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Objective: Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context. Methods: MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in β-cells of adult mice using Ins1-CreER+. Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER. Results: With a Km ≈3.5 mM, glucose rapidly (T1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with β-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi. Conclusion: The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing β-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating β-cell failure.

The discovery of the insulin hormone over 100 years ago, and its subsequent therapeutic application, marked a key landmark in the history of medicine and medical research. The many roles insulin plays in cell metabolism and growth have been revealed by extensive investigations into the structure and function of insulin, the insulin tyrosine kinase receptor (IR), as well as the signalling cascades, which occur upon insulin binding to the IR. In this review, the insulin gene mutations identified as causing disease and the structural implications of these mutations will be discussed. Over 100 studies were evaluated by one reviewing author, and over 70 insulin gene mutations were identified. Mutations may impair insulin gene transcription and translation, preproinsulin trafficking and proinsulin sorting, or insulin-IR interactions. A better understanding of insulin gene mutations and the resultant pathophysiology can give essential insight into the molecular mechanisms underlying impaired insulin biosynthesis and insulin-IR interaction. [ABSTRACT FROM AUTHOR]

Purpose: Pancreatic β-cell failure is a central hallmark of the pathogenesis of diabetes mellitus; however, the molecular basis underlying chronic inflammation-caused β-cell failure remains unclear. This study reported here specifically assessed the association between miR-25/miR-92b family and β-cell failure in diabetes. Methods: IL-1β and two additional ER stress activators, palmitate and tunicamycin were applied to evaluate the expression level miR-25 by Taqman® RT-PCR. Glucose- and potassium-stimulated insulin secretion assays were performed to assess β-cell function. Dual-luciferase activity, and western blotting assays were utilized for miR-25 target gene verification. CCK-8 and TUNEL staining were used to evaluate β-cell viability and apoptosis. Results: miRNA ChIP identified the increased level of miR-25 in INS-1 cells by IL-1β treatment. Expression levels of miR-25 were significantly upregulated with the treatment of IL-1β, palmitate or tunicamycin in both INS-1 cells and human islets. Ectopic elevation of miR-25 recapitulated most featured β-cell defects caused by IL-1β, including inhibition of insulin biosynthesis and increased β-cell apoptosis. These detrimental effects of miR-25 relied on its seed sequence recognition and repressed expression of its target genes Neurod1 and Mcl1. The miR-25/NEUROD1 axis reduced insulin biosynthesis via transcriptional regulation of β-cell specific genes. The miR-25/MCL1 axis caused β-cell apoptosis in a CASPASE-3/PARP1-dependent manner. Comparable impairments were generated by miR-92b and miR-25, emphasizing the redundant biological roles of miRNA family members with the same seed sequence. Conclusion: MiR-25/miR-92b family plays a major role in β-cell failure occurring under inflammation and diabetes states. [ABSTRACT FROM AUTHOR]

The discovery of the insulin hormone over 100 years ago, and its subsequent therapeutic application, marked a key landmark in the history of medicine and medical research. The many roles insulin plays in cell metabolism and growth have been revealed by extensive investigations into the structure and function of insulin, the insulin tyrosine kinase receptor (IR), as well as the signalling cascades, which occur upon insulin binding to the IR. In this review, the insulin gene mutations identified as causing disease and the structural implications of these mutations will be discussed. Over 100 studies were evaluated by one reviewing author, and over 70 insulin gene mutations were identified. Mutations may impair insulin gene transcription and translation, preproinsulin trafficking and proinsulin sorting, or insulin-IR interactions. A better understanding of insulin gene mutations and the resultant pathophysiology can give essential insight into the molecular mechanisms underlying impaired insulin biosynthesis and insulin-IR interaction.

Purpose of Review: Diabetes mellitus (DM) due to toxic misfolding of proinsulin variants provides a monogenic model of endoplasmic reticulum (ER) stress. The mutant proinsulin syndrome (also designated MIDY; Mutant INS-gene-induced Diabetes of Youth or Maturity-onset diabetes of the young 10 (MODY10)) ordinarily presents as permanent neonatal-onset DM, but specific amino-acid substitutions may also present later in childhood or adolescence. This review highlights structural mechanisms of proinsulin folding as inferred from phenotype-genotype relationships. Recent Findings: MIDY mutations most commonly add or remove a cysteine, leading to a variant polypeptide containing an odd number of thiol groups. Such variants are associated with aberrant intermolecular disulfide pairing, ER stress, and neonatal β-cell dysfunction. Non-cysteine-related (NCR) mutations (occurring in both the B and A domains of proinsulin) define distinct determinants of foldability and vary in severity. The range of ages of onset, therefore, reflects a "molecular rheostat" connecting protein biophysics to quality-control ER checkpoints. Because in most mammalian cell lines even wild-type proinsulin exhibits limited folding efficiency, molecular barriers to folding uncovered by NCR MIDY mutations may pertain to β-cell dysfunction in non-syndromic type 2 DM due to INS-gene overexpression in the face of peripheral insulin resistance. Summary: Recent studies of MIDY mutations and related NCR variants, combining molecular and cell-based approaches, suggest that proinsulin has evolved at the edge of non-foldability. Chemical protein synthesis promises to enable comparative studies of "non-foldable" proinsulin variants to define key steps in wild-type biosynthesis. Such studies may create opportunities for novel therapeutic approaches to non-syndromic type 2 DM. [ABSTRACT FROM AUTHOR]

The increased prevalence of diabetes and the growing popularity of non-invasive methods of recombinant human insulin uptake, such as oral insulin, have increased insulin demand, further limiting the affordability of insulin. Over 40 years have passed since the development of engineered microorganisms that replaced the animal pancreas as the primary source of insulin. To stay ahead of the need for insulin in the present and the future, a few drawbacks with the existing expression systems need to be alleviated, including the inclusion body formation, the use of toxic inducers, and high process costs. To address these bottlenecks and improve insulin production, a variety of techniques are being used in bacteria, yeasts, transgenic plants and animals, mammalian cell lines, and cell-free expression systems. Different approaches for the production of insulin, including two-chain, proinsulin or mini-proinsulin, preproinsulin coupled with fusion protein, chaperone, signal peptide, and purification tags, are explored in upstream, whereas downstream processing takes into account the recovery of intact protein in its bioactive form and purity. This article focuses on the strategies used in the upstream and downstream phases of the bioprocess to produce recombinant human insulin. This review also covers a range of analytical methods and tools employed in investigating the genuity of recombinant human insulin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: While insulin has been central to the pathophysiology and treatment of patients with diabetes for the last 100 years, it has only been since 2007 that genetic variation in the INS gene has been recognised as a major cause of monogenic diabetes. Both dominant and recessive mutations in the INS gene are now recognised as important causes of neonatal diabetes and offer important insights into both the structure and function of insulin. It is also recognised that in rare cases, mutations in the INS gene can be found in patients with diabetes diagnosed outside the first year of life. Scope of Review: This review examines the genetics and clinical features of monogenic diabetes resulting from INS gene mutations from the first description in 2007 and includes information from 389 patients from 292 families diagnosed in Exeter with INS gene mutations. We discuss the implications for diagnosing and treating this subtype of monogenic diabetes. Major Conclusions: The dominant mutations in the INS gene typically affect the secondary structure of the insulin protein, usually by disrupting the 3 disulfide bonds in mature insulin. The resulting misfolded protein results in ER stress and beta-cell destruction. In contrast, recessive INS gene mutations typically result in no functional protein being produced due to reduced insulin biosynthesis or loss-of-function mutations in the insulin protein. There are clinical differences between the two genetic aetiologies, between the specific mutations, and within patients with identical mutations.

Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context. MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in β-cells of adult mice using Ins1-CreER+. Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER. With a K m ≈3.5 mM, glucose rapidly (T 1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T 1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with β-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi. The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing β-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating β-cell failure. Regulation of β - cell function by glucose activation of CREB3L2. Glucose stimulates flux through the hexosamine biosynthesis pathway (HBP), enhancing O-GlcNAcylation and thus impairing degradation of full-length (FL) CREB3L2 by the proteasome. Independently, glucose also activates mTORC1 to enhance the conversion of FL-CREB3L2 to its transcriptionally active form, P60. This leads to enhanced expression of a select cohort of genes including many controlling steps in the early secretory pathway. Thus, CREB3L2 helps co-ordinate the synthesis of proinsulin with its incorporation into nascent insulin secretory granules (ISG)s. This mitigates against induction of the terminal ER stress marker, CHOP, and contributes to β-cell compensation in response to obesity. [Display omitted] • Glucose rapidly activates CREB3L2 in β-cells independently of ER stress. • Activation by glucose is mediated by O-GlcNAcylation and mTORC1. • Insulin secretion is impaired in vivo and ex vivo by deletion of CREB3L2 in β-cells. • CREB3L2 regulates genes of the early secretory pathway and β-cell compensation. • Glucose co-ordinates the synthesis of insulin with its export from the ER via CREB3L2. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Insulin biosynthesis and secretion by pancreatic β cells are critical for the maintenance of blood glucose homeostasis. Here, we show that the expression of glutathione S-transferase omega-1 (GSTO1) is upregulated in the primary islet cells of diabetic Goto-Kakizaki (GK) rats. Knocking out GSTO1 upregulated insulin transcripts and increased the insulin content in both INS-1 cells and primary islet cells. In contrast, overexpression of GSTO1 reduced the insulin content. Furthermore, knocking out GSTO1 increased the expression of pancreatic duodenal homeobox-1 (PDX1) at both the transcription and protein levels. These results indicate that GSTO1 may be involved in the regulation of insulin biosynthesis by modulating the transcriptional expression of PDX1. • GSTO1 expression was upregulated in the primary islet cells of diabetic Goto-Kakizaki (GK) rats. • Knocking out GSTO1 upregulated insulin transcription and increased the insulin content in pancreatic β cells. • Overexpression of GSTO1 reduced the insulin content in the INS-1 cells. • In the absence of GSTO1, PDX1 was upregulated at both the transcription and protein levels. [ABSTRACT FROM AUTHOR]

Aims: We compared the protective effects of sodium glucose co‐transporter (SGLT) 2 inhibitor luseogliflozin on pancreatic β‐cells between early and advanced stages of diabetes and between short‐ and long‐term use. Materials and methods: Diabetic db/db mice were treated with luseogliflozin for 2 weeks in an early stage of diabetes (7‐9 weeks of age) and an advanced stage of diabetes (16‐18 weeks) for a longer period of time (7‐18 weeks). We performed various morphological analyses of pancreatic islets and examined gene expression profiles in islets after such treatment. Results: In diabetic db/db mice, insulin biosynthesis and secretion were markedly increased by luseogliflozin in an early stage of diabetes but not in an advanced stage. In addition, β‐cell mass was preserved by luseogliflozin only in an early stage. Furthermore, when db/db mice were treated with luseogliflozin for a longer period of time, starting from an early stage, β‐cell function and mass were markedly preserved even after a longer period of time compared to untreated db/db mice. Conclusion: Luseogliflozin exerts more protective effects in an early stage of diabetes compared to an advanced stage, and longer‐term use of luseogliflozin exerts more beneficial effects on pancreatic β‐cells compared to short‐term use. [ABSTRACT FROM AUTHOR]

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic β-cells remains largely unknown. Here, we first examined β-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 β-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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Over the last 20 years, proinsulin C‐peptide emerged as an important player in various biological events. Much time and effort has been spent in exploring all functional features of C‐peptide and recording its implications in Diabetes mellitus. Only a few studies, though, have addressed C‐peptide oligomerization and link this procedure with Diabetes. The aim of our work was to examine the aggregation propensity of C‐peptide, utilizing Transmission Electron Microscopy, Congo Red staining, ATR‐FTIR, and X‐ray fiber diffraction at a 10 mg ml−1 concentration. Our experimental work clearly shows that C‐peptide self‐assembles into amyloid‐like fibrils and therefore, the aggregation propensity of C‐peptide is a characteristic novel feature that should be related to physiological and also pathological conditions. [ABSTRACT FROM AUTHOR]

Traditionally a disease of adults, type 2 diabetes (T2D) has been increasingly diagnosed in youth, particularly among adolescents and young adults of minority ethnic groups. Especially, during the recent COVID-19 pandemic, obesity and prediabetes have surged not only in minority ethnic groups but also in the general population, further raising T2D risk. Regarding its pathogenesis, a gradually increasing insulin resistance due to central adiposity combined with a progressively defective β-cell function are the main culprits. Especially in youth-onset T2D, a rapid β-cell activity decline has been observed, leading to higher treatment failure rates, and early complications. In addition, it is well established that both the quantity and quality of food ingested by individuals play a key role in T2D pathogenesis. A chronic imbalance between caloric intake and expenditure together with impaired micronutrient intake can lead to obesity and insulin resistance on one hand, and β-cell failure and defective insulin production on the other. This review summarizes our evolving understanding of the pathophysiological mechanisms involved in defective insulin secretion by the pancreatic islets in youth- and adult-onset T2D and, further, of the role various micronutrients play in these pathomechanisms. This knowledge is essential if we are to curtail the serious long-term complications of T2D both in pediatric and adult populations.

Over the last 20 years, intensive research has been focused on the specific mechanisms mediating the pancreatic β-cell injury. Both the decreased viability and the dysfunction of β-cells have become the key factors in the development of diabetes mellitus. Thus, it is of utmost importance to elucidate the discrete pathological changes in pancreatic β-cells within the context of the various lipotoxicity models. The goal of these studies is to generate evidence to improve not only the clinical treatment for diabetics, but also modulate the diet and activities of groups at high risk for diabetes. In this regard, we review the role of lipotoxicity in pancreatic β-cell injury and identify potential therapeutic targets in this cell model.

A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS -gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved. [ABSTRACT FROM AUTHOR]

To maintain copious insulin granule stores in the face of ongoing metabolic demand, pancreatic beta cells must produce large quantities of proinsulin, the insulin precursor. Proinsulin biosynthesis can account for up to 30–50% of total cellular protein synthesis of beta cells. This puts pressure on the beta cell secretory pathway, especially the endoplasmic reticulum (ER), where proinsulin undergoes its initial folding, including the formation of three evolutionarily conserved disulfide bonds. In normal beta cells, up to 20% of newly synthesized proinsulin may fail to reach its native conformation, suggesting that proinsulin is a misfolding-prone protein. Misfolded proinsulin molecules can either be refolded to their native structure or degraded through ER associated degradation (ERAD) and autophagy. These degraded molecules decrease proinsulin yield but do not otherwise compromise beta cell function. However, under certain pathological conditions, proinsulin misfolding increases, exceeding the genetically determined threshold of beta cells to handle the misfolded protein load. This results in accumulation of misfolded proinsulin in the ER – a causal factor leading to beta cell failure and diabetes. In patients with Mutant INS -gene induced diabetes of Youth (MIDY), increased proinsulin misfolding due to insulin gene mutations is the primary defect operating as a “first hit” to beta cells. Additionally, increased proinsulin misfolding can be secondary to an unfavorable ER folding environment due to genetic and/or environmental factors. Under these conditions, increased wild-type proinsulin misfolding becomes a “second hit” to the ER and beta cells, aggravating beta cell failure and diabetes. In this article, we describe our current understanding of the normal proinsulin folding pathway in the ER, and then review existing links between proinsulin misfolding, ER dysfunction, and beta cell failure in the development and progression of type 2, type 1, and some monogenic forms of diabetes. [ABSTRACT FROM AUTHOR]

Background: Leydig cells produce testosterone and insulin-like 3, two hormones essential for male sex differentiation and reproductive function. The orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor type II (COUP-TFII), and the zinc finger factor GATA4 are two transcription factors involved in Leydig cell differentiation, gene expression, and function., Objectives: Several Leydig cell gene promoters contain binding motifs for both GATA factors and nuclear receptors. The goal of the present study is to determine whether GATA4 and COUP-TFII cooperate to regulate gene expression in Leydig cells., Materials and Methods: The transcriptomes from GATA4- and COUP-TFII-depleted MA-10 Leydig cells were analyzed using bioinformatic tools. Functional cooperation between GATA4 and COUP-TFII, and other related family members, was assessed by transient transfections in Leydig (MA-10 and MLTC-1) and fibroblast (CV-1) cell lines on several gene promoters. Recruitment of GATA4 and COUP-TFII to gene promoters was investigated by chromatin immunoprecipitation. Co-immunoprecipitation was used to determine whether GATA4 and COUP-TFII interact in MA-10 Leydig cells., Results: Transcriptomic analyses of GATA4- and COUP-TFII-depleted MA-10 Leydig cells revealed 44 commonly regulated genes including the anti-Müllerian hormone receptor type (Amhr2) gene. GATA4 and COUP-TFII independently activated the Amhr2 promoter, and their combination led to a stronger activation. A GC-rich element, located in the proximal Amhr2 promoter was found to be essential for GATA4- and COUP-TFII-dependent activation as well as for the COUP-TFII/GATA4 cooperation. COUP-TFII and GATA4 directly interacted in MA-10 Leydig cell extracts. Chromatin immunoprecipitation revealed that GATA4 and COUP-TFII are recruited to the proximal Amhr2 promoter, which contains binding sites for both factors in addition to the GC-rich element. Cooperation between COUP-TFII and GATA6, but not GATA1 and GATA3, was also observed., Discussion and Conclusion: Our results establish the importance of physical and functional cooperation between COUP-TFII/GATA4 in the regulation of gene expression in MA-10 Leydig cells, and more specifically the Amhr2 gene., (© 2022 American Society of Andrology and European Academy of Andrology.)

The progression of diabetes is accompanied by increasing demand to the beta-cells to produce and secrete more insulin, requiring complex beta-cell adaptations. Functionally active and ubiquitous non-coding RNAs (nc RNAs) have the capacity to take part in such adaptations as they have been shown to be key regulatory molecules in various biological processes. In the pancreatic islets, the function of nc RNAs and their contribution to disease development is beginning to be understood. Here, we review the different classes of nc RNAs, such as long non-coding RNAs (lnc RNAs) and micro RNAs (mi RNAs), and their potential contribution to insulin secretion. A special focus will be on mi RNAs and their regulatory function in beta-cell physiology and insulin exocytosis. As important players in gene regulation, nc RNAs have huge potential in opening innovative therapeutic avenues against diabetes and associated complications. [ABSTRACT FROM AUTHOR]

Highlights: [•] Hyperoxia is mainly negative for function and viability of pancreatic islets. [•] Insulin secretion, oxygen consumption and mitochondrial complexes are decreased. [•] Negative effects can be clinically relevant for oxygen treatment during prematurity. [ABSTRACT FROM AUTHOR]

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 β 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.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Abstract: Over the last 20 years, intensive research has been focused on the specific mechanisms mediating the pancreatic β-cell injury. Both the decreased viability and the dysfunction of β-cells have become the key factors in the development of diabetes mellitus. Thus, it is of utmost importance to elucidate the discrete pathological changes in pancreatic β-cells within the context of the various lipotoxicity models. The goal of these studies is to generate evidence to improve not only the clinical treatment for diabetics, but also modulate the diet and activities of groups at high risk for diabetes. In this regard, we review the role of lipotoxicity in pancreatic β-cell injury and identify potential therapeutic targets in this cell model. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5′-UTR of insulin mRNA. For this purpose, human islets were incubated for 2h in the presence of low (1.67mM) or high glucose (16.7mM). Nitrosative stress was induced by addition of 1mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5′-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7mM glucose but not at 1.67mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5′-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40–100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic β-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5′-UTR of the human insulin mRNA. [Copyright &y& Elsevier]

Abstract: MicroRNAs are small noncoding RNAs that have been highly conserved during evolution and have been implicated to play an important role in many diseases, including diabetes. Several reports indicated the function of miRNAs in insulin production. However, the mechanisms by which miRNAs regulate this process remain poorly understood. Here we found that the expression of miR-15a was up-regulated in the presence of high glucose for 1h, whereas prolonged periods of high glucose exposure resulted in depressed expression of miR-15a, and the change in expression levels of miR-15a coincided with insulin biosynthesis. Moreover, ectopic expression of miR-15a promoted insulin biosynthesis in MIN6 cells, whereas its repression was sufficient to inhibit insulin biosynthesis. Further, we verified that miR-15a directly targeted and inhibited uncoupling protein-2 (UCP-2) gene expression. miR-15a mimics inhibited UCP-2 3′UTR luciferase reporter activity. Western blot analysis showed that miR-15a inhibited endogenous UCP-2 protein levels, and resulted in the increase in oxygen consumption and reduced ATP generation. This study suggests miR-15a is a mediator of β cell function and insulin biosynthesis, thus offering a new target for the development of preventive or therapeutic agents against diabetes. [ABSTRACT FROM AUTHOR]

Abstract: The p24 protein family have multiple functions in protein transport in the early secretory pathway. In this study we examined the role of p24 proteins in insulin transport. Several members were detected in insulinoma cell lines and rat islets and expression levels positively correlated with insulin abundance, particularly for p24δ1 and p24β1. Knocking down p24δ1 in insulinoma cell lines, which also resulted in the concomitant knock-down of other family members, impaired glucose-stimulated insulin secretion, decreased total cellular insulin content and reduced proinsulin biosynthesis. There was no effect on overall protein biosynthesis or ER stress. These results suggest that p24δ1 and possibly other p24 family proteins are required for normal insulin biosynthesis and subsequent secretion in pancreatic β-cells. [Copyright &y& Elsevier]

Abstract: Islet β-cell dysfunction is a characteristic and the main cause of hyperglycaemia of Type 2 diabetes. Understanding the mechanisms that cause β-cell dysfunction will lead to better therapeutic outcomes for patients with Type 2 diabetes. Chronic fatty acid exposure of susceptible islet β-cells causes dysfunction and death and this is associated with increased reactive oxygen species production leading to oxidative stress and increased endoplasmic reticulum stress. We present the hypothesis that metabolic deceleration can reduce both oxidative and endoplasmic reticulum stress and lead to improved β-cell function and viability when exposed to a deleterious fat milieu. This is illustrated by the C57BL/6J mouse which is characterised by reduced insulin secretion and glucose intolerance associated with a mutation in nicotinamide nucleotide transhydrogenase (Nnt) but is resistant to obesity induced diabetes. On the other hand the DBA/2 mouse has comparatively higher insulin secretion and better glucose tolerance associated with increased Nnt activity but is susceptible to obesity-induced diabetes, possibly as a result of increased oxidative stress. We therefore suggest that in states of excess nutrient load, a reduced ability to metabolise this load may protect both the function and viability of β-cells. Strategies that reduce metabolic flux when β-cells are exposed to nutrient excess need to be considered when treating Type 2 diabetes. [Copyright &y& Elsevier]

The Zucker fatty (ZF) rat subjected to 60% pancreatectomy (Px) develops moderate diabetes by 3 weeks. We determined whether a progressive fall in beta cell mass and/or beta cell dysfunction contribute to beta cell failure in this type 2 diabetes model. Partial (60%) or sham Px was performed in ZF and Zucker lean (ZL) rats. At 3 weeks post-surgery, beta cell mass and proliferation, proinsulin biosynthesis, pancreatic insulin content, insulin secretion, and islet glucose and lipid metabolism were measured. ZL-Px rats maintained normal glycaemia and glucose-stimulated insulin secretion (GSIS) despite incomplete recovery of beta cell mass possibly due to compensatory enhanced islet glucose metabolism and lipolysis. ZF-Px rats developed moderate hyperglycaemia (14 mmol/l), hypertriacylglycerolaemia and relative hypoinsulinaemia. Despite beta cell mass recovery and normal arginine-induced insulin secretion, GSIS and pancreatic insulin content were profoundly lowered in ZF-Px rats. Proinsulin biosynthesis was not reduced. Compensatory increases in islet glucose metabolism above those observed in ZF-Sham rats were not seen in ZF-Px rats. Triacylglycerol content was not increased in ZF-Px islets, possibly due to lipodetoxification by enhanced lipolysis and fatty acid oxidation. Fatty acid accumulation into monoacylglycerol and diacylglycerol was increased in ZF-Px islets together with a 4.5-fold elevation in stearoyl-CoA desaturase mRNA expression. Falling beta cell mass, reduced proinsulin biosynthesis and islet steatosis are not implicated in early beta cell failure and glucolipotoxicity in ZF-Px rats. Rather, severe beta cell dysfunction with a specific reduction in GSIS and marked depletion of beta cell insulin stores with altered lipid partitioning underlie beta cell failure in this animal model of type 2 diabetes. [ABSTRACT FROM AUTHOR]

While insulin has been central to the pathophysiology and treatment of patients with diabetes for the last 100 years, it has only been since 2007 that genetic variation in the INS gene has been recognised as a major cause of monogenic diabetes. Both dominant and recessive mutations in the INS gene are now recognised as important causes of neonatal diabetes and offer important insights into both the structure and function of insulin. It is also recognised that in rare cases, mutations in the INS gene can be found in patients with diabetes diagnosed outside the first year of life. This review examines the genetics and clinical features of monogenic diabetes resulting from INS gene mutations from the first description in 2007 and includes information from 389 patients from 292 families diagnosed in Exeter with INS gene mutations. We discuss the implications for diagnosing and treating this subtype of monogenic diabetes. The dominant mutations in the INS gene typically affect the secondary structure of the insulin protein, usually by disrupting the 3 disulfide bonds in mature insulin. The resulting misfolded protein results in ER stress and beta-cell destruction. In contrast, recessive INS gene mutations typically result in no functional protein being produced due to reduced insulin biosynthesis or loss-of-function mutations in the insulin protein. There are clinical differences between the two genetic aetiologies, between the specific mutations, and within patients with identical mutations. • Dominant and recessive mutations in the insulin (INS) gene are important causes of neonatal diabetes. • Associated phenotypes are variable in terms of age at diabetes onset, birth weight and treatment requirements. • Dominant mutations affect the secondary structure of the insulin protein, resulting in beta-cell ER stress and destruction. • Recessive mutations result in reduced insulin biosynthesis or loss-of-function mutations of the insulin protein. • The studies of these forms of diabetes offer important insights into the structure, biosynthesis and secretion of insulin. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the effect of oleanolic acid, a plant-derived triterpenoid, on insulin secretion and content in pancreatic β-cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS-1 832/13 cells and enhanced acute glucose-stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin-4 under glucose-stimulated conditions, yet neither Ca2+ nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti-diabetic properties of this natural product. [Copyright &y& Elsevier]

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with β cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially β cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the lns2 gene, leading to loss of β cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder. [ABSTRACT FROM AUTHOR]

S 21403 (mitiglinide) is a new drug for type 2 diabetes mellitus (T2DM). Its action on insulin release and biosynthesis was investigated in several experimental systems utilizing pancreas from normal and T2DM animals.At high concentrations (10 μM), S 21403, like classical sulphonylurea, induced insulin release in the absence of glucose. In contrast, at therapeutic (0.1–1.0 μM) concentrations, S 21403 amplified insulin secretion glucose dose-dependently and with similar magnitude in normal and diabetic GK rat islets.In perfused GK rat pancreas, S 21403 induced normal kinetics of insulin secretion including first-phase response.The effect of S 21403 was strongly modulated by physiological factors. Thus, 0.1 μM adrenaline inhibited S 21403-induced insulin release. There was marked synergism between S 21403 and arginine in GK rat islets, combination of the two normalizing insulin secretion.In primary islet cultures from normal rats or prediabetic Psammomys obesus, prolonged exposure to S 21403 did not induce further depletion of insulin stores under normal or ‘glucotoxic’ conditions.Proinsulin biosynthesis was not affected by 2-h exposure of rat or prediabetic P. obesus islets to 1 μM S 21403. Yet, 24-h exposure of rat islets to S 21403 resulted in 30% increase in proinsulin biosynthesis at 8.3 mM glucose.Amplification by S 21403 of glucose-induced insulin secretion in diabetic GK β-cells with restoration of first-phase response, a strong synergistic interaction with arginine and marked inhibition by adrenaline, make it a prime candidate for successful oral antidiabetic agent.British Journal of Pharmacology (2005) 146, 872–881. doi:10.1038/sj.bjp.0706374; published online 12 September 2005 [ABSTRACT FROM AUTHOR]

Pancreatic β-cell dysfunction and insulin resistance are observed in type 2 diabetes. Under diabetic conditions, oxidative stress and ER stress are induced in various tissues, leading to activation of the JNK pathway. This JNK activation suppresses insulin biosynthesis and interferes with insulin action. Indeed, suppression of the JNK pathway in diabetic mice improves insulin resistance and ameliorates glucose tolerance. Thus, the JNK pathway plays a central role in pathogenesis of type 2 diabetes and may be a potential target for diabetes therapy. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: The aim of this study was to investigate molecular mechanisms of glucose-induced changes in islets of Langerhans by analysing global changes in protein patterns of islets exposed to elevated glucose concentrations. Methods: Islets were isolated from C57BL/6J mice and used either directly or after exposure to 11 mmol/l glucose for 24 h. Islet protein profiles were obtained by two-dimensional gel electrophoresis, and protein spots were identified by peptide mass fingerprinting using mass spectrometry. Results: Two-dimensional gels of freshly isolated islets and islets exposed to 11 mmol/l glucose contained 1,074 and 1,254 spots, respectively. The number of differentially expressed spots was 379, with 20 spots appearing as new proteins in islets exposed to 11 mmol/l glucose. We identified 124 spots corresponding to 77 protein entries and generated a reference map from freshly isolated islets. Actin, alpha enolase, cytokeratin 8, endoplasmin, glucose- regulated proteins, heat shock proteins, peroxiredoxins, prohormone convertase 2, protein disulphide isomerase, superoxide dismutase, tubulin, and V-type H+-ATPase (V1 subunit A) were upregulated in islets exposed to 11 mmol/l glucose. In contrast, exocrine proteins and secretagogin were downregulated in these islets compared with in freshly isolated islets. Conclusions/interpretation: The islet proteome approach revealed simultaneous changes in protein patterns of islets exposed to elevated glucose concentrations, indicating enhanced insulin synthesis, granular mobilisation and maturation, and increased stress response. The changes may be of relevance for the understanding of altered islet function in the hyperglycaemic state. It is expected that the islet reference map will become an important tool for dissecting multifactorial islet processes. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. A local angiotensin-generating system has been found in the exocrine pancreas. This study aimed, primarily, to investigate the existence of a local angiotensin-generating system in the pancreatic islets and, secondly, to elucidate its role in regulating insulin secretion. Methods. Real-time RT-PCR and western blot were used to investigate if angiotensin-generating components are present in the mouse pancreatic islets, which are subject to regulation by islet transplantation. The localisation of AT1-receptors in islets was investigated by immunohistochemistry. Batch-type incubations of isolated islets were applied for studying the influence of angiotensin II on the glucose-stimulated insulin release, glucose oxidation and (pro)insulin, and total protein biosynthesis. Results. Major components, namely angiotensinogen, ACE, AT1- and AT2-receptors, were expressed in endogenous islets. AT1-receptors were localised to pancreatic beta cells. Exposure of the isolated islets to angiotensin II induced a dose-dependent inhibition of glucose-stimulated insulin release and inhibited (pro)insulin biosynthesis. This inhibitory action was fully preventable by pretreatment of the islets with losartan, an AT1-receptor antagonist. We also investigated if the expression of these components was changed after islet transplantation. Notably, a markedly increased expression of mRNA for the AT1-receptor was observed in islets retrieved from 4-week-old syngeneic islet transplants, a finding that was confirmed at the protein level. Conclusion/interpretation. These data indicate the existence of an islet angiotensin-generating system of potential importance in the physiological regulation of glucose-induced insulin secretion, thus diabetes mellitus. The increased expression of the AT1-receptor in islet transplants could have relevance to islet-graft function. [ABSTRACT FROM AUTHOR]

The C-peptide links the insulin A and B chains in proinsulin, providing thereby a means to promote their efficient folding and assembly in the endoplasmic reticulum during insulin biosynthesis. It then facilitates the intracellular transport, sorting, and proteolytic processing of proinsulin into biologically active insulin in the maturing secretory granules of the β cells. These manifold functions impose significant constraints on the C-peptide structure that are conserved in evolution. After cleavage of proinsulin, the intact C-peptide is stored with insulin in the soluble phase of the secretory granules and is subsequently released in equimolar amounts with insulin, providing a useful independent indicator of insulin secretion. This brief review highlights many aspects of its roles in biosynthesis, as a prelude to consideration of its possible additional role(s) as a physiologically active peptide after its release with insulin into the circulation in vivo. [ABSTRACT FROM AUTHOR]

Glitazones are known to modulate fatty acid-induced effects on insulin secretion in the pancreatic β-cell. The present study focused on combined effects of troglitazone and oleate on preproinsulin (PPI) biosynthesis. Insulin-producing INS-1 cells were incubated for 4 hr at 11.2 mM glucose in the presence (O+) or absence (O−) of 200 μM oleate with (T+) or without (T−) 10 μM troglitazone. After cell lysis, cytoplasmic RNA was extracted and employed for Northern blotting and corresponding in vitro translation. Compared with untreated controls (CTRL=O−/T−), the cellular content of PPI-mRNA from cells which had been simultaneously treated by troglitazone and oleate (O+/T+) was significantly diminished (O+/T+=75±10%×CTRL; P=0.015). The PPI-mRNA content from those cells which had been exclusively exposed either to oleate (O+/T−) or troglitazone (O−/T+) did not significantly differ from that of the untreated controls. In spite of that decreased PPI-mRNA content, in vitro translation revealed the highest yield of newly synthesized PPI in RNA samples from those cells which had been simultaneously exposed to oleate and troglitazone before (O+/T+=1.6±0.3×CTRL; P=0.01). It is concluded that troglitazone and oleate synergistically affect the translational rate at the level of the PPI-mRNA molecule. [Copyright &y& Elsevier]

Evidence for “lipotoxicity,” i.e., negative effects of fatty acids (FA) on pancreatic β-cells is incomplete. Here, we tested whether non-toxic concentrations of FA potentiate toxic effects of interleukin-1β (IL-1β). Culture of INS-1E clonal β-cells for 2–6 days with 70 μM docosahexaenoic acid (DHA), eicosapentaenoic acid, arachidonic acid, 0.1 mM linoleic acid, or 0.1–0.2 mM oleic acid exerted no or minor negative effects on cell viability (MTT assay). Viability was reduced by 0.5 ng/ml IL-1β. All tested FA significantly aggravated this effect after 6 days of culture. IL-1β also exerted negative effects on cellular insulin content and DHA and oleic acid aggravated these effects. l-NAME, an inhibitor of constitutive nitric oxide (NO) synthase, reduced the negative effects of IL-1β per se but did not abolish the potentiating effects of FA. Conclusion: FA potentiate toxic effects of IL-1β on β-cells by mechanisms that include NO-independent ones. [Copyright &y& Elsevier]

Aims/hypothesis: Syntaxin-1A (Syn-1A) is known to play a negative regulatory role in insulin secretion but the precise mechanisms for its action are not clear. Syn-2, –3 and –4 are also present in islet beta cells but their functions are not known. Here, we investigated the role of these syntaxins in the insulin secretory process. Methods: We examined the following effects of Syn-1, –2, –3 and –4 expression in insulinoma beta-cell lines. Endogenous insulin secretion was measured by batch radioimmunoassay (RIA) and single cell patch clamp capacitance measurements. The l-type Ca2+ channel activity was studied by patch clamp electrophysiology. Insulin gene transcription was examined by Northern blotting and measurement of insulin gene promoter activity by the co-expression of cyan fluorescent protein-labelled rat insulin promoter. Results: Syn-1A or –3, but not Syn-2 or –4 overexpression, inhibited K+-induced insulin release as determined by RIA (49.7 ± 5.5 % and 49.1 ± 6.2 %, respectively) and electrophysiologic membrane capacitance measurements (68.0 ± 21.0 % and 58.0 ± 13.2 %, respectively). Overexpressed Syn-1A and –3, but not Syn-2, inhibited Ca2+ channel current amplitude by 39.5 ± 11.6 % and 52.7 ± 6.0 %, respectively. Of note, overexpression of Syn-1A and –3 also reduced single cell (by confocal microscopy) and total cellular endogenous insulin content (by RIA) by 24.8 ± 4.2 % and 31.8 ± 3.9 %, respectively. This correlated to a reduction in endogenous insulin mRNA by 24.5 ± 4.2 % and 25.7 ± 4.2 %, respectively. This inhibition of insulin biosynthesis is mainly at the level of insulin gene transcription as demonstrated by an inhibition of insulin gene promoter activity (53.3 ± 9.15 % and 39.0 ± 6.8 %, respectively). Conclusions/interpretation: These results demonstrate that Syn-1A and –3 possess strong inhibitory actions on both insulin exocytosis and insulin biosynthesis whereas Syn-2 and –4 do not inhibit the insulin secretory process. [Diabetologia (2002) 45: 231–241] [ABSTRACT FROM AUTHOR]

Blood glucose is inadequately controlled in diabetes mellitus, causing various inflammation-related complications. This study aimed to investigate responses to an oral sucrose/lipid challenge in the context of glucose metabolism after consumption of Mori ramulus (MR) extract. In this study on healthy subjects, the optimal dose and safety of MR were confirmed in a preliminary pilot trial (n = 24), meanwhile, blood glucose, insulin, and inflammatory marker levels were detected via an oral sucrose/lipid tolerance test in the main trial (n = 36). In the main study, the blood glucose response was significantly decreased after 240 min in the MR group. Compared to the placebo group, the treatment group exhibited plasma insulin levels that were significantly increased at 120 min and decreased at 240 min. In conclusion, a single MR extract dose protects against inflammation induced by high-fat/sugar to maintain normal insulin secretion and thus helps to maintain postprandial blood glucose levels via an inflammatory mechanism., (Copyright © 2021. Published by Elsevier Masson SAS.)

Introduction: Adipose tissue (AT) expandability may be facilitated by adiponectin and suppressed by orosomucoid, and reduced AT expandability may be associated with first-degree relatives of type 2 diabetes. We tested the hypothesis that orosomucoid may be associated not only with adiponectin and adipose tissue insulin resistance but also with a family history of type 2 diabetes (FHD). Research Design and Methods. Anthropometric and metabolic variables, adipokines, and measures of inflammatory and insulin resistance were cross-sectionally investigated in 153 young normal weight Japanese women. Stepwise multivariate linear regression analyses were used to identify the most important determinants of orosomucoid., Results: Orosomucoid was higher in women with positive ( n = 57) compared to women with negative FHD and was associated positively with FHD (both p = 0.01). Orosomucoid also showed positive associations with fasting glucose ( p < 0.001), free fatty acids ( p = 0.001), and HbA1c ( p = 0.007), whereas there was no association with fasting insulin and serum lipids. In addition, orosomucoid was associated inversely with adiponectin ( p = 0.02) and positively with adipose tissue-insulin resistance index (AT-IR, the product of fasting insulin and free fatty acids; p = 0.001) but not with homeostasis model assessment-insulin resistance, leptin, and high-sensitivity C-reactive protein. In multivariate analyses, AT-IR (standardized β , 0.22; p = 0.003), serum adiponectin (standardized β , -0.163; p = 0.032), FHD+ (standardized β , 0.178; p = 0.029), and HbA1c (standardized β , 0.213; p = 0.005) emerged as independent determinants of orosomucoid and explained 15.2% of its variability., Conclusions: These results are the first to demonstrate that orosomucoid is associated not only with adipose tissue-insulin resistance and adiponectin but also with FHD., Competing Interests: The authors declare that there is no duality of interest associated with this manuscript., (Copyright © 2022 Mari Honda et al.)

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 Cav 1.2 expression may point out a potential pathway of circadian rhythm affecting insulin secretion., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Studies have reported that miRNAs regulate β-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.