectively, than in control mice. At inhibitory glucose concentrations (10?mmol/l), islets from Pask ^?/? mice secreted 2.04?±?0.2-fold (p?p?PASK overexpression inhibited glucagon secretion from these cells and human islets. Extracellular insulin (20?nmol/l) inhibited glucagon secretion from control and PASK-deficient alpha-TC1-9 cells. PASK-depleted alpha-TC1-9 cells and pancreatic embryonic explants displayed increased expression of the preproglucagon (Gcg) and AMP-activated protein kinase (AMPK)-alpha2 (Prkaa2) genes, implying a possible role for AMPK-alpha2 downstream of PASK in the control of glucagon gene expression and release.

Conclusions/interpretation

PASK is involved in the regulation of glucagon secretion by glucose and may be a useful target for the treatment of type 2 diabetes.     

<Emphasis Type="Italic">HDAC7</Emphasis> is overexpressed in human diabetic islets and impairs insulin secretion in rat islets and clonal beta cells

Aims/hypothesis

Pancreatic beta cell dysfunction is a prerequisite for the development of type 2 diabetes. Histone deacetylases (HDACs) may affect pancreatic endocrine function and glucose homeostasis through alterations in gene regulation. Our aim was to investigate the role of HDAC7 in human and rat pancreatic islets and clonal INS-1 beta cells (INS-1?832/13).

Methods

To explore the role of HDAC7 in pancreatic islets and clonal beta cells, we used RNA sequencing, mitochondrial functional analyses, microarray techniques, and HDAC inhibitors MC1568 and trichostatin A.

Results

Using RNA sequencing, we found increased HDAC7 expression in human pancreatic islets from type 2 diabetic compared with non-diabetic donors. HDAC7 expression correlated negatively with insulin secretion in human islets. To mimic the situation in type 2 diabetic islets, we overexpressed Hdac7 in rat islets and clonal beta cells. In both, Hdac7 overexpression resulted in impaired glucose-stimulated insulin secretion. Furthermore, it reduced insulin content, mitochondrial respiration and cellular ATP levels in clonal beta cells. Overexpression of Hdac7 also led to changes in the genome-wide gene expression pattern, including increased expression of Tcf7l2 and decreased expression of gene sets regulating DNA replication and repair as well as nucleotide metabolism. In accordance, Hdac7 overexpression reduced the number of beta cells owing to enhanced apoptosis. Finally, we found that inhibiting HDAC7 activity with pharmacological inhibitors or small interfering RNA-mediated knockdown restored glucose-stimulated insulin secretion in beta cells that were overexpressing Hdac7.

Conclusions/interpretation

Taken together, these results indicate that increased HDAC7 levels caused beta cell dysfunction and may thereby contribute to defects seen in type 2 diabetic islets. Our study supports HDAC7 inhibitors as a therapeutic option for the treatment of type 2 diabetes.

     

Production and function of IL-12 in islets and beta cells

Aims/hypothesis

IL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells.

Methods

In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ).

Results

The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines.

Conclusions/interpretation

These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.     

Identification of an intracellular metabolic signature impairing beta cell function in the rat beta cell line INS-1E and human islets

Aims/hypothesis

Chronic hyperglycaemia promotes the progressive failure of pancreatic beta cells in patients with type 2 diabetes mellitus, a clinically highly relevant phenomenon known as glucotoxicity. The intracellular metabolic consequences of a chronically high availability of glucose in beta cells are, as yet, poorly understood in its full complexity.

Methods

An unbiased metabolite profiling analysis (GC-time-of-flight-MS) was used to identify the time course of core metabolite patterns in rat beta cell line INS-1E during exposure to high glucose concentrations and its relation to insulin expression.

Results

We report here that pentose phosphate pathway (PPP) metabolites accumulate remarkably during chronic but not acute glucose treatment, indicating altered processing of glucose through the pentose phosphate pathway. Subsequent functional studies in INS-1E cells and human islets revealed that a disturbance in this pathway contributes to decreases in insulin gene expression and a lack of glucose-stimulated insulin secretion. These effects were found to depend on the activation of extracellular-regulated-kinase (ERK1/2). Long-term inhibition of 6-phosphogluconic acid dehydrogenase resulted in accumulation of PPP metabolites, induced ERK1/2 activation independently of high glucose and impaired beta cell function. In turn, inhibition of ERK1/2 overstimulation during chronic glucose exposure partly inhibited metabolite accumulation and restored beta cell function.

Conclusions/interpretation

Based on unbiased metabolite analyses, the data presented here provide novel targets, namely the inhibition of PPP metabolite accumulation towards the therapeutic goal to preserve and potentially improve beta cell function in diabetes.     

The loss of Sirt1 in mouse pancreatic beta cells impairs insulin secretion by disrupting glucose sensing

Aims/hypothesis

Sirtuin 1 (SIRT1) has emerged as a key metabolic regulator of glucose homeostasis and insulin secretion. Enhanced SIRT1 activity has been shown to be protective against diabetes, although the mechanisms remain largely unknown. The aim of this study was to determine how SIRT1 regulates insulin secretion in the pancreatic beta cell.

Methods

Pancreatic beta cell-specific Sirt1 deletion was induced by tamoxifen injection in 9-week-old Pdx1CreER:floxSirt1 mice (Sirt1BKO). Controls were injected with vehicle. Mice were assessed metabolically via glucose challenge, insulin tolerance tests and physical variables. In parallel, Sirt1 short interfering RNA-treated MIN6 cells (SIRT1KD) and isolated Sirt1BKO islets were used to investigate the effect of SIRT1 inactivation on insulin secretion and gene expression.

Results

OGTTs showed impaired glucose disposal in Sirt1BKO mice due to insufficient insulin secretion. Isolated Sirt1BKO islets and SIRT1KD MIN6 cells also exhibited impaired glucose-stimulated insulin secretion. Subsequent analyses revealed impaired α-ketoisocaproic acid-induced insulin secretion and attenuated glucose-induced Ca²⁺ influx, but normal insulin granule exocytosis in Sirt1BKO beta cells. Microarray studies revealed a large cluster of mitochondria-related genes, the expression of which was dysregulated in SIRT1KD MIN6 cells. Upon further analysis, we demonstrated an explicit defect in mitochondrial function: the inability to couple nutrient metabolism to mitochondrial membrane hyperpolarisation and reduced oxygen consumption rates.

Conclusions/interpretation

Taken together, these findings indicate that in beta cells the deacetylase SIRT1 regulates the expression of specific mitochondria-related genes that control metabolic coupling, and that a decrease in beta cell Sirt1 expression impairs glucose sensing and insulin secretion.