Showing papers on "Proinsulin published in 2018"

Pseudotime Ordering of Single Human β-Cells Reveals States of Insulin Production and Unfolded Protein Response.

Abstract: Proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum (ER) a stressful event. Pancreatic β-cells overcome ER stress by activating the unfolded protein response (UPR) and reducing insulin production. This suggests that β-cells transition between periods of high insulin biosynthesis and UPR-mediated recovery from cellular stress. We now report the pseudotime ordering of single β-cells from humans without diabetes detected by large-scale RNA sequencing. We identified major states with 1) low UPR and low insulin gene expression, 2) low UPR and high insulin gene expression, or 3) high UPR and low insulin gene expression. The latter state was enriched for proliferating cells. Stressed human β-cells do not dedifferentiate and show little propensity for apoptosis. These data suggest that human β-cells transition between states with high rates of biosynthesis to fulfill the body's insulin requirements to maintain normal blood glucose levels and UPR-mediated recovery from ER stress due to high insulin production.

Biosynthesis, structure, and folding of the insulin precursor protein.

Abstract: Insulin synthesis in pancreatic β-cells is initiated as preproinsulin Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic β-cells Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes

IRE1–XBP1 pathway regulates oxidative proinsulin folding in pancreatic β cells

Abstract: In mammalian pancreatic β cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed β cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic β cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic β cells.

Biosynthesis, structure, and folding of the insulin precursor protein

Abstract: Insulin synthesis in pancreatic β-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic β-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes.

Misfolded proinsulin in the endoplasmic reticulum during development of beta cell failure in diabetes

Abstract: The endoplasmic reticulum (ER) is broadly distributed throughout the cytoplasm of pancreatic beta cells, and this is where all proinsulin is initially made. Healthy beta cells can synthesize 6000 proinsulin molecules per second. Ordinarily, nascent proinsulin entering the ER rapidly folds via the formation of three evolutionarily conserved disulfide bonds (B7-A7, B19-A20, and A6-A11). A modest amount of proinsulin misfolding, including both intramolecular disulfide mispairing and intermolecular disulfide-linked protein complexes, is a natural by-product of proinsulin biosynthesis, as is the case for many proteins. The steady-state level of misfolded proinsulin-a potential ER stressor-is linked to (1) production rate, (2) ER environment, (3) presence or absence of naturally occurring (mutational) defects in proinsulin, and (4) clearance of misfolded proinsulin molecules. Accumulation of misfolded proinsulin beyond a certain threshold begins to interfere with the normal intracellular transport of bystander proinsulin, leading to diminished insulin production and hyperglycemia, as well as exacerbating ER stress. This is most obvious in mutant INS gene-induced Diabetes of Youth (MIDY; an autosomal dominant disease) but also likely to occur in type 2 diabetes owing to dysregulation in proinsulin synthesis, ER folding environment, or clearance.

Islet prohormone processing in health and disease.

Abstract: Biosynthesis of peptide hormones by pancreatic islet endocrine cells is a tightly orchestrated process that is critical for metabolic homeostasis Like neuroendocrine peptides, insulin and other islet hormones are first synthesized as larger precursor molecules that are processed to their mature secreted products through a series of proteolytic cleavages, mediated by the prohormone convertases Pc1/3 and Pc2, and carboxypeptidase E Additional posttranslational modifications including C-terminal amidation of the β-cell peptide islet amyloid polypeptide (IAPP) by peptidyl-glycine α-amidating monooxygenase (Pam) may also occur Genome-wide association studies (GWAS) have showed genetic linkage of these processing enzymes to obesity, β-cell dysfunction, and type 2 diabetes (T2D), pointing to their important roles in metabolism and blood glucose regulation In both type 1 diabetes (T1D) and T2D, and in the face of metabolic or inflammatory stresses, islet prohormone processing may become impaired; indeed elevated proinsulin:insulin (PI:I) ratios are a hallmark of the β-cell dysfunction in T2D Recent studies suggest that genetic or acquired defects in proIAPP processing may lead to the production and secretion of incompletely processed forms of proIAPP that could contribute to T2D pathogenesis, and additionally that impaired processing of both PI and proIAPP may be characteristic of β-cell dysfunction in T1D In islet α-cells, the prohormone proglucagon is normally processed to bioactive glucagon by Pc2 but may express Pc1/3 under certain conditions leading to production of GLP-1(7-36NH2 ) A better understanding of how β-cell processing of PI and proIAPP, as well as α-cell processing of proglucagon, are impacted by genetic susceptibility and in the face of diabetogenic stresses, may lead to new therapeutic approaches for improving islet function in diabetes

Revealing the specificity of regulatory T cells in murine autoimmune diabetes.

Abstract: Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαβ pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9–23 and proinsulin. Consistently, insulin B:9–23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28−/− recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.

Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells

Abstract: The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells. Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets. An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking. The SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection.

Proinsulin C-peptide is an autoantigen in people with type 1 diabetes.

Abstract: Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells, found within the islets of Langerhans in the pancreas, are destroyed by islet-infiltrating T cells. Identifying the antigenic targets of beta-cell reactive T cells is critical to gain insight into the pathogenesis of T1D and develop antigen-specific immunotherapies. Several lines of evidence indicate that insulin is an important target of T cells in T1D. Because many human islet-infiltrating CD4+ T cells recognize C-peptide-derived epitopes, we hypothesized that full-length C-peptide (PI33-63), the peptide excised from proinsulin as it is converted to insulin, is a target of CD4+ T cells in people with T1D. CD4+ T cell responses to full-length C-peptide were detected in the blood of: 14 of 23 (>60%) people with recent-onset T1D, 2 of 15 (>13%) people with long-standing T1D, and 1 of 13 (<8%) HLA-matched people without T1D. C-peptide-specific CD4+ T cell clones, isolated from six people with T1D, recognized epitopes from the entire 31 amino acids of C-peptide. Eighty-six percent (19 of 22) of the C-peptide-specific clones were restricted by HLA-DQ8, HLA-DQ2, HLA-DQ8trans, or HLA-DQ2trans, HLA alleles strongly associated with risk of T1D. We also found that full-length C-peptide was a much more potent agonist of some CD4+ T cell clones than an 18mer peptide encompassing the cognate epitope. Collectively, our findings indicate that proinsulin C-peptide is a key target of autoreactive CD4+ T cells in T1D. Hence, full-length C-peptide is a promising candidate for antigen-specific immunotherapy in T1D.

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion

Abstract: A rare loss-of-function variant p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8) enriched in Western Finland protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, especially compared with individuals matched for the genotype of a common T2D risk variant in SLC30A8, p.Arg325. In genome-edited human IPS-derived β-like cells, we establish that the p.Arg138* variant results in reduced SLC30A8 expression due to haploinsufficiency. In human β-cells loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function, which was also seen in isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aiming at maintaining insulin secretion capacity in T2D.

Tau ablation in mice leads to pancreatic β cell dysfunction and glucose intolerance

Abstract: The microtubule-associated protein tau is highly expressed in pancreatic islets. Abnormally phosphorylated tau aggregates assemble into neurofibrillary tangles linked to Alzheimer's disease pathology and has also been found in islets of patients with type 2 diabetes. However, the significance of tau in islet function remains relatively unexplored. Therefore, we investigated the role of tau on β cell function and glucose homeostasis using tau knockout (tauKO) mice. TauKO mice were hyperglycemic and glucose intolerant at an early age. Islet insulin content was reduced and proinsulin levels were significantly elevated in tauKO mice, resulting in impaired glucose-stimulated insulin secretion. Loss of tau also resulted in increased epididymal fat mass and leptin levels, reduced glucose production, and insulin resistance at later ages, leading to complete onset of diabetes. Transgenic expression of human tau in islets was unable to rescue those defects in glucose regulation, indicating structural and/or functional differences between mouse and human tau. Cumulatively, these results suggest an important role for tau in the proper maintenance of pancreatic β cell function and glucose homeostasis.-Wijesekara, N., Goncalves, R. A., Ahrens, R., De Felice, F. G., Fraser, P. E. Tau ablation in mice leads to pancreatic β cell dysfunction and glucose intolerance.

β-Cell secretory defects are present in pancreatic insufficient cystic fibrosis with 1-hour oral glucose tolerance test glucose ≥155 mg/dL

Abstract: BACKGROUND Patients with pancreatic insufficient cystic fibrosis (PI-CF) meeting standard criteria for normal glucose tolerance display impaired β-cell secretory capacity and early-phase insulin secretion defects. We sought evidence of impaired β-cell secretory capacity, a measure of functional β-cell mass, among those with early glucose intolerance (EGI), defined as 1-hour oral glucose tolerance test (OGTT) glucose ≥155 mg/dL (8.6 mmol/L). METHODS A cross-sectional study was conducted in the Penn and CHOP Clinical & Translational Research Centers. PI-CF categorized by OGTT as normal (PI-NGT: 1-hour glucose <155 mg/dL and 2-hour <140 mg/dL [7.8 mmol/L]; n = 13), PI-EGI (1-hour ≥155 mg/dL and 2-hour <140 mg/dL; n = 13), impaired (PI-IGT: 2-hour ≥140 and <200 mg/dL [11.1 mmol/L]; n = 8), and diabetic (cystic fibrosis-related diabetes, CFRD: 2-hour ≥200 mg/dL; n = 8) participated. Post-prandial glucose tolerance and insulin secretion, and β-cell secretory capacity and demand were derived from mixed-meal tolerance tests (MMTTs), and glucose-potentiated arginine (GPA) tests, respectively. RESULTS PI-EGI had elevated post-prandial glucose with reduced early-phase insulin secretion during MMTT compared to PI-NGT (P < .05). PI-EGI also exhibited impaired acute insulin and C-peptide responses to GPA (P < .01 vs PI-NGT), measures of β-cell secretory capacity. Proinsulin secretory ratios were higher under hyperglycemic clamp conditions in PI-IGT and CFRD (P < .05 vs PI-NGT), and correlated with 1-hour glucose in PI-CF (P < .01). CONCLUSIONS PI-CF patients with 1-hour OGTT glucose ≥155 mg/dL already manifest impaired β-cell secretory capacity with associated early-phase insulin secretion defects. Avoiding hyperglycemia in patients with EGI may be important for preventing excessive insulin demand indicated by disproportionately increased proinsulin secretion.

Increased β-Cell Workload Modulates Proinsulin-to-Insulin Ratio in Humans.

Abstract: Increased proinsulin secretion, which characterizes type 2 diabetes and insulin resistance, may be due to an intrinsic, primitive defect in proinsulin processing or be secondary to increased demand on β-cells (hyperinsulinemia secondary to insulin resistance). An alternative way to investigate the relation between relative hyperproinsulinemia and increased secretory demand is to study the dynamic changes in the proinsulin-to-insulin ratio after partial pancreatectomy, a model of acute increased β-cell workload on the remaining pancreas. To pursue this aim, patients without diabetes, scheduled for partial pancreatectomy, underwent 4-h mixed-meal tests and hyperinsulinemic-euglycemic clamps before and after surgery. After acute β-cell mass reduction, no changes were observed in the fasting proinsulin-to-insulin ratio, whereas the fold change in the proinsulin-to-insulin ratio significantly increased over time after the meal. Further, our data demonstrate that whole-body insulin resistance is associated with underlying defects in proinsulin secretion, which become detectable only in the presence of increased insulin secretion demand.

Characterization and optimization of two-chain folding pathways of insulin via native chain assembly

Abstract: Until recently the total synthesis of insulin, with its characteristic heterodimeric structure crosslinked by two interchain and one intrachain disulfide (SS) bridge, remained largely an unsolved challenge. By optimizing the synthesis and directed disulfide crosslinking of the two chains, and by applying biomimetic monocomponent proinsulin approaches, efficient insulin syntheses have been realized. Here we report the optimization and characterisation of an alternative strategy, oxidative native chain assembly. In this method unprotected A- and B-chains assemble oxidatively under thermodynamic control to afford bovine pancreatic insulin in 39% yield. Folding is found to proceed predominantly via structured 1SS* and 2SS* intermediates with a common interchain CysA20‒CysB19 disulfide. These results suggest that native chain assembly, long considered inefficient, may represent a reasonable strategy to access insulin variants. This is supported by the synthesis of human insulin and human type-II relaxin in yields of up to 49 and 47%, respectively, although the application to human insulin ValA16 variant is unsuccessful. The synthesis and folding pathways of insulin and related proteins are of wide interest. Here the authors characterise the major two-chain oxidative folding pathways of bovine pancreatic insulin, and develop synthetic conditions applicable to related foldable insulin variants

Effects of proinsulin misfolding on β-cell dynamics, differentiation and function in diabetes

Abstract: ER stress due to proinsulin misfolding has an important role in the pathophysiology of rare forms of permanent neonatal diabetes (PNDM) and probably also of common type 1 (T1D) and type 2 diabetes (T2D) Accumulation of misfolded proinsulin in the ER stimulates the unfolded protein response (UPR) that may eventually lead to apoptosis through a process called the terminal UPR However, the β-cell ER has an incredible ability to cope with accumulation of misfolded proteins; therefore, it is not clear whether in common forms of diabetes the accumulation of misfolded proinsulin exceeds the point of no return in which terminal UPR is activated Many studies showed that the UPR is altered in both T1D and T2D; however, the observed changes in the expression of different UPR markers are inconsistent and it is not clear whether they reflect an adaptive response to stress or indeed mediate the β-cell dysfunction of diabetes Herein, we critically review the literature on the effects of proinsulin misfolding and ER stress on β-cell dysfunction and loss in diabetes with emphasis on β-cell dynamics, and discuss the gaps in understanding the role of proinsulin misfolding in the pathophysiology of diabetes

β-Cell Control of Insulin Production During Starvation-Refeeding in Male Rats.

Abstract: Mammalian metabolism has evolved to adapt to changes in nutrient status. Insulin, the key anabolic hormone, facilitates intracellular storage of nutrient fuels and plays a pivotal role in the transition away from catabolism upon refeeding. Although circulating insulin relative to nutrient levels has been well characterized during fasting and refeeding, how pancreatic β-cell biology caters to acute changes in insulin demand has not been sufficiently addressed. Here, we examined the dynamics of (pro)insulin production and associated changes in β-cell ultrastructure during refeeding after a 72-hour fast in male rats. We found that fasted β-cells had marked degranulation, which inversely coordinated with the upregulation of autophagolysomal and lysosomal organelles. There was also expanded Golgi that correlated with enhanced (pro)insulin biosynthetic capacity but, conversely, blunted in vivo insulin secretion. Within 4 to 6 hours of refeeding, proinsulin biosynthesis, cellular ultrastructure, in vivo insulin secretion, and glucose tolerance normalized to levels near those of fed control animals, indicating a rapid replenishment of normal insulin secretory capacity. Thus, during a prolonged fast, the β-cell protects against hypoglycemia by markedly reducing insulin secretory capacity in vivo but is simultaneously poised to efficiently increase (pro)insulin production upon refeeding to effectively return normal insulin secretory capacity within hours.

Exercise Increases Insulin Sensitivity and Skeletal Muscle AMPK Expression in Systemic Lupus Erythematosus: A Randomized Controlled Trial.

Abstract: Systemic lupus erythematosus (SLE) patients may show increased insulin resistance (IR) when compared with their healthy peers. Exercise training has been shown to improve insulin sensitivity in other insulin-resistant populations, but it has never been tested in SLE. Therefore, the aim of the present study was to assess the efficacy of a moderate-intensity exercise training program on insulin sensitivity and potential underlying mechanisms in SLE patients with mild/inactive disease. A 12-wk, randomized-controlled trial was conducted. Nineteen SLE patients were randomly assigned into two groups: trained (SLE-TR, n=9) and non-trained (SLE-NT, n=10). Before and after 12 weeks of the exercise training program, patients underwent a meal test (MT), from which surrogates of insulin sensitivity and beta-cell function were determined. Muscle biopsies were performed after the MT for the assessment of total and membrane GLUT4 and proteins related to insulin signaling (Akt and AMPK). SLE-TR showed, when compared with SLE-NT, significant decreases in fasting insulin (-39% vs. +14%, p=0.009, effect size [ES]=-1.0) and in the insulin response to MT (-23% vs. +21%, p=0.007, ES=-1.1), HOMA-IR (-30% vs. +15%, p=0.005, ES=-1.1), a tendency towards decreased proinsulin response to MT (-19% vs. +6%, p=0.07, ES=-0.-9) and increased glucagon response to MT (+3% vs. -3%, p=0.09, ES=0.6), and significant increases in the Matsuda index (+66% vs. -31%, p=0.004, ES=0.9) and fasting glucagon (+4% vs. -8%, p=0.03, ES=0.7). No significant differences between SLT-TR and SLT-NT were observed in fasting glucose, glucose response to MT and insulinogenic index (all p>0.05). SLE-TR showed a significant increase in AMPK Thr 172 phosphorylation when compared to SLE-NT (+73% vs. -12%, p=0.014, ES=1.3), whereas no significant differences between groups were observed in Akt Ser 473 phosphorylation, total and membrane GLUT4 expression and GLUT4 translocation (all p>0.05). In conclusion, a 12-week moderate-intensity aerobic exercise training program improved insulin sensitivity in SLE patients with mild/inactive disease. This effect appears to be partially mediated by the increased insulin-stimulated skeletal muscle AMPK phosphorylation. Registered at clinicaltrial.gov as NCT01515163.

TCF7L2 correlation in both insulin secretion and postprandial insulin sensitivity.

Abstract: The TCF7L2 rs7903146 variant is strongly associated with type 2 diabetes mellitus (T2DM). However, the mechanisms involved in this association remain unknown and may include extrapancreatic effects. The aim of this study was to perform a metabolic characterization of T2DM patients with and without the TCF7L2 rs7903146 risk T allele and analyze some influences of the TCF7L2 genotype on glucose metabolism. Patients with T2DM (n = 162) were genotyped for the TCF7L2 rs7903146 single nucleotide polymorphism. Individuals with CT/TT and CC genotypes were compared regarding basal serum levels of glucose, glycosylated hemoglobin A1C, HDL, uric acid, insulin, and C-peptide. A subset of 56 individuals was evaluated during a 500-calorie mixed-meal test with measurements of glucose, insulin, proinsulin, C-peptide and glucagon. Additional secondary assessments included determination of insulinogenic index (IGI30), and insulin sensitivity (%S) and resistance (IR) by Homeostatic model assessment (HOMA). Patients with the CT/TT genotype showed lower baseline plasma concentrations of C-peptide when compared with those with the CC genotype. Of the 56 individuals who participated in the mixed-meal test, 26 and 30 had the CC and CT/TT genotypes, respectively. CT/TT subjects, compared with CC individuals, had higher post prandial plasma levels of insulin and C-peptide at 30–120 min (p < 0.05) and proinsulin at 45–240 min (p < 0.05). Interestingly CT/TT individuals presented at baseline higher %S (p = 0.021), and lower IR (p = 0.020) than CC individuals. No significant differences in IGI30 values were observed between groups. The T2DM individuals carrying the rs7903146 T allele of the TCF7L2 gene presented higher IR pattern in response to a mix-meal test, different of beta cell function at baseline assessed by C-peptide levels which was lower, and Homa-IR was lower when comparing with non-carriers.

PICK1 is essential for insulin production and the maintenance of glucose homeostasis.

Abstract: Protein interacting with C-kinase 1 (PICK1) is a peripheral membrane protein that controls insulin granule formation, trafficking, and maturation in INS-1E cells. However, global Pick1-knockout mice showed only a subtle diabetes-like phenotype. This raises the possibility that compensatory effects from tissues other than pancreatic beta cells may obscure the effects of insulin deficiency. To explore the role of PICK1 in pancreatic islets, we generated mice harboring a conditional Pick1 allele in a C57BL/6J background. The conditional Pick1-knockout mice exhibited impaired glucose tolerance, profound insulin deficiency, and hyperglycemia. In vitro experiments showed that the ablation of Pick1 in pancreatic beta cells selectively decreased the initial rapid release of insulin and the total insulin levels in the islets. Importantly, the specific ablation of Pick1 induced elevated proinsulin levels in the circulation and in the islets, accompanied by a reduction in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3). The deletion of Pick1 triggered the specific elimination of chromogranin B in pancreatic beta cells, which is believed to control granule formation and release. Collectively, these data demonstrate the critical role of PICK1 in secretory granule biogenesis, proinsulin processing, and beta cell function. We conclude that the beta cell-specific deletion of Pick1 in mice led to hyperglycemia and eventually to diabetes.

Impaired Processing of Prohormones in Secretogranin III-Null Mice Causes Maladaptation to an Inadequate Diet and Stress.

Abstract: Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.

Proinsulin-mediated induction of type 1 diabetes in HLA-DR4-transgenic mice.

Abstract: Antigen-specific immunotherapy of autoimmune disease currently remains the only potentially curative approach. However, translation of promising pre-clinical results into successful clinical application has proven challenging. In part, this is because pre-clinical findings in mouse models have to be redesigned for human application due to differences in MHC II. To reduce the gap between pre-clinical and clinical studies, we have created a novel mouse model that expresses human HLA-DR4, but no endogenous MHC on antigen-presenting cells. Moreover, human B7.1 (CD80) is expressed in the pancreatic islets under the control of the rat insulin promoter. Although this model does not develop diabetes spontaneously, it is susceptible to the induction of type 1 diabetes by challenging mice with overlapping peptides derived from murine proinsulin-2 in adjuvant. Unlike the NOD model of spontaneous type 1 diabetes, but akin to the human condition, this model does not have a gender bias. Furthermore, similar to the human condition, the disease is characterised by a diverse leucocyte infiltration of the pancreatic islets and the formation of anti-proinsulin auto-antibodies. The model that we report here offers detailed insights into type-1 diabetes and is expected to prove instrumental when studying the mechanism of action in translational, antigen-specific immunotherapy.

A novel INS mutation in a family with maturity-onset diabetes of the young: Variable insulin secretion and putative mechanisms.

Abstract: Insulin gene (INS) mutations cause a rare form of maturity-onset diabetes of the young (MODY), a heterogeneous group of autosomal dominant diabetes with at least 14 confirmed causative genes. Here, we describe a family with MODY due to a novel INS mutation, detected using massively parallel sequencing (MPS). The proband presented aged 11 years with mild diabetic ketoacidosis. She was negative for IA2 and GAD antibodies. She had a strong family history of diabetes affecting both her two siblings and her mother, none of whom had ketosis but who were considered to have type 1 diabetes and managed on insulin, and her maternal grandfather, who was managed for decades on sulfonylureas. Of note, her younger sister had insulin deficiency but an elevated fasting proinsulin:insulin ratio of 76% (ref 5%-30%). Sanger sequencing of HNF4A, HNF1A, and HNF1B in the proband was negative. Targeted MPS using a custom-designed amplicon panel sequenced on an Illumina MiSeq detected a heterozygous INS mutation c.277G>A (p.Glu93Lys). Sanger sequencing confirmed the variant segregated with diabetes within the family. Structural analysis of this variant suggested disruption of a critical hydrogen bond between insulin and the insulin receptor; however, the clinical picture in some individuals also suggested abnormal insulin processing and insulin deficiency. This family has a novel INS mutation and demonstrated variable insulin deficiency. MPS represents an efficient method of MODY diagnosis in families with rarer gene mutations.

Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec.

Abstract: Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity.

Early Intensive Insulin Use May Preserve β-Cell Function in Neonatal Diabetes Due to Mutations in the Proinsulin Gene.

Abstract: Although mutations in the proinsulin gene (INS) are the second most common cause of neonatal diabetes mellitus, the natural history of β-cell death and the most appropriate treatments remains unknown. We describe the management and outcome of two sisters with INS-mediated diabetes (S1 and S2) and suggest that more intensive insulin treatment of S2 may have resulted in better clinical outcomes. S1 was diagnosed with diabetes after presenting with serum glucose of 404 mg/dL (22.4 mmol/L) and started multiple daily insulin injections at age 4 months, followed by continuous subcutaneous insulin infusion (CSII) at age 42 months. S1 had positive genetic testing at age 4 months for the GlyB8Ser or Gly32Ser mutation in proinsulin. S2 had positive research-based genetic testing, age 1 month, before she had consistently elevated blood glucose levels. Continuous glucose monitoring revealed abnormal excursions to 200 mg/dL. Low-dose insulin therapy was initiated at age 2.5 months via CSII. At age-matched time points, S2 had higher C-peptide levels, lower hemoglobin A1c values, and lower estimated doses of insulin as compared with S1. Earlier, more intensive insulin treatment was associated with higher C-peptide levels, decreased insulin dosing, and improved glycemic control. Initiating exogenous insulin before overt hyperglycemia and maintaining intensive insulin management may reduce the demand for endogenous insulin production and may preserve β-cell function. Studies accumulating data on greater numbers of participants will be essential to determine whether these associations are consistent for all INS gene mutations.

Changes in insulin, glucagon and ER stress precede immune activation in type 1 diabetes

Abstract: It is unknown whether there is a gene signature in pancreas which is associated with type 1 diabetes (T1D). We performed partial pancreatectomies on 30-day preinsulitic, diabetes-prone BioBreeding (BBdp) rats to prospectively identify factors involved in early prediabetes. Microarrays of the biopsies revealed downregulation of endoplasmic reticulum (ER) stress, metabolism and apoptosis. Based on these results, additional investigations compared gene expression in control (BBc) and BBdp rats age ~8, 30 and 60 days using RT-qPCR. Neonates had increased ER stress gene expression in pancreas. This was associated with decreased insulin, cleaved caspase-3 and Ins1 whereas Gcg and Pcsk2 were increased. The increase in ER stress was not sustained at 30 days and decreased by 60 days. In parallel, the liver gene profile showed a similar signature in neonates but with an early decrease of the unfolded protein response (UPR) at 30 days. This suggested that changes in the liver precede those in the pancreas. Tnf and Il1b expression was increased in BBdp pancreas in association with increased caspase-1, cleaved caspase-3 and decreased proinsulin area. Glucagon area was increased in both 30-day and 60-day BBdp rats. Increased colocalization of BIP and proinsulin was observed at 60 days in the pancreas, suggesting insulin-related ER dysfunction. We propose that dysregulated metabolism leads to ER stress in neonatal rats long before insulitis, creating a microenvironment in both pancreas and liver that promotes autoimmunity.

Sertoli Cells Engineered to Express Insulin to Lower Blood Glucose in Diabetic Mice

Abstract: Long-term survival of allo- and xenotransplanted immune-privileged Sertoli cells (SCs) is well documented suggesting that SCs can be used to deliver foreign proteins for cell-based gene therapy. The aim of this study was to use a lentivirus carrying proinsulin cDNA to achieve stable expression and lowering of blood glucose levels (BGLs). A SC line transduced with the lentivirus (MSC-LV-mI) maintained stable insulin expression in vitro. These MSC-LV-mI cells were transplanted and grafts were analyzed for cell survival, continued proinsulin mRNA, and insulin protein expression. All grafts contained MSC-LV-mI cells that expressed proinsulin mRNA and insulin protein. Transplantation of MSC-LV-mI cells into diabetic mice significantly lowered BGLs for 4 days after transplantation. Interestingly, in three transplanted SCID mice and one transplanted BALB/c mouse, the BGLs again significantly lowered by day 50 and 70, respectively. This is the first time SC transduced with a lentiviral vector was able to stably express insulin and lower BGLs. In conclusion, a SC line can be modified to stably express therapeutic proteins (e.g., insulin), thus taking us one step further in the use of SCs as an immune-privileged vehicle for cell-based gene therapy.