Cells Deploy a Two-Pronged Strategy to Rectify Misfolded Proinsulin Aggregates.

Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded proinsulin accumulation remain incompletely understood. Here, we report that Akita mutant proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). We show that enhanced RTN-dependent clearance of aggregated Akita proinsulin helps to restore ER export of WT proinsulin, which can promote WT insulin production, potentially alleviating MIDY. We also find that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates.

Nutrient-dependent regulation of ß-cell proinsulin content.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that ß-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.

miR-200b-3p Induces the Formation of Insulin-Producing Cells from Umbilical Cord Mesenchymal Stem Cells by Targeting ZEB2.

Studies have reported that miRNAs regulate ß-cell differentiation, pancreatic development, and insulin secretion. However, the biological function of miRNAs during the formation of insulin-producing cells (IPCs) from umbilical cord-derived mesenchymal stem cells (UCMSCs) is poorly understood. Herein, the role and mechanism of miR-200b-3p during UCMSC differentiation into IPCs were investigated. UCMSCs were induced for IPC differentiation. An animal model was established by transplanting UCMSC-derived IPCs into streptozotocin-induced diabetic mice. Cell surface markers of undifferentiated UCMSCs and the expression of proinsulin and Pdx-1 in UCMSC-derived IPCs were measured by flow cytometry analysis. The interaction between miR-200b-3p and zinc finger E-box binding homeobox 2 (ZEB2) 3' untranslated region (UTR) was confirmed by luciferase reporter assay. Insulin secretion in UCMSC-derived IPCs was measured by enzyme-linked immunosorbent assay (ELISA). Islet marker (insulin and Pdx-1) levels were evaluated using immunofluorescence staining. In this study, undifferentiated UCMSCs had MSC phenotype and the potential for osteogenesis and adipogenesis. UCMSC-derived IPCs displayed glucose responsive insulin secretion and expressed insulin, Pdx-1 and proinsulin. miR-200b-3p was overexpressed in UCMSC-derived IPCs. Mechanically, miR-200b-3p targeted ZEB2. ZEB2 knockdown reversed the inhibitory effect of miR-200b-3p downregulation on IPC differentiation from UCMSCs in vitro. Moreover, miR-200b-3p silencing inhibited the anti-hypoglycemic effects and insulinogenesis of UCMSC-derived IPCs grafts in the kidney capsule of diabetic mice. Overall, miR-200b-3p induces the formation of IPCs from UCMSCs by targeting ZEB2.

The CLOCK protein regulates insulin secretion related with L-type calcium channels in rat pancreatic beta cells.

BACKGROUND: Circadian locomotor output cycles kaput protein (CLOCK) plays a crucial role in glucose homeostasis and controlling insulin secretion. However, the mechanism of the CLOCK regulating rhythmic insulin secretion has not been fully understood. METHODS: Rhythmic expression of the CLOCK in rat pancreatic beta cell was detected. INS-1 cells were transfected with siRNAs to knockdown the CLOCK before the cells were incubated with different concentrations of glucose. Insulin secretion was analyzed by ELISA method. Expression of the L-type calcium channel protein (Cav1.2, Cacna1c) was determined both in the CLOCK-knockdown cells and the control cells. Calcium influx was probed by fluorescent. Chromatin immunoprecipitation (ChIP) test and dual-luciferase reporter gene experiments were applied to verify the relationship between the CLOCK and Cav1.2. RESULTS: The CLOCK is abundantly expressed in rat pancreatic beta cells. Transcription level of the CLOCK showed rhythmicity in the beta cells. Compared to the control group, insulin release was significantly impaired with 25 mM glucose incubation in the CLOCK-knockdown group, but not showed with 2.5 mM glucose incubation. The expression of Cav1.2 and the influx of calcium were significantly decreased in the CLOCK-knockdown group with 25 mM glucose incubation. ChIP test indicted that the CLOCK bound to -444∼-454 region of the Cacna1c promoter of the INS-1 cells, but the binding was significantly reduced following the CLOCK-knockdown. Luciferase experiment was in accordance with the finding of ChIP. CONCLUSIONS: The CLOCK mediating Cav1.2 expression may point out a potential pathway of circadian rhythm affecting insulin secretion.

Engineering-inspired approaches to study ß-cell function and diabetes.

Strategies to mitigate the pathologies from diabetes range from simply administering insulin to prescribing complex drug/biologic regimens combined with lifestyle changes. There is a substantial effort to better understand ß-cell physiology during diabetes pathogenesis as a means to develop improved therapies. The convergence of multiple fields ranging from developmental biology to microfluidic engineering has led to the development of new experimental systems to better study complex aspects of diabetes and ß-cell biology. Here we discuss the available insulin-secreting cell types used in research, ranging from primary human ß-cells, to cell lines, to pluripotent stem cell-derived ß-like cells. Each of these sources possess inherent strengths and weaknesses pertinent to specific applications, especially in the context of engineered platforms. We then outline how insulin-expressing cells have been used in engineered platforms and how recent advances allow for better mimicry of in vivo conditions. Chief among these conditions are ß-cell interactions with other endocrine organs. This facet is beginning to be thoroughly addressed by the organ-on-a-chip community, but holds enormous potential in the development of novel diabetes therapeutics. Furthermore, high throughput strategies focused on studying ß-cell biology, improving ß-cell differentiation, or proliferation have led to enormous contributions in the field and will no doubt be instrumental in bringing new diabetes therapeutics to the clinic.

Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin.

The conserved endoplasmic reticulum (ER) membrane protein TRAPα (translocon-associated protein, also known as signal sequence receptor 1, SSR1) has been reported to play a critical but unclear role in insulin biosynthesis. TRAPα/SSR1 is one component of a four-protein complex including TRAPß/SSR2, TRAPγ/SSR3, and TRAPδ/SSR4. The TRAP complex topologically has a small exposure on the cytosolic side of the ER via its TRAPγ/SSR3 subunit, whereas TRAPß/SSR2 and TRAPδ/SSR4 function along with TRAPα/SSR1 largely on the luminal side of the ER membrane. Here, we have examined pancreatic ß-cells with deficient expression of either TRAPß/SSR2 or TRAPδ/SSR4, which does not perturb mRNA expression levels of other TRAP subunits, or insulin mRNA. However, deficient protein expression of TRAPß/SSR2 and, to a lesser degree, TRAPδ/SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin. Deficient TRAPß/SSR2 or TRAPδ/SSR4 is not associated with any apparent defect of exocytotic mechanism but rather by a decreased abundance of the proinsulin and insulin that accompanies glucose-stimulated secretion. Amino acid pulse labeling directly establishes that much of the steady-state deficiency of intracellular proinsulin can be accounted for by diminished proinsulin biosynthesis, observed in a pulse-labeling as short as 5 minutes. The proinsulin and insulin levels in TRAPß/SSR2 or TRAPδ/SSR4 null mutant ß-cells are notably recovered upon re-expression of the missing TRAP subunit, accompanying a rebound of proinsulin biosynthesis. Remarkably, overexpression of TRAPα/SSR1 can also suppress defects in ß-cells with diminished expression of TRAPß/SSR2, strongly suggesting that TRAPß/SSR2 is needed to support TRAPα/SSR1 function.

IGF-1R/YAP signaling pathway is involved in collagen V-induced insulin biosynthesis and secretion in rat islet INS-1 cells.

PURPOSE: Type V collagen (collagen V) is one of the important components of extracellular matrix (ECM) in pancreas. We previously reported that pre-coating collagen V on the culture dishes enhanced insulin production in INS-1 rat pancreatic ß cells. In this study, we investigate the underlying mechanism. RESULTS: Insulin biosynthesis and secretion are both increased in INS-1 cells cultured on collagen V-coated dishes, accompanied by the reduced nuclear translocation of Yes-associated protein (YAP), a transcriptional co-activator. YAP, the downstream effector of Hippo signaling pathway, plays an important role in the development and function of pancreas. Inhibition of YAP activation by verteporfin further up-regulates insulin biosynthesis and secretion. Silencing large tumor suppressor (LATS), a core component of Hippo pathway which inhibits activity of YAP by phosphorylation, by siRNA transfection inhibits both insulin biosynthesis and secretion. In the present study, the protein level of insulin-like growth factor 1 receptor (IGF-1 R), detected as the upstream molecule of YAP, is reduced in the INS-1 cells cultured on the dishes coated with collagen V. The silencing of IGF-1 R by siRNA transfection further enhances insulin biosynthesis and secretion. IGF-1 treatment reduces collagen V-induced up-regulation of insulin biosynthesis and secretion, accompanying the increased nuclear YAP. CONCLUSION: Inhibition of IGF-1 R/YAP signal pathway is involved in collagen V-induced insulin biosynthesis and secretion in INS-1 cells.

Identification of C2CD4A as a human diabetes susceptibility gene with a role in ß cell insulin secretion.

Fine mapping and validation of genes causing ß cell failure from susceptibility loci identified in type 2 diabetes genome-wide association studies (GWAS) poses a significant challenge. The VPS13C-C2CD4A-C2CD4B locus on chromosome 15 confers diabetes susceptibility in every ethnic group studied to date. However, the causative gene is unknown. FoxO1 is involved in the pathogenesis of ß cell dysfunction, but its link to human diabetes GWAS has not been explored. Here we generated a genome-wide map of FoxO1 superenhancers in chemically identified ß cells using 2-photon live-cell imaging to monitor FoxO1 localization. When parsed against human superenhancers and GWAS-derived diabetes susceptibility alleles, this map revealed a conserved superenhancer in C2CD4A, a gene encoding a ß cell/stomach-enriched nuclear protein of unknown function. Genetic ablation of C2cd4a in ß cells of mice phenocopied the metabolic abnormalities of human carriers of C2CD4A-linked polymorphisms, resulting in impaired insulin secretion during glucose tolerance tests as well as hyperglycemic clamps. C2CD4A regulates glycolytic genes, and notably represses key ß cell "disallowed" genes, such as lactate dehydrogenase A We propose that C2CD4A is a transcriptional coregulator of the glycolytic pathway whose dysfunction accounts for the diabetes susceptibility associated with the chromosome 15 GWAS locus.

Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic ß-cells by activating Phospholipase C signal pathway in diabetic mice.

In early stage of diabetes, insulin secretion from pancreatic ß-cells is increased to deal with the elevated blood glucose. Previous studies have reported that islet-produced carbon monoxide (CO) is associated with increased glucose-stimulated insulin secretion from ß-cells. However, this compensatory mechanism by which CO may act to enhance ß-cell function remain unclear. In this study, we revealed that CO promoted intracellular calcium ([Ca2+]i) elevation and glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells in leptin receptor deficient db/db mice but not in C57 mice. The stimulatory effects of CO on ß-cell function in db/db mice was blocked by inhibition of Phospholipase C (PLC) signaling pathway. We further demonstrated that CO triggered [Ca2+]i transients and enhanced GSIS in C57 islets when ß-cells overexpressed with PLCγ1 and PLCδ1, but not PLCß1. On the other hand, reducing PLCγ1 and PLCδ1 expressions in db/db islets dramatically attenuated the stimulatory effects of CO on ß-cell function, whereas interfering PLCß1 expression had no effects on CO-induced ß-cell function enhancement. Our findings showing that CO elevated [Ca2+]i and enhanced GSIS by activating PLC signaling through PLCγ1 and PLCδ1 isoforms in db/db pancreatic ß-cells may suggest an important mechanism by which CO promotes ß-cell function to prevent hyperglycemia. Our study may also provide new insights into the therapy for type II diabetes and offer a potential target for therapeutic applications of CO.

Effect of alginate matrix engineered to mimic the pancreatic microenvironment on encapsulated islet function.

Islet transplantation is emerging as a therapeutic option for type 1 diabetes, albeit, only a small number of patients meeting very stringent criteria are eligible for the treatment because of the side effects of the necessary immunosuppressive therapy and the relatively short time frame of normoglycemia that most patients achieve. The challenge of the immune-suppressive regimen can be overcome through microencapsulation of the islets in a perm-selective coating of alginate microbeads with poly-l-lysine or poly- l-ornithine. In addition to other issues including the nutrient supply challenge of encapsulated islets a critical requirement for these cells has emerged as the need to engineer the microenvironment of the encapsulation matrix to mimic that of the native pancreatic scaffold that houses islet cells. That microenvironment includes biological and mechanical cues that support the viability and function of the cells. In this study, the alginate hydrogel was modified to mimic the pancreatic microenvironment by incorporation of extracellular matrix (ECM). Mechanical and biological changes in the encapsulating alginate matrix were made through stiffness modulation and incorporation of decellularized ECM, respectively. Islets were then encapsulated in this new biomimetic hydrogel and their insulin production was measured after 7 days in vitro. We found that manipulation of the alginate hydrogel matrix to simulate both physical and biological cues for the encapsulated islets enhances the mechanical strength of the encapsulated islet constructs as well as their function. Our data suggest that these modifications have the potential to improve the success rate of encapsulated islet transplantation.

Large scale purification and characterization of A21 deamidated variant-most prominent post translational modification (PTM) for insulins which is also widely observed in insulins pharmaceutical manufacturing and storage.

Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and suboptimal product quality. In addition, these variants may undergo immune reactions, henceforth need to be appropriately controlled to ensure consistent product quality and patient safety. Deamidation of insulin is the most common post-translational modification occurring in insulin and insulin analogues. AsnA21 desamido variant is also the most prominent product variant formed during human insulin manufacturing process and/or during the storage. Often, this deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of purified deamidated material is always being challenging due to highly similar mass, ionic, hydrophobic properties, and high structural similarity of the variant compared to the parent product. Present work demonstrates the simplified and efficient scalable process for generation of AsnA21 deamidated variant in powder form with ~96% purity. The mixed-mode property of anion exchange resin PolyQuat was utilized to purify the deamidated impurity with high recovery. Subsequent reversed-phase high performance liquid chromatography (RP-HPLC) step was introduced for concentration of product in bind elute mode. Elution pool undergone isoelectric precipitation and lyophilisation. The lyophilized product allows users for convenient use of the deamidated impurity for intended purposes. Detailed characterization by Mass spectrometry revealed deamidation is at AsnA21 and further confirmed that, structural and functional characterization as well as the biological activity of isolated variant is equivalent to insulin.

Haplotypes of the genes (GCK and G6PC2) underlying the glucose/glucose-6-phosphate cycle are associated with pancreatic beta cell glucose sensitivity in patients with newly diagnosed type 2 diabetes from the VNDS study (VNDS 11).

BACKGROUND: Elevated fasting plasma glucose has been associated with increased risk for development of type 2 diabetes (T2D). The balance between glucokinase (GCK) and glucose-6-phosphate catalytic subunit 2 (G6PC2) activity are involved in glucose homeostasis through glycolytic flux, and subsequent insulin secretion. AIM: In this study, we evaluated the association between the genetic variability of G6PC2 and GCK genes and T2D-related quantitative traits. METHODS: In 794 drug-naïve, GADA-negative, newly diagnosed T2D patients (VNDS; NTC01526720) we performed: genotyping of 6 independent tag-SNPs within GCK gene and 5 tag-SNPs within G6PC2 gene; euglycaemic insulin clamp to assess insulin sensitivity; OGTT to estimate beta-cell function (derivative and proportional control; DC, PC) by mathematical modeling. Genetic association analysis has been conducted using Plink software. RESULTS: Two SNPs within GCK gene (rs882019 and rs1303722) were associated to DC in opposite way (both p < 0.004). Two G6PC2 variants (rs13387347 and rs560887) were associated to both parameters of insulin secretion (DC and PC) and to fasting C-peptide levels (all p < 0.038). Moreover, subjects carrying the A allele of rs560887 showed higher values of 2h-plasma glucose (2hPG) (p = 0.033). Haplotype analysis revealed that GCK (AACAAA) haplotype was associated to decreased fasting C-peptide levels, whereas, the most frequent haplotype of G6PC2 (GGAAG) was associated with higher fasting C-peptide levels (p = 0.001), higher PC (ß = 6.87, p = 0.022) and the lower 2hPG (p = 0.012). CONCLUSION: Our findings confirmed the role of GCK and G6PC2 in regulating the pulsatility in insulin secretion thereby influencing insulin-signaling and leading to a gradual modulation in glucose levels in Italian patients with newly diagnosed T2D.

The RB gene family controls the maturation state of the EndoC-ßH2 human pancreatic ß-cells.

The functional maturation of human pancreatic ß-cells remains poorly understood. EndoC-ßH2 is a human ß-cell line with a reversible immortalized phenotype. Removal of the two oncogenes, SV40LT and hTERT introduced for its propagation, stops proliferation, triggers cell size increase and senescence, promotes mitochondrial activity and amplifies several ß-cell traits and functions. Overall, these events recapitulate several aspects of functional ß-cell maturation. We report here that selective depletion of SV40LT, but not of hTERT, is sufficient to revert EndoC-ßH2 immortalization. SV40LT inhibits the activity of the RB family members and of P53. In EndoC-ßH2 cells, the knock-down of RB itself, and, to a lesser extent, of its relative P130, precludes most events triggered by SV40LT depletion. In contrast, the knock-down of P53 does not prevent reversion of immortalization. Thus, an increase in RB and P130 activity, but not in P53 activity, is required for functional maturation of EndoC-ßH2 cells upon SV40LT-depletion. In addition, RB and/or P130 depletion in SV40LT-expressing EndoC-ßH2 cells decreases cell size, stimulates proliferation, and decreases the expression of key ß-cell genes. Thus, despite SV40LT expression, EndoC-ßH2 cells have a residual RB activity, which when suppressed reverts them to a more immature phenotype. These results show that the expression and activity levels of RB family members, especially RB itself, regulate the maturation state of EndoC-ßH2 cells.

Exploring Insulin Production Following Alveolar Islet Transplantation (AIT).

Recent studies have demonstrated the feasibility of islet implantation into the alveoli. However, until today, there are no data on islet behavior and morphology at their transplant site. This study is the first to investigate islet distribution as well insulin production at the implant site. Using an ex vivo postmortem swine model, porcine pancreatic islets were isolated and aerosolized into the lung using an endoscopic spray-catheter. Lung tissue was explanted and bronchial airways were surgically isolated and connected to a perfusor. Correct implantation was confirmed via histology. The purpose of using this new lung perfusion model was to measure static as well as dynamic insulin excretions following glucose stimulation. Alveolar islet implantation was confirmed after aerosolization. Over 82% of islets were correctly implanted into the intra-alveolar space. The medium contact area to the alveolar surface was estimated at 60 +/- 3% of the total islet surface. The new constructed lung perfusion model was technically feasible. Following static glucose stimulation, insulin secretion was detected, and dynamic glucose stimulation revealed a biphasic insulin secretion capacity during perfusion. Our data indicate that islets secrete insulin following implantation into the alveoli and display an adapted response to dynamic changes in glucose. These preliminary results are encouraging and mark a first step toward endoscopically assisted islet implantation in the lung.

Isoprenoid Derivatives of Lysophosphatidylcholines Enhance Insulin and GLP-1 Secretion through Lipid-Binding GPCRs.

Insulin plays a significant role in carbohydrate homeostasis as the blood glucose lowering hormone. Glucose-induced insulin secretion (GSIS) is augmented by glucagon-like peptide (GLP-1), a gastrointestinal peptide released in response to ingesting nutriments. The secretion of insulin and GLP-1 is mediated by the binding of nutrients to G protein-coupled receptors (GPCRs) expressed by pancreatic ß-cells and enteroendocrine cells, respectively. Therefore, insulin secretagogues and incretin mimetics currently serve as antidiabetic treatments. This study demonstrates the potency of synthetic isoprenoid derivatives of lysophosphatidylcholines (LPCs) to stimulate GSIS and GLP-1 release. Murine insulinoma cell line (MIN6) and enteroendocrinal L cells (GLUTag) were incubated with LPCs bearing geranic acid (1-GA-LPC), citronellic acid (1-CA-LPC), 3,7-dimethyl-3-vinyloct-6-enoic acid (GERA-LPC), and (E)-3,7,11-trimethyl- 3-vinyldodeca-6,10-dienoic acid (1-FARA-LPC). Respective free terpene acids were also tested for comparison. Besides their insulin- and GLP-1-secreting capabilities, we also investigated the cytotoxicity of tested compounds, the ability to intracellular calcium ion mobilization, and targeted GPCRs involved in maintaining lipid and carbohydrate homeostasis. We observed the high cytotoxicity of 1-GERA-LPC and 1-FARA-LPC in contrast 1-CA-LPC and 1-GA-LPC. Moreover, 1-CA-LPC and 1-GA-LPC demonstrated the stimulatory effect on GSIS and 1-CA-LPC augmented GLP-1 secretion. Insulin and GLP-1 release appeared to be GPR40-, GPR55-, GPR119- and GPR120-dependent.

Schisandrin C Affects Glucose-Stimulated Insulin Secretion in Pancreatic ß-Cells and Glucose Uptake in Skeletal Muscle Cells.

The aim of our study was to investigate the effect of three lignans (schisandrol A, schisandrol B, and schisandrin C) on insulin secretion in rat INS-1 pancreatic ß-cells and glucose uptake in mouse C2C12 skeletal muscle cells. Schisandrol A and schisandrin C enhanced insulin secretion in response to high glucose levels with no toxic effects on INS-1 cells. The effect of schisandrin C was superior to that of gliclazide (positive control), a drug commonly used to treat type 2 diabetes (T2D). In addition, western blot analysis showed that the expression of associated proteins, including peroxisome proliferator-activated receptor γ (PPARγ), pancreatic and duodenal homeobox 1 (PDX-1), phosphatidylinositol 3-kinase (PI3K), Akt, and insulin receptor substrate-2 (IRS-2), was increased in INS-1 cells after treatment with schisandrin C. In addition, insulin secretion effect of schisandrin C were enhanced by the Bay K 8644 (L-type Ca2+ channel agonist) and glibenclamide (K+ channel blocker), were abolished by the nifedipine (L-type Ca2+ channel blocker) and diazoxide (K+ channel activator). Moreover, schisandrin C enhanced glucose uptake with no toxic effects on C2C12 cells. Western blot analysis showed that the expression of associated proteins, including insulin receptor substrate-1 (IRS-1), AMP-activated protein kinase (AMPK), PI3K, Akt, glucose transporter type 4 (GLUT-4), was increased in C2C12 cells after treatment with schisandrin C. Schisandrin C may improve hyperglycemia by enhancing insulin secretion in pancreatic ß-cells and improving glucose uptake into skeletal muscle cells. Our findings may provide evidence that schisandrin C may be beneficial in devising novel anti-T2D strategies.

Systolic Dysfunction of the Heart in Type 1 Diabetes Mellitus.

Impaired insulin synthesis is accompanied by hyperglycemia and the development of diabetic cardiomyopathy. Echocardiography and left-ventricular catheterization were employed for studying the contractile function of the left ventricle in 2 weeks after administration of streptozotocin (60 mg/kg). The results obtained by both methods were similar and indicated the development of systolic dysfunction with a 27% decrease in cardiac output. The invasive study showed that the maximum rate of left-ventricular pressure development, the contractility index, and systolic left-ventricular pressure were within the normal range, but the peak ejection rate was reduced by 28%. BP was normal, but the vascular stiffness index was increased by about 1.5 times and inversely correlated with the peak ejection rate (r=-0.69). The results showed that systolic dysfunction in type 1 diabetes model was due to reduced ejection from the left ventricle at normal rate of left-ventricular pressure development.

Presumption of innocence for beta cells: why are they vulnerable autoimmune targets in type 1 diabetes?

It is increasingly appreciated that the pathogenic mechanisms of type 1 diabetes involve both the autoimmune aggressors and their beta cell targets, which engage in a conflicting dialogue within and possibly outside the pancreas. Indeed, autoimmune CD8+ T cells, which are the final mediators of beta cell destruction, circulate at similar frequencies in type 1 diabetic and healthy individuals. Hence a universal state of 'benign' islet autoimmunity exists, and we hypothesise that its progression to type 1 diabetes may at least partially rely on a higher vulnerability of beta cells, which play a key, active role in disease development and/or amplification. We posit that this autoimmune vulnerability is rooted in some features of beta cell biology: the stress imposed by the high rate of production of insulin and other granule proteins, their dense vascularisation and the secretion of their products directly into the bloodstream. Gene variants that may predispose individuals to this vulnerability have been identified, e.g. MDA5, TYK2, PTPN2. They interact with environmental cues, such as viral infections, that may drive this genetic potential towards exacerbated local inflammation and progressive beta cell loss. On top of this, beta cells set up compensatory responses, such as the unfolded protein response, that become deleterious in the long term. The relative contribution of immune and beta cell drivers may vary and phenotypic subtypes (endotypes) are likely to exist. This dual view argues for the use of circulating biomarkers of both autoimmunity and beta cell stress for disease staging, and for the implementation of both immunomodulatory and beta cell-protective therapeutic strategies. Graphical abstract.

The making of insulin in health and disease.

The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes. Graphical abstract.

Deciphering genetic signatures by whole exome sequencing in a case of co-prevalence of severe renal hypouricemia and diabetes with impaired insulin secretion.

BACKGROUND: Renal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic ß-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism. CASE PRESENTATION: We describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8-7.0 mg/dl), 41.6 µmol/l (226-416 µmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 µg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic ß-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation. CONCLUSION: We found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic ß-cell functions that deserve further scrutiny.