Glucagon-like peptide-1 analogues in monogenic syndromic obesity: Real-world data from a large cohort of Alström syndrome patients.

AIM: To examine the real-world efficacy of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) in monogenic obesity in patients with Alström syndrome (ALMS). METHODS: We screened 72 UK adult patients with ALMS and offered treatment to 34 patients meeting one of the following criteria: body mass index of 25 kg/m2 or higher, insulin resistance, suboptimal glycaemic control on antihyperglycaemic medications or non-alcoholic fatty liver disease. RESULTS: In total, 30 patients, with a mean age of 31 ± 11 years and a male to-female ratio of 2:1, completed 6 months of treatment with GLP-1 RAs either in the form of semaglutide or exenatide. On average, treatment with GLP-1 RAs reduced body weight by 5.4 ± 1.7 (95% confidence interval [CI] 3.6-7) kg and HbA1c by 12 ± 3.3 (95% CI 8.7-15.3) mmol/mol, equating to 6% weight loss (P < .01) and 1.1% absolute reduction in HbA1c (P < .01). Significant improvements were also observed in serum total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and alanine aminotransferase. The improvement of metabolic variables in our cohort of monogenic syndromic obesity was comparable with data for polygenic obesity, irrespective of weight loss. CONCLUSIONS: Data from our centre highlight the non-inferiority of GLP-1 RAs in monogenic syndromic obesity to the available GLP-1 RA-use data in polygenic obesity, therefore, these agents can be considered as a treatment option in patients with ALMS, as well as other forms of monogenic obesity.

Adipocyte-specific deletion of Tcf7l2 induces dysregulated lipid metabolism and impairs glucose tolerance in mice.

AIMS/HYPOTHESIS: Transcription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/ß-catenin signalling pathway implicated in type 2 diabetes risk through genome-wide association studies. Although its expression is critical for adipocyte development, the potential roles of changes in adipose tissue TCF7L2 levels in diabetes risk are poorly defined. Here, we investigated whether forced changes in Tcf7l2 expression in adipocytes affect whole body glucose or lipid metabolism and crosstalk between disease-relevant tissues. METHODS: Tcf7l2 was selectively ablated in mature adipocytes in C57BL/6J mice using Cre recombinase under Adipoq promoter control to recombine Tcf7l2 alleles floxed at exon 1 (referred to as aTCF7L2 mice). aTCF7L2 mice were fed normal chow or a high-fat diet for 12 weeks. Glucose and insulin sensitivity, as well as beta cell function, were assessed in vivo and in vitro. Levels of circulating NEFA, selected hormones and adipokines were measured using standard assays. RESULTS: Reduced TCF7L2 expression in adipocytes altered glucose tolerance and insulin secretion in male but not in female mice. Thus, on a normal chow diet, male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance at 16 weeks (p = 0.03) and increased fat mass (1.4 ± 0.1-fold, p = 0.007) but no changes in insulin secretion. In contrast, male homozygote knockout (aTCF7L2hom) mice displayed normal body weight but impaired oral glucose tolerance at 16 weeks (p = 0.0001). These changes were mechanistically associated with impaired in vitro glucose-stimulated insulin secretion (decreased 0.5 ± 0.1-fold vs control mice, p = 0.02) and decreased levels of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (0.6 ± 0.1-fold and 0.4 ± 0.1-fold vs control mice, p = 0.04 and p < 0.0001, respectively). Circulating levels of plasma NEFA and fatty acid binding protein 4 were increased by 1.3 ± 0.1-fold and 1.8 ± 0.3-fold vs control mice (p = 0.03 and p = 0.05, respectively). Following exposure to a high-fat diet for 12 weeks, male aTCF7L2hom mice exhibited reduced in vivo glucose-stimulated insulin secretion (0.5 ± 0.1-fold vs control mice, p = 0.02). CONCLUSIONS/INTERPRETATION: Loss of Tcf7l2 gene expression selectively in adipocytes leads to a sexually dimorphic phenotype, with impairments not only in adipocytes, but also in pancreatic islet and enteroendocrine cells in male mice only. Our findings suggest novel roles for adipokines and incretins in the effects of diabetes-associated variants in TCF7L2, and further illuminate the roles of TCF7L2 in glucose homeostasis and diabetes risk. Graphical abstract.

The pore-forming subunit MCU of the mitochondrial Ca2+ uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice.

AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.

Transcription factor-7-like 2 (TCF7L2) gene acts downstream of the Lkb1/Stk11 kinase to control mTOR signaling, ß cell growth, and insulin secretion.

Variants in the transcription factor-7-like 2 (TCF7L2/TCF4) gene, involved in Wnt signaling, are associated with type 2 diabetes. Loss of Tcf7l2 selectively from the ß cell in mice has previously been shown to cause glucose intolerance and to lower ß cell mass. Deletion of the tumor suppressor liver kinase B1 (LKB1/STK11) leads to ß cell hyperplasia and enhanced glucose-stimulated insulin secretion, providing a convenient genetic model for increased ß cell growth and function. The aim of this study was to explore the possibility that Tcf7l2 may be required for the effects of Lkb1 deletion on insulin secretion in the mouse ß cell. Mice bearing floxed Lkb1 and/or Tcf7l2 alleles were bred with knockin mice bearing Cre recombinase inserted at the Ins1 locus (Ins1Cre), allowing highly ß cell-selective deletion of either or both genes. Oral glucose tolerance was unchanged by the further deletion of a single Tcf7l2 allele in these cells. By contrast, mice lacking both Tcf7l2 alleles on this background showed improved oral glucose tolerance and insulin secretion in vivo and in vitro compared with mice lacking a single Tcf7l2 allele. Biallelic Tcf7l2 deletion also enhanced ß cell proliferation, increased ß cell mass, and caused changes in polarity as revealed by the "rosette-like" arrangement of ß cells. Tcf7l2 deletion also increased signaling by mammalian target of rapamycin (mTOR), augmenting phospho-ribosomal S6 levels. We identified a novel signaling mechanism through which a modifier gene, Tcf7l2, lies on a pathway through which LKB1 acts in the ß cell to restrict insulin secretion.

Pancreatic alpha cell-selective deletion of Tcf7l2 impairs glucagon secretion and counter-regulatory responses to hypoglycaemia in mice.

AIMS/HYPOTHESIS: Transcription factor 7-like 2 (TCF7L2) is a high mobility group (HMG) box-containing transcription factor and downstream effector of the Wnt signalling pathway. SNPs in the TCF7L2 gene have previously been associated with an increased risk of type 2 diabetes in genome-wide association studies. In animal studies, loss of Tcf7l2 function is associated with defective islet beta cell function and survival. Here, we explore the role of TCF7L2 in the control of the counter-regulatory response to hypoglycaemia by generating mice with selective deletion of the Tcf7l2 gene in pancreatic alpha cells. METHODS: Alpha cell-selective deletion of Tcf7l2 was achieved by crossing mice with floxed Tcf7l2 alleles to mice bearing a Cre recombinase transgene driven by the preproglucagon promoter (PPGCre), resulting in Tcf7l2AKO mice. Glucose homeostasis and hormone secretion in vivo and in vitro, and islet cell mass were measured using standard techniques. RESULTS: While glucose tolerance was unaffected in Tcf7l2AKO mice, glucose infusion rates were increased (AUC for glucose during the first 60 min period of hyperinsulinaemic-hypoglycaemic clamp test was increased by 1.98 ± 0.26-fold [p < 0.05; n = 6] in Tcf7l2AKO mice vs wild-type mice) and glucagon secretion tended to be lower (plasma glucagon: 0.40 ± 0.03-fold vs wild-type littermate controls [p < 0.01; n = 6]). Tcf7l2AKO mice displayed reduced fasted plasma glucose concentration. Glucagon release at low glucose was impaired in islets isolated from Tcf7l2AKO mice (0.37 ± 0.02-fold vs islets from wild-type littermate control mice [p < 0.01; n = 6). Alpha cell mass was also reduced (72.3 ± 20.3% [p < 0.05; n = 7) in Tcf7l2AKO mice compared with wild-type mice. CONCLUSIONS/INTERPRETATION: The present findings demonstrate an alpha cell-autonomous role for Tcf7l2 in the control of pancreatic glucagon secretion and the maintenance of alpha cell mass and function.

Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression.

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis.

Selective disruption of Tcf7l2 in the pancreatic ß cell impairs secretory function and lowers ß cell mass.

Type 2 diabetes (T2D) is characterized by ß cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired ß cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the ß cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in ß cells from the earliest expression of the Ins1 gene (∼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (∼20%) and Glp1r (∼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual ß cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ∼30% decrease in ß cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of ß cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.

Cell type-specific deletion in mice reveals roles for PAS kinase in insulin and glucagon production.

AIMS/HYPOTHESIS: Per-Arnt-Sim kinase (PASK) is a nutrient-regulated domain-containing protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of PASK in the control of islet hormone release, by generating mice with selective deletion of the Pask gene in pancreatic beta or alpha cells. METHODS: Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1 (Cre); PaskBKO) or alpha (Ppg (Cre) [also known as Gcg]; PaskAKO) cell-selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression and islet cell mass were measured using standard techniques. RESULTS: Ins1 (Cre)-based recombination led to efficient beta cell-targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p < 0.05) compared with controls in PaskBKO mice, as well as in global Pask-null mice (38%, p < 0.05). PaskBKO mice displayed normal body weight and fasting glycaemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high-fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycaemic clamps. Although alpha cell mass was increased (21.9%, p < 0.05), glucagon release at low glucose was impaired (p < 0.05) in PaskAKO islets. CONCLUSIONS/INTERPRETATION: The findings demonstrate cell-autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differences between the glycaemic phenotype of global vs cell type-specific null mice suggest important roles for tissue interactions in the control of glycaemia by PASK.

Sulfation pathways in the maintenance of functional beta-cell mass and implications for diabetes.

Diabetes Type 1 and Type 2 are widely occurring diseases. In spite of a vast amount of biomedical literature about diabetic processes in general, links to certain biological processes are only becoming evident these days. One such area of biology is the sulfation of small molecules, such as steroid hormones or metabolites from the gastrointestinal tract, as well as larger biomolecules, such as proteins and proteoglycans. Thus, modulating the physicochemical propensities of the different sulfate acceptors, resulting in enhanced solubility, expedited circulatory transit, or enhanced macromolecular interaction. This review lists evidence for the involvement of sulfation pathways in the maintenance of functional pancreatic beta-cell mass and the implications for diabetes, grouped into various classes of sulfated biomolecule. Complex heparan sulfates might play a role in the development and maintenance of beta-cells. The sulfolipids sulfatide and sulfo-cholesterol might contribute to beta-cell health. In beta-cells, there are only very few proteins with confirmed sulfation on some tyrosine residues, with the IRS4 molecule being one of them. Sulfated steroid hormones, such as estradiol-sulfate and vitamin-D-sulfate, may facilitate downstream steroid signaling in beta-cells, following de-sulfation. Indoxyl sulfate is a metabolite from the intestine, that causes kidney damage, contributing to diabetic kidney disease. Finally, from a technological perspective, there is heparan sulfate, heparin, and chondroitin sulfate, that all might be involved in next-generation beta-cell transplantation. Sulfation pathways may play a role in pancreatic beta-cells through multiple mechanisms. A more coherent understanding of sulfation pathways in diabetes will facilitate discussion and guide future research.

Beta cell lipotoxicity in the development of type 2 diabetes: the need for species-specific understanding.

The propensity to develop type 2 diabetes (T2D) is known to have both environmental and hereditary components. In those with a genetic predisposition to T2D, it is widely believed that elevated concentrations of circulatory long-chain fatty acids (LC-FFA) significantly contribute towards the demise of insulin-producing pancreatic ß-cells - the fundamental feature of the development of T2D. Over 25 years of research support that LC-FFA are deleterious to ß-cells, through a process termed lipotoxicity. However, the work underpinning the theory of ß-cell lipotoxicity is mostly based on rodent studies. Doubts have been raised as to whether lipotoxicity also occurs in humans. In this review, we examine the evidence, both in vivo and in vitro, for the pathogenic effects of LC-FFA on ß-cell viability and function in humans, highlighting key species differences. In this way, we aim to uncover the role of lipotoxicity in the human pathogenesis of T2D and motivate the need for species-specific understanding.

Nucleo-cytosolic shuttling of FoxO1 directly regulates mouse Ins2 but not Ins1 gene expression in pancreatic beta cells (MIN6).

The Forkhead box transcription factor FoxO1 regulates metabolic gene expression in mammals. FoxO1 activity is tightly controlled by phosphatidylinositol 3-kinase (PI3K) signaling, resulting in its phosphorylation and nuclear exclusion. We sought here to determine the mechanisms involved in glucose and insulin-stimulated nuclear shuttling of FoxO1 in pancreatic ß cells and its consequences for preproinsulin (Ins1, Ins2) gene expression. Nuclear-localized endogenous FoxO1 translocated to the cytosol in response to elevated glucose (3 versus 16.7 mM) in human islet ß cells. Real-time confocal imaging of nucleo-cytosolic shuttling of a FoxO1-EGFP chimera in primary mouse and clonal MIN6 ß cells revealed a time-dependent glucose-responsive nuclear export, also mimicked by exogenous insulin, and blocked by suppressing insulin secretion. Constitutively active PI3K or protein kinase B/Akt exerted similar effects, while inhibitors of PI3K, but not of glycogen synthase kinase-3 or p70 S6 kinase, blocked nuclear export. FoxO1 overexpression reversed the activation by glucose of pancreatic duodenum homeobox-1 (Pdx1) transcription. Silencing of FoxO1 significantly elevated the expression of mouse Ins2, but not Ins1, mRNA at 3 mM glucose. Putative FoxO1 binding sites were identified in the distal promoter of rodent Ins2 genes and direct binding of FoxO1 to the Ins2 promoter was demonstrated by chromatin immunoprecipitation. A 915-bp glucose-responsive Ins2 promoter was inhibited by constitutively active FoxO1, an effect unaltered by simultaneous overexpression of PDX1. We conclude that nuclear import of FoxO1 contributes to the suppression of Pdx1 and Ins2 gene expression at low glucose, the latter via a previously unsuspected and direct physical interaction with the Ins2 promoter.

Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells.

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic ß cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in ß cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in ß cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.

Dynamic changes in cytosolic and mitochondrial ATP levels in pancreatic acinar cells.

BACKGROUND & AIMS: Previous studies of pancreatic acinar cells characterized the effects of Ca(2+)-releasing secretagogues and substances, inducing acute pancreatitis on mitochondrial Ca(2+), transmembrane potential, and NAD(P)H, but dynamic measurements of the crucial intracellular adenosine triphosphate (ATP) levels have not been reported. Here we characterized the effects of these agents on ATP levels in the cytosol and mitochondria. METHODS: ATP levels were monitored using cytosolic- or mitochondrial-targeted luciferases. RESULTS: Inhibition of oxidative phosphorylation produced a substantial decrease in cytosolic ATP comparable to that induced by inhibition of glycolysis. Cholecystokinin-8 (CCK) increased cytosolic ATP in spite of accelerating ATP consumption. Acetylcholine, caerulein, and bombesin had similar effect. A bile acid, taurolithocholic acid 3-sulfate (TLC-S); a fatty acid, palmitoleic acid (POA); and palmitoleic acid ethyl ester (POAEE) reduced cytosolic ATP. The ATP decrease in response to these substances was observed in cells with intact or inhibited oxidative phosphorylation. TLC-S, POA, and POAEE reduced mitochondrial ATP, whereas physiological CCK increased mitochondrial ATP. Supramaximal CCK produced a biphasic response composed of a small initial decline followed by a stronger increase. CONCLUSIONS: Both glycolysis and oxidative phosphorylation make substantial contributions to ATP production in acinar cells. Ca(2+)-releasing secretagogues increased ATP level in the cytosol and mitochondria of intact isolated cells. TLC-S, POA, and POAEE reduced cytosolic and mitochondrial ATP. When cells rely on nonoxidative ATP production, secretagogues as well as TLC-S, POA, and POAEE all diminish cytosolic ATP levels.

Sodium-potassium ATPase 1 subunit is a molecular partner of Wolframin, an endoplasmic reticulum protein involved in ER stress.

Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene encoding an endoplasmic reticulum (ER) membrane protein, Wolframin. Although its precise functions are unknown, Wolframin deficiency increases ER stress, impairs cell cycle progression and affects calcium homeostasis. To gain further insight into its function and identify molecular partners, we used the WFS1-C-terminal domain as bait in a yeast two-hybrid screen with a human brain cDNA library. Na+/K+ ATPase beta1 subunit was identified as an interacting clone. We mapped the interaction to the WFS1 C-terminal and transmembrane domains, but not the N-terminal domain. Our mapping data suggest that the interaction most likely occurs in the ER. We confirmed the interaction by co-immunoprecipitation in mammalian cells and with endogenous proteins in JEG3 placental cells, neuroblastoma SKNAS and pancreatic MIN6 beta cells. Na+/K+ ATPase beta1 subunit expression was reduced in plasma membrane fractions of human WFS1 mutant fibroblasts and WFS1 knockdown MIN6 pancreatic beta-cells compared with wild-type cells; Na+/K+ ATPase alpha1 subunit expression was also reduced in WFS-depleted MIN6 beta cells. Induction of ER stress in wild-type cells only partly accounted for the reduced Na+/K+ ATPase beta1 subunit expression observed. We conclude that the interaction may be important for Na+/K+ ATPase beta1 subunit maturation; loss of this interaction may contribute to the pathology seen in Wolfram syndrome via reductions in sodium pump alpha1 and beta1 subunit expression in pancreatic beta-cells.

ChREBP regulates Pdx-1 and other glucose-sensitive genes in pancreatic ß-cells.

Carbohydrate responsive element-binding protein (ChREBP) is a transcription factor whose expression and activity are increased in pancreatic ß-cells maintained at elevated glucose concentrations. We show here that ChREBP inactivation in clonal pancreatic MIN6 ß-cells results in an increase in Pdx-1 expression at low glucose and to a small, but significant, increase in Ins2, GcK and MafA gene expression at high glucose concentrations. Conversely, adenovirus-mediated over-expression of ChREBP in mouse pancreatic islets results in decreases in Pdx-1, MafA, Ins1, Ins2 and GcK mRNA levels. These data suggest that strategies to reduce ChREBP activity might protect against ß-cell dysfunction in type 2 diabetes.

Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization.

Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature beta-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in beta-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 beta-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51+/-0.2% decrease in Ser-269 phosphorylation in MIN6 beta-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.

Metabolic and Functional Heterogeneity in Pancreatic ß Cells.

Metabolic and secretory heterogeneity are fundamental properties of pancreatic islet ß cells. Emerging data suggest that stable differences in the transcriptome and proteome of individual cells may create cellular hierarchies, which, in turn, establish coordinated functional networks. These networks appear to govern the secretory activity of the whole islet and be affected in some forms of diabetes mellitus. Functional imaging, for example, of intracellular calcium dynamics, has led to the demonstration of "small worlds" behavior, and the identification of highly connected "hub" (or "leader") cells and of follower populations subservient to them. Subsequent inactivation of members of either population, for example, using optogenetic approaches or photoablation, has confirmed the importance of hub cells as possible pacemakers. Hub cells appear to be enriched for the glucose phosphorylating enzyme glucokinase and for genes encoding other enzymes involved in glucose metabolism compared to follower cells. Recent findings have shown the relevance of cellular hierarchy in islets from multiple species including human, mouse and fish, and shown that it is preserved in vivo in the context of the fully vascularized and innervated islet. Importantly, connectivity is impaired by insults, which mimic the diabetic milieu, including high glucose and/or fatty levels, and by the ablation of genes associated with type 2 diabetes risk in genome-wide association studies. We discuss here the evidence for the existence of these networks and their failure in disease settings. We also briefly survey the challenges in understanding their properties.

Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion.

Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in ß cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.

Convolutional neural networks for reconstruction of undersampled optical projection tomography data applied to in vivo imaging of zebrafish.

Optical projection tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of projection angles using the Radon transform, as is typically implemented with optically cleared samples of the mm-to-cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts-reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to compressed sensing with 40% fewer projections. We further show that diverse training data classes, for example, ex vivo mouse tissue data, can be used for CNN-based reconstructions of OPT data of other species including live zebrafish.