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Diabetes causes macrovascular and microvascular complications such as peripheral neuropathy. Glutamate regulates insulin secretion in pancreatic β-cells, and its increased activity in the central nervous system is associated with peripheral neuropathy in animal models of diabetes. One strategy to modulate glutamatergic activity consists in the pharmacological manipulation of metabotropic glutamate receptors (mGluRs), which, compared to the ionotropic receptors, allow for a fine-tuning of neurotransmission that is compatible with therapeutic interventions. mGluRs are a family of eight G-protein coupled receptors classified into three groups (I-III) based on sequence homology, transduction mechanisms, and pharmacology. Activation of group II and III or inhibition of group I represents a strategy to counteract the glutamatergic hyperactivity associated with diabetic neuropathy. In this review article, we will discuss the role of glutamate receptors in the release of insulin and the development/treatment of diabetic neuropathy, with particular emphasis on their manipulation to prevent the glutamatergic hyperactivity associated with diabetic neuropathy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, financial or otherwise., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the potential benefits of close-looped insulin delivery systems in regulating glycemic homeostasis and effectively alleviating diabetes, they still encounter challenges such as limited effectiveness in preventing low glycemic episodes due to sluggish glucose response, and issues with the instability of enzymes and carriers. In this study, dually-crosslinked and glucose oxidase (GOx)-immobilized insulin nanogels (DC-NGs@Ins) are developed for rapid-responsive and sustained hypoglycemic therapy. The DC-NGs@Ins with the phenylborate ester linker enabled the insulin release in a close-looped fashion, and moreover, immobilized GOx-generated hydrogen peroxide (H 2 O 2 ) by consuming the glucose, which can further bind to phenylborate ester for enhancing glucose response and accelerating the insulin release. The dually-crosslinked structure (phenylboronic ester and UV-crosslinking) effectively minimized the initial burst release of insulin, thus preventing the potential risk of hypoglycemia. More interestingly, GOx immobilized in the nanogels mitigated GOx leakage and enhanced its multiple utilization compared to free GOx. In vivo study demonstrated that DC-NGs@Ins effectively maintained glycemic levels (BGLs) below 200 mg dL -1 for at least 8 h compared to singly-crosslinked nanogels (SC-NGs@Ins). Therefore, this intelligent insulin delivery system shows potential applications in diabetes treatment., (© 2024 Wiley‐VCH GmbH.)

Expanding the functions and applications of DNA by integrating noncanonical bases and structures into biopolymers is a continuous scientific effort. An adenine-rich strand (A-strand) is introduced as functional scaffold revealing, in the presence of the low-molecular-weight cofactor cyanuric acid (CA, p K a 6.9), supramolecular hydrogel-forming efficacies demonstrating multiple pH-responsiveness. At pH 1.2, the A-strand transforms into a parallel A-motif duplex hydrogel cross-linked by AH + -H + A units due to the protonation of adenine (p K a 3.5). At pH 5.2, and in the presence of coadded CA, a helicene-like configuration is formed between adenine and protonated CA, generating a parallel A-CA triplex cross-linked hydrogel. At pH 8.0, the hydrogel undergoes transition into a liquid state by deprotonation of CA cofactor units and disassembly of A-CA triplex into its constituent components. Density functional theory calculations and molecular dynamics simulations, supporting the structural reconfigurations of A-strand in the presence of CA, are performed. The sequential pH-stimulated hydrogel states are rheometrically characterized. The hydrogel framework is loaded with fluorescein-labeled insulin, and the pH-stimulated release of insulin from the hydrogel across the pH barriers present in the gastrointestinal tract is demonstrated. The results provide principles for future application of the hydrogel for oral insulin administration for diabetes.

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca 2+ -dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca 2+ ] i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca 2+ ] i microdomains around single or clustered Ca 2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca 2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca 2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca 2+ spikes or incidentally overlap with subplasmalemmal Ca 2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca 2+ microdomains around voltage-gated Ca 2+ channels., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: This study aimed to present an innovative method for synthesizing pH-thermo-glucose responsive poly(NIPA- co -DMAEMA)-PBA hydrogel nanoparticles via single-step aqueous free radical polymerization., Methods: The synthesis process involved free radical polymerization in an aqueous solution, and the resulting nanoparticles were characterized for their physical and chemical properties by 1 H NMR, Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Insulin-loaded poly(NIPA- co -DMAEMA)-PBA hydrogel nanoparticles were prepared and evaluated for their insulin capture and release properties at different pH and temperature, in addition to different glucose concentrations, with the release profile of insulin quantitatively evaluated using the Bradford method., Results: 1 H NMR results confirmed successful PBA incorporation, and DLS outcomes consistently indicated a transition to a more hydrophobic state above the Lower Critical Solution Temperature (LCST) of NIPA and DMAEMA. While pH responsiveness exhibited variation, insulin release generally increased with rising pH from acidic to neutral conditions, aligning with the anticipated augmentation of anionic PBA moieties and increased hydrogel hydrophilicity. Increased insulin release in the presence of glucose, particularly for formulations with the lowest mol % PBA, along with a slight increase for the highest mol % PBA formulation when increasing glucose from 1 to 4 mg/mL, supported the potential of this approach for nanoparticle synthesis tailored for glucose-responsive insulin release., Conclusions: This work successfully demonstrates a novel method for synthesizing responsive hydrogel nanoparticles and underscores their potential for controlled insulin release in response to glucose concentrations. The observed pH-dependent insulin release patterns and the influence of PBA content on responsiveness highlight the versatility and promise of this nanoparticle synthesis approach for applications in glucose-responsive drug delivery systems., Graphical Abstract: Poly(NIPA) nanoparticles containing PBA moieties are normally synthesized in two or more steps in the presence of organic solvents. Here we propose a new method for the synthesis of multiresponsive hydrogel poly(NIPA- co -DMAEMA)-PBA nanoparticles in aqueous medium in a single reaction to provide a fast and effective strategy for the production of glucose-responsive multi-systems in aqueous media from free radical polymerization., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s), under exclusive licence to Tehran University of Medical Sciences 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

β 3 -Adrenoceptors mediate several functions in rodents that could be beneficial for the treatment of obesity and type 2 diabetes. This includes promotion of insulin release from the pancreas, cellular glucose uptake, lipolysis, and thermogenesis in brown adipose tissue. In combination, they lead to a reduction of body weight in several rodent models including ob/ob mice and Zucker diabetic fatty rats. These findings stimulated drug development programs in various pharmaceutical companies, and at least nine β 3 -adrenoceptor agonists have been tested in clinical trials. However, all of these projects were discontinued due to the lack of clinically relevant changes in body weight. Following a concise historical account of discoveries leading to such drug development programs we discuss species differences that explain why β 3 -adrenoceptors are not a meaningful drug target for the treatment of obesity and type 2 diabetes in humans.

In order to investigate the chronic effects of basic amino acids (BAA) on β-cell metabolism and insulin secretion, INS-1 β-cells were randomly assigned to cultures in standard medium (Con), standard medium plus 10 mM L-Arginine (Arg), standard medium plus 10 mM L-Histidine (His) or standard medium plus 10 mM L-Lysine (Lys) for 24 h. Results showed that insulin secretion was decreased by the Arg treatment but was increased by the His treatment relative to the Con group ( p < 0.05). Higher BAA concentrations reduced the high glucose-stimulated insulin secretions ( p < 0.001), but only Lys treatment increased the intracellular insulin content than that in the Con group ( p < 0.05). Compared with Arg and Lys, the His treatment increased the mitochondrial key enzyme gene expressions including Cs , mt-Atp6 , mt-Nd4l and Ogdh , and caused a greater change in the metabolites profiling ( p < 0.05). The most significant pathways affected by Arg, His and Lys were arginine and proline metabolism, aminoacyl-tRNA biosynthesis and pyrimidine metabolism, respectively. Regression analysis screened 7 genes and 9 metabolites associated with insulin releases during BAA stimulations ( p < 0.05). Together, different BAAs exerted dissimilar effects on β-cell metabolism and insulin outputs.

Background: In vitro studies with human fetal islets of different gestational ages (GA) would be a great tool to generate information on the developmental process of the islets as this would help to recontextualize diabetes research and clinical practice. Pancreatic islets from human cadavers and other animal species are extensively researched to explore their suitability for islet transplantation procedure, one of the upcoming treatment strategies for insulin-dependent diabetes mellitus. Although human fetal islets are also considered for islet transplantation, ethical issues and limited knowledge constraints their use. The fetal islets could be explored to address the information lacunae on the maturity process of pancreatic islets and the endocrine-exocrine signaling mechanisms., Aim: This study aimed to assess the feasibility of isolating viable islets and study the cytoarchitecture of the fetal pancreas of GA 22-29 weeks, not reported otherwise., Methodology: Pancreas obtained from the aborted fetuses of GA 22-29 weeks were subjected to collagenase digestion and were further cultured to determine the viability in vitro. Parameters assessed were expression of markers for endocrine cell lineages and insulin release to glucose challenge., Results: Islets were viable in vitro and islets were shown to maintain cues for post-digestion re-aggregation and expansion in culture. The immunofluorescent staining showed islets of varying sizes, homogenous cell clusters aggregating to form heterogenous cell clusters, otherwise not reported for this GA. On stimulation with different concentrations of glucose (2.8 and 28 mM), the fetal islets in the culture exhibited insulin release, and this response confirmed their viability in vitro., Conclusion: Our findings showed that viable islets could be isolated and cultured in vitro for further in-depth studies to explore their proliferative potential as well as for the identification of pancreatic progenitors, a good strategy to take forward., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Rout et al.)

Successful management of type 2 diabetes mellitus (T2DM), a complex and chronic disease, requires a combination of anti-hyperglycemic and anti-inflammatory agents. Here, we have conceptualized and tested an integrated "closed-loop mimic" in the form of a glucose-responsive microgel (GRM) based on chitosan, comprising conventional insulin (INS) and curcumin-laden nanoparticles (nCUR) as a potential strategy for effective management of the disease. In addition to mimicking the normal, on-demand INS secretion, such delivery systems display an uninterrupted release of nCUR to combat the inflammation, oxidative stress, lipid metabolic abnormality, and endothelial dysfunction components of T2DM. Additives such as gum arabic (GA) led to a fivefold increased INS loading capacity compared to GRM without GA. The GRMs showed excellent in vitro on-demand INS release, while a constant nCUR release is observed irrespective of glucose concentrations. Thus, this study demonstrates a promising drug delivery technology that can simultaneously, and at physiological/pathophysiological relevance, deliver two drugs of distinct physicochemical attributes in the same formulation., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Holarrhena pubescens is widely used in Indian and Chinese medicine in the treatment of diabetes. The current work determined the oral hypoglycemic and antidiabetic effects of seed extract in rats. The probable mechanism of action was evaluated in-vitro by α - glucosidase inhibition, glucose metabolism in insulinoma (INS-1) cells to reflect secretion of insulin, and protein glycation inhibition. Its potential for herb-drug interaction was evaluated in the cytochrome P450 3A4 (CYP3A4) inhibition assay. The seed extract increased serum insulin levels and reduced serum blood glucose levels in the oral glucose tolerance test. It also reduced the serum glucose levels in streptozocin-induced diabetes. The extract also inhibited α -glucosidase enzyme activity and demonstrated that it can increase the secretion of insulin from INS to 1-rat insulinoma cell line cells in-vitro in a concentration-dependent manner. However, it had a very weak inhibitory effect on protein glycation and it did not affect the activity of CYP3A4. The results of the study showed that H. pubescens seed extract increases insulin secretion and inhibits glucose absorption both in-vivo and in-vitro with a weak protein glycation inhibitory effect. The herb is devoid of CYP3A4 inhibitory effect indicating that it may not have pharmacokinetic interaction with the drug metabolized by this enzyme., Competing Interests: 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., (© 2023 The Author(s).)

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH 2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp 4 →Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 μM) compared with the naturally occurring peptide. The substitution Ala 18 →Lys and the double substitution Asp 4 →Lys and Ala 18 →Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC 50 values in the range of 12-20 μM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC 50  = 20 μM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca 2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 μM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes., (© 2022 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

We developed insulin loaded biomimetic microsphere by laccase-mediated crosslinking using a microfluidic device in the water-in-oil emulsion system as an injectable vehicle for the repair of sciatic tissue. Aqueous polymeric solution of phenol-substituted hyaluronic acid (HAPh) and collagen (ColPh) containing insulin and laccase flowed from the inner channel into oil flow within an outer channel which leads formation of hydrogel microsphere. The physical properties of prepared specimens including swelling rate, mechanical resistance and the prolonged release rate of microspheres proved applicability of fabricated vehicles for tissue engineering and drug delivery systems. The growth profile and behavior of cells in microspheres indicated cytocompatibility of the method and prepared vehicles for microtissue development. Histopathological examination revealed a significant increase in axonal regeneration, and remyelination process in injured sciatic nerve following treatment with HAPh/ColPh microspheres containing insulin compared to control groups. Also, the functional characteristic of sciatic tissue showed that the presence of biomimetic microsphere and insulin simultaneously had improved sciatic tissue functions including functional sciatic index (SFI) values, reaction to hot plate and muscle weight of rats. In summary, the results proved that composite biomimetic microspheres containing insulin effectively improved nerve regeneration in the rat model., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

Hormones and neurotransmitters are released from (neuro)endocrine cells by regulated exocytosis of secretory granules. During exocytosis, the granule membrane fuses with the plasma membrane, which allows release of the stored content into the bloodstream or the surrounding tissue. Here, we give a detailed description of two complementary methods to observe and quantify exocytosis in single cells: high-resolution TIRF microscopy and patch-clamp capacitance recordings. Precise stimulation of exocytosis is achieved by local pressure application or voltage-clamp depolarizations. While the chapter is focused on insulin-secreting cells as an accessible and disease-relevant model system, the methodology is applicable to a wide variety of secretory cells including chromaffin and PC12 cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Previous work demonstrated that arginine is one of the strongest insulin secretagogues. However, knowledge of the mechanisms linking chronic arginine metabolism with β-cell function and insulin secretion is relatively limited. After preliminary selection of concentration according to the cell proliferation, the MIN6 pancreatic β-cells were randomly assigned to culture in 0.04 mM (low-arginine, LA), 0.4 mM (standard-arginine, SA), or 8 mM arginine (high-arginine, HA) for 24 h. Following the treatment, a combination of transcriptomics and metabolomics, together with a series of molecular biological tests were performed to investigate the responses of β-cells to varied arginine availability. Our results showed that HA treatment reduced the chronic insulin releases, and LA and HA treatments decreased the glucose-stimulated insulin secretions (GSIS) of β-cells relative to the SA group (p < .05). Transcriptomics analysis indicated that LA administration significantly inhibited oxidative phosphorylation and ATP metabolic process but promoted DNA repair and mRNA processing in β-cells, while HA administration affected ammonium ion metabolic process and mRNA export (p < .05). Both LA and HA regulated the expressions of genes involved in DNA replication, cell-cycle phase transition, and response to oxidative stress (p < .05). Protein-protein interaction and transcription factor analyses suggested that Trp53 and Nr4a2 genes may play key roles during arginine stimulation. On the contrary, metabolomics analysis demonstrated that the differentially expressed metabolites (DEM) of MIN6 β-cells induced by LA were mainly enriched in glycerophospholipid metabolism, linoleic acid metabolism, and purine metabolism, while most DEMs between LA vs. SA comparison belonged to amino acid metabolism. When combined the three groups, co-expression analysis suggested that insulin secretions had strong associations with L-pyroglutamic acid, L-glutamate, and creatine concentrations, while intracellular insulin contents were mainly correlated to L-arginine, argininosuccinic acid, and phosphorylcholine. At last, integrated analysis of transcriptomics and metabolomics showed that glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids, and amino acid metabolism were the most relevant pathways in β-cells exposed to abnormal arginine supply. This descriptive bioinformatics analysis suggested that the disturbed carbohydrate, lipid, and amino acid metabolisms, as well as the increased apoptosis and elevated oxidative stress, contributed to the reduced insulin secretion and lower GSIS in β-cells induced by LA or HA treatments, while some underlying mechanisms need to be further explored., (© 2022 Federation of American Societies for Experimental Biology.)

Glucagon-like peptide-1 (GLP-1) receptor agonists are effective treatments for type 2 diabetes as they stimulate insulin release and promote weight loss through appetite suppression. Their main side effect is nausea. All approved GLP-1 agonists are full agonists across multiple signalling pathways. However, selective engagement with specific intracellular effectors, or biased agonism, has been touted as a means to improve GLP-1 agonists therapeutic efficacy. In this review, I critically examine how GLP-1 receptor-mediated intracellular signalling is linked to physiological responses and discuss the implications of recent studies investigating the metabolic effects of biased GLP-1 agonists. Overall, there is little conclusive evidence that beneficial and adverse effects of GLP-1 agonists are attributable to distinct, nonoverlapping signalling pathways. Instead, G protein-biased GLP-1 agonists appear to achieve enhanced anti-hyperglycaemic efficacy by avoiding GLP-1 receptor desensitisation and downregulation, partly via reduced β-arrestin recruitment. This effect seemingly applies more to insulin release than to appetite regulation and nausea, possible reasons for which are discussed. At present, most evidence derives from cellular and animal studies, and more human data are required to determine whether this approach represents a genuine therapeutic advance. LINKED ARTICLES: This article is part of a themed issue on GLP1 receptor ligands (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.4/issuetoc., (© 2021 The Author. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Objective: Type 2 diabetes with obesity is regarded as an incurable, progressive disease with many complications. The hypothesis was tested that glycated haemoglobin (HbA1c) and the insulin release curve can be restored by traceable systematic methods., Methods: 122 people with diabesity were investigated before and after three and 6 months of traceable systematic management methods. Basal body mass index (BMI), fatty liver, HbA1c, and insulin release curve were measured., Results: After 3 months of traceable systematic management, BMI decreased from 30.76 ± 0.48 to 21.86 ± 0.09 kg/m2 (p < 0.001) and remained stable during the last 3 months (21.82 ± 0.09 kg/m2 at 6 months). Colour Doppler ultrasound showed non-alcoholic fatty liver disease (NAFLD) in all diabesity participants at baseline. At 3 months, only one participant had low-grade fatty liver, and fatty liver was reversed in other participants (p < 0.001). The number and grade of fatty liver at 6 months were the same as at 3 months. Fasting plasma glucose decreased and continued to decrease thereafter (p < 0.001). Two-hour postprandial plasma glucose decreased and continued to decline until 6 months (p < 0.001). HbA1c also decreased and maintained this level at 6 months. At baseline, the peak value of insulin release was 1,141.09 ± 43.02 pmol/L at 2 h after meals, and the early phase of insulin secretion was lost. After 3 months of management, the insulin concentration was 621.62 ± 19.32 pmol/L at 2 h after meals. After 6 months, the value decreased, and the early phase of insulin secretion recovered., Conclusions: Normalization of the insulin release curve in type 2 diabetes was achieved by traceable systematic methods. This was associated with recovery from NAFLD. Diabesity is reversible by traceable systematic management., (© 2022 S. Karger AG, Basel.)

Pancreatic β-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell-specific knockout (KO) mice for all 3 isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in β-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

It is suggested that clock genes link the circadian rhythm to glucose and lipid metabolism. In this study, we explored the role of the clock gene Bmal1 in the hypothalamic paraventricular nucleus (PVN) in glucose metabolism. The Sim1 - Cre -mediated deletion of Bmal1 markedly reduced insulin secretion, resulting in impaired glucose tolerance. The pancreatic islets' responses to glucose, sulfonylureas (SUs) and arginine vasopressin (AVP) were well maintained. To specify the PVN neuron subpopulation targeted by Bmal1, the expression of neuropeptides was examined. In these knockout (KO) mice, the mRNA expression of Avp in the PVN was selectively decreased, and the plasma AVP concentration was also decreased. However, fasting suppressed Avp expression in both KO and Cre mice. These results demonstrate that PVN BMAL1 maintains Avp expression in the PVN and release to the circulation, possibly providing islet β-cells with more AVP. This action helps enhance insulin release and, consequently, glucose tolerance. In contrast, the circadian variation of Avp expression is regulated by feeding, but not by PVN BMAL1.

Islets organoids would have value in the cell replacement therapy for diabetes apart from usual personalized drug screening routes. Generation of a large number of Islets like clusters, with ability to respond to glucose stimulation appears to be an ideal choice. In this study we have generated islet organoids with the ability to respond to glucose stimulation by insulin release. The source of the cells was an iPSC cell line differentiated into the pancreatic progenitors. These cells were assembled in matrigel or cross-linked collagen scaffold and compared for their efficacy to release insulin upon stimulation with glucose. The assembled organoids were examined by immunohistochemistry and expression of the relevant marker genes. The organoids showed expression of islet like markers in both - matrigel and crosslinked collagen scaffold. The islet organoids in both the cases showed release of insulin upon stimulation with glucose. The crosslinked collagen scaffold is quite stable and supports islet cells growth and function., (© 2021. The Author(s).)

Alginate composite hydrogels that exhibit highly sensitive stimuli-responsive behavior were used for signal-stimulated release of pre-loaded insulin. The alginate pores, particularly located at the periphery, were blocked by interpenetration of polyvinyl alcohol (PVA) cross-linked with 1,3-benzenediboronic acid (IPN), thus, significantly reducing uncontrolled leakage of the entrapped biomolecules. The beads were loaded with insulin and various enzymes mimicking different Boolean logic gates (AND, OR, NOR, IMP, INHIB). The enzymes were activated with biologically relevant input signals applied in four logic combinations: 0,0; 1,0; 0,1; 1,1, having the production of H 2 O 2 as the result of the biocatalytic reactions. The "successful" combination of the input signals leading to the H 2 O 2 production was different for different logic gates, following the corresponding truth tables of the logic gates. When H 2 O 2 was produced, boronate ester bonds were oxidized and the IPN was irreversibly degraded, thus re-opening the original pores of the hydrogel. This process allowed release of insulin from the alginate beads. The smart soft material that we have developed tackled well-known limitations of these systems and it may prove valuable in future medical diagnostics or treatments., (© 2021 Wiley-VCH GmbH.)

Aims/hypothesis: ZBED6 (zinc finger, BED-type containing 6) is known to regulate muscle mass by suppression of Igf2 gene transcription. In insulin-producing cell lines, ZBED6 maintains proliferative capacity at the expense of differentiation and beta cell function. The aim was to study the impact of Zbed6 knockout on beta cell function and glucose tolerance in C57BL/6 mice., Methods: Beta cell area and proliferation were determined in Zbed6 knockout mice using immunohistochemical analysis. Muscle and fat distribution were assessed using micro-computed tomography. Islet gene expression was assessed by RNA sequencing. Effects of a high-fat diet were analysed by glucose tolerance and insulin tolerance tests. ZBED6 was overexpressed in EndoC-βH1 cells and human islet cells using an adenoviral vector. Beta cell cell-cycle analysis, insulin release and mitochondrial function were studied in vitro using propidium iodide staining and flow cytometry, ELISA, the Seahorse technique, and the fluorescent probes JC-1 and MitoSox., Results: Islets from Zbed6 knockout mice showed lowered expression of the cell cycle gene Pttg1, decreased beta cell proliferation and decreased beta cell area, which occurred independently from ZBED6 effects on Igf2 gene expression. Zbed6 knockout mice, but not wild-type mice, developed glucose intolerance when given a high-fat diet. The high-fat diet Zbed6 knockout islets displayed upregulated expression of oxidative phosphorylation genes and genes associated with beta cell differentiation. In vitro, ZBED6 overexpression resulted in increased EndoC-βH1 cell proliferation and a reduced glucose-stimulated insulin release in human islets. ZBED6 also reduced mitochondrial JC-1 J-aggregate formation, mitochondrial oxygen consumption rates (OCR) and mitochondrial reactive oxygen species (ROS) production, both at basal and palmitate + high glucose-stimulated conditions. ZBED6-induced inhibition of OCR was not rescued by IGF2 addition. ZBED6 reduced levels of the mitochondrial regulator PPAR-γ related coactivator 1 protein (PRC) and bound its promoter/enhancer region. Knockdown of PRC resulted in a lowered OCR., Conclusions/interpretation: It is concluded that ZBED6 is required for normal beta cell replication and also limits excessive beta cell mitochondrial activation in response to an increased functional demand. ZBED6 may act, at least in part, by restricting PRC-mediated mitochondrial activation/ROS production, which may lead to protection against beta cell dysfunction and glucose intolerance in vivo., (© 2021. The Author(s).)

As a calcineurin inhibitor, tacrolimus is commonly used as a first‑line immunosuppressant in organ transplant recipients. Post‑transplantation diabetes mellitus (PTDM) is a common complication following kidney transplantation and is associated with immunosuppressant drugs, such as tacrolimus. PTDM caused by tacrolimus may be related to its influence on insulin secretion and insulin resistance. However, the specific mechanism has not been fully elucidated. The aim of the present study was to investigate whether the PI3K/Akt/mTOR signaling pathway served an important role in the pathogenesis of PTDM induced by tacrolimus. In the present study, the Cell Counting Kit‑8 assay was used to measure the effect of tacrolimus on the viability of Min6 mouse insulinoma cells. The effects of tacrolimus on the insulin secretion and the activity of caspase‑3 of Min6 cells stimulated by glucose exposure were measured by ELISA. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using WST‑8 and thiobarbituric acid assays, respectively. The effects of tacrolimus on the mRNA expression levels of PI3K, Akt and mTOR were detected by reverse transcription‑quantitative PCR (RT‑qPCR), whereas the protein expression levels of PI3K, Akt, mTOR, phosphorylated (p)‑AKT and p‑mTOR in Min6 cells were assessed using western blotting. The present data indicated that, compared with the control group, 5, 25 and 50 ng/ml tacrolimus treatment could inhibit the insulin secretion of Min6 cells stimulated by glucose solution, and 50 ng/ml tacrolimus could notably decrease the stimulation index (P<0.05). Moreover, 50 ng/ml tacrolimus markedly increased the activity of caspase‑3 by 175.1% (P<0.05), it also decreased the SOD activity (P<0.01) and increased MDA levels (P<0.05). The RT‑qPCR results demonstrated that the mRNA expression levels of PI3K, Akt and mTOR were downregulated by 25 and 50 ng/ml tacrolimus (P<0.01). Furthermore, the western blotting results suggested that tacrolimus had no significant effects on the expression levels of total PI3K, Akt and mTOR proteins (P>0.05), but 25 and 50 ng/ml tacrolimus could significantly inhibit the expression levels of p‑Akt and p‑mTOR (P<0.01). In conclusion, tacrolimus decreased the activity and insulin secretion of pancreatic β cells and induced the apoptosis of islet β cells by inhibiting the mRNA expression levels of PI3K, Akt and mTOR and reducing the phosphorylation of Akt and mTOR proteins in the PI3K/Akt/mTOR signaling pathway, which may ultimately lead to the occurrence of diabetes mellitus, and may be considered as one of the specific mechanisms of PTDM caused by tacrolimus.

Objective: Elevated intracellular cAMP concentrations potentiate insulin secretion from pancreatic β cells. Phosphodiesterase 3B (PDE3B) is highly expressed in these cells and plays a role in the regulation of insulin secretion., Materials and Methods: In this study, effects of amrinone, an inhibitor of PDE3B on insulin release from isolated pancreatic islets, were determined., Results: Exposure of islets to amrinone for 15, 30 and 90 min markedly increased secretion induced by 6.7 mM glucose. Amrinone enhanced also secretion stimulated by 6.7 mM glucose and DB-cAMP, an activator of PKA. It was also demonstrated that amrinone potentiated insulin secretion induced by 6.7 mM glucose in the combination with PMA (activator of PKC) or acetylcholine. However, the insulin-secretory response to glucose and glibenclamide was unchanged by amrinone., Conclusions: These results indicate that amrinone is capable of increasing insulin secretion; however, its action is restricted.

Diabetes mellitus (DM) is one of the principal manifestations of metabolic syndrome and its prevalence with modern lifestyle is increasing incessantly. Chronic hyperglycemia can induce several vascular complications that were referred to be the major cause of morbidity and mortality in DM. Although several therapeutic targets have been identified and accessed clinically, the imminent risk of DM and its prevalence are still ascending. Substantial pieces of evidence revealed that histone deacetylase (HDAC) isoforms can regulate various molecular activities in DM via epigenetic and post-translational regulation of several transcription factors. To date, 18 HDAC isoforms have been identified in mammals that were categorized into four different classes. Classes I, II, and IV are regarded as classical HDACs, which operate through a Zn-based mechanism. In contrast, class III HDACs or Sirtuins depend on nicotinamide adenine dinucleotide (NAD + ) for their molecular activity. Functionally, most of the HDAC isoforms can regulate β cell fate, insulin release, insulin expression and signaling, and glucose metabolism. Moreover, the roles of HDAC members have been implicated in the regulation of oxidative stress, inflammation, apoptosis, fibrosis, and other pathological events, which substantially contribute to diabetes-related vascular dysfunctions. Therefore, HDACs could serve as the potential therapeutic target in DM towards developing novel intervention strategies. This review sheds light on the emerging role of HDACs/isoforms in diabetic pathophysiology and emphasized the scope of their targeting in DM for constituting novel interventional strategies for metabolic disorders/complications.

Aims: Previous studies suggest that small intestinal bacterial overgrowth (SIBO) is associated with type 2 diabetes. However, few studies have evaluated the association between SIBO and beta-cell function in type 2 diabetes. The aim of this study was to evaluate whether beta-cell function was associated with SIBO., Materials and Methods: One hundred four patients with type 2 diabetes were included in this study. Based on the presence of SIBO, the patients were divided into SIBO-positive and SIBO-negative groups. Oral glucose tolerance tests were performed. Insulin sensitivity was measured using 1/homeostasis model assessment of insulin resistance (1/HOMA-IR) and the insulin sensitivity index (ISIM). Insulin release was calculated by HOMA-β, early-phase insulin secretion index InsAUC 30 /GluAUC 30 , and total-phase insulin secretion index InsAUC 120 /GluAUC 120 ., Results: Compared with the SIBO-negative group, patients in the SIBO-positive group showed a higher glucose level at 120 minutes, HbA1c, 1/HOMA-IR, and ISIM and a lower HOMA-β level, early-phase InsAUC 30 /GluAUC 30 , and total-phase InsAUC 120 /GluAUC 120 . Multiple linear regression analysis showed that body mass index, glucose at 0 minutes, and SIBO were independently associated with the early-phase and total-phase insulin secretion., Conclusion: SIBO may be involved in lower levels of insulin release and worse glycemic control.

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gβ5-R7, a protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit β 5 ( Gnb5 ) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5 -/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K + -evoked membrane depolarization. These results indicate that Gβ5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gβ5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release., (© 2020 Wang et al.)

Ghrelin, a 28-amino acid peptide, is a strong growth hormone secretagogue and a regulator of food intake. In addition, ghrelin is thought to play a role in insulin secretion and in glucose homeostasis. A lot of contradictory data have been reported in the literature regarding the co-localization of ghrelin with other hormones in the islet of Langerhans, its role in insulin secretion and attenuation of type 2 diabetes mellitus. In this study, we investigate the effect of chronic ghrelin treatment on glucose, body weight and insulin level in normal and streptozotocin-induced diabetic male Wistar rats. We have also examined the distribution pattern and co-localization of ghrelin with insulin in pancreatic islet cells using immunohistochemistry and immune-electron microscopy and the ability of ghrelin to stimulate insulin release from the CRL11065 beta cell line. Control, non-diabetic groups received intraperitoneal injection of normal saline, while treated groups received intraperitoneal injection of 5 µg/kg body weight of ghrelin (amino acid chain 24-51) on a daily basis for a duration of four weeks. Our results show that the administration of ghrelin increases the number of insulin-secreting beta cells and serum insulin level in both normal and diabetic rats. We also demonstrated that ghrelin co-localizes with insulin in pancreatic islet cells and that the pattern of ghrelin distribution is altered after the onset of diabetes. Moreover, ghrelin at a dose of 10-6M and 10-12M increased insulin release from the CRL11065 beta cell line. In summary, ghrelin co-localizes with insulin in the secretory granules of pancreatic beta cells and enhances insulin production., Competing Interests: The authors have no competing interests whatsoever. The authors also confirm that they are responsible for the content of the manuscript.

A key feature of type 2 diabetes (T2D) is that beta-cells of the pancreatic islets fail to release sufficient amounts of insulin to overcome peripheral insulin resistance. Glucose-stimulated insulin secretion (GSIS) is regulated by the activity of numerous neurotransmitters, hormones and paracrine factors that act by stimulating specific G protein-coupled receptors expressed by pancreatic beta-cells. Studies with both mouse and human islets suggest that acetylcholine (ACh) acts on beta-cell M 3 muscarinic receptors (M3Rs) to promote GSIS. In mouse islets, beta-cell M3Rs are thought to be activated by ACh released from parasympathetic nerve endings. Interestingly, studies with human pancreatic islets suggest that ACh is synthesized, stored and released by alpha-cells, which, in human pancreatic islets, are intermingled with beta-cells. Independent of the source of pancreatic islet ACh, recent studies indicate that beta-cell M3Rs represent a potential target for drugs capable of promoting insulin release for therapeutic purposes. In this review, we will provide an overview about signaling pathways and molecules that regulate the activity of beta-cell M3Rs. We will also discuss a novel pharmacological strategy to stimulate the activity of these receptors to reduce the metabolic impairments associated with T2D., Competing Interests: Declaration of Competing Interest None of the authors has any conflicts of interest regarding this submission., (Published by Elsevier B.V.)

Pancreatic β-cells recognize blood glucose changes and release insulin that is a peptide hormone responsible for stable glycemia. Diabetes, a chronic disorder of insulin insufficiency, leads to disturbed glucose homeostasis and multi-organ problems. Glucose and insulin are key markers in the follow-up and control of this disease. Mitochondrial metabolism of pancreatic beta cells is a crucial part of glucose-stimulated cascade of insulin secretion. Effective factors on β-cells mitochondrial function in production of compounds such as tricarboxylic acid intermediates, glutamate, nicotinamide adenine dinucleotide phosphate, and reactive oxygen species can have great effects on the secretion of insulin under diabetes. This review enhances our knowledge of factors influencing mitochondrial function as a key mediator of glucose-induced insulin release that accordingly will be helpful to further our understanding of the mechanisms implicated in the progressive beta cell failure that results in diabetes.

The tolerability and efficacy of low-frequency, low-intensity therapeutic ultrasound-induced insulin release was investigated in a pre-clinical in vivo murine model. The treatment groups received a single 5-min continuous sonication at 1 MHz and 1.0 W/cm 2 . Insulin and glucagon levels in the serum were determined using enzyme-linked immunosorbent assay. The pancreas was excised and sectioned for histologic analysis. In terminal studies, we observed a moderate (∼50 pM) but significant increase in blood insulin concentration in vivo immediately after sonication compared with a decrease of approximately 60 pM in sham animals (n < 6, p < 0.005). No difference was observed in the change in glucose or glucagon concentrations between groups. Comparisons of hematoxylin and eosin-stained terminal and survival pancreatic tissue revealed no visible differences or evidence of damage. This study is the first step in assessing the translational potential of therapeutic ultrasound as a treatment for early stages of type 2 diabetes., (Copyright © 2019. Published by Elsevier Inc.)

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection.

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu 13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1β and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal β-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10 -6  M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M 3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic β cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured β cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by β-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in β cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents., Competing Interests: The authors declare no conflict of interest.

Evidence suggests that low concentration perinatal exposure to environmental contaminants, such as organophosphate (OP) is associated with later life insulin resistance and type 2 diabetes. The aim of this work was to investigate whether chronic maternal OP exposure exacerbates metabolic dysfunctions in early-overfed rats. During pregnancy and lactational periods, dams received OP by gavage. To induce neonatal overnutrition at postnatal day 3, pups were standardized to 9 or 3 per nest. At 90-days-old, glucose-insulin homeostasis and insulin release from pancreatic islets were analyzed. While both OP exposure and overfeeding alone did induce diabetogenic phenotypes in adulthood, there was no exacerbation in rats that experienced both. Unexpectedly, the group that experienced both had improved adiposity, metabolic parameters, attenuated insulin release from isolated islets in the presence of glucose and low function of muscarinic acetylcholine receptor M3, as well as an attenuation of beta cell mass hyperplasia. High levels of butyrylcholinesterase and low levels of insulin in milk may contribute to the OP-induced developmental programming. Our study showed that maternal OP exposure may program insulin release as well as endocrine pancreas structure, thus affecting metabolism in adulthood. Our data suggest that while perinatal OP exposure alone increases the risk for later life T2D, it actually reverses many of the programmed metabolic dysfunction that is induced by postnatal overfeeding. These surprising results may suggest that low-dose administration of acetylcholinesterase inhibitors could be of utility in preventing detrimental developmental programming that is caused by early-life overnutrition., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have previously described the photoactivated depot (PAD) approach for the light-stimulated release of therapeutic proteins such as insulin. The aim of this method is to release insulin from a shallow dermal depot in response to blood glucose information, using transcutaneous irradiation. Our first approach utilized a photocleavable group that linked insulin to an insoluble but injectable polymer bead. The bead conferred insolubility, ensuring that the injected material stayed at the site of injection, until light cleaved the link, and allowed insulin to be absorbed systemically. While this proved to be effective, the use of a polymer to ensure insolubility introduces two major design problems: (1) low concentration of insulin, as a majority of the material is composed of polymer, and (2) upon release of the insulin, the polymer has to be cleared from the system. To address these two problems, in this work, we have pursued "hydrophobic tags", photocleavable small nonpolar molecules that confer insolubility to the modified insulin prior to irradiation without the bulk or need for biodegradation required of polymers. We developed a combined solid- and solution-phase synthetic approach that allowed us to incorporate a range of small nonpolar moieties, including peptides, into the final depot materials. The resulting materials are >90% w/w insulin and have sharply decreased solubilities relative to unmodified insulin (≤1000 × lower). We demonstrated that they can be milled into low micron-sized particles that can be readily injected through a 31G needle. These suspensions can be prepared at an effective concentration of 20 mM insulin, a concentration at which 120 μL contains 7 days of insulin for a typical adult. Finally, upon photolysis, the insoluble particles release soluble, native insulin in a predictable fashion. These combined properties make these new modified insulins nearly ideal as candidates for PAD materials.

Venom from mammals, amphibians, snakes, arachnids, sea anemones and insects provides diverse sources of peptides with different potential medical applications. Several of these peptides have already been converted into drugs and some are still in the clinical phase. Diabetes type 2 is one of the diseases with the highest mortality rate worldwide, requiring specific attention. Diverse drugs are available (e.g., Sulfonylureas) for effective treatment, but with several adverse secondary effects, most of them related to the low specificity of these compounds to the target. In this context, the search for specific and high-affinity compounds for the management of this metabolic disease is growing. Toxins isolated from animal venom have high specificity and affinity for different molecular targets, of which the most important are ion channels. This review will present an overview about the electrical activity of the ion channels present in pancreatic β cells that are involved in the insulin secretion process, in addition to the diversity of peptides that can interact and modulate the electrical activity of pancreatic β cells. The importance of prospecting bioactive peptides for therapeutic use is also reinforced.

The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin-releasing activities of seven phylloseptin-TR peptides and plasticin-TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin-1.1TR and phylloseptin-3.1TR, showed greatest cytotoxic potency against A549, MDA-MB231, and HT-29 human tumor-derived cells and against mouse erythrocytes. Phylloseptin-4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF-α and IL-1β by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL-10. Phylloseptin-2.1TR and phylloseptin-3.3TR were the most effective at stimulating the production of IL-10. The noncytotoxic peptide, plasticin-TR, inhibited production of TNF-α and IL-1β but was without effect on IL-10 production. The results of CD spectroscopy suggest that the different properties of plasticin-TR compared with the immunostimulatory activities of the previously characterized plasticin-L1 from Leptodactylus laticeps may arise from greater ability of plasticin-TR to oligomerize and adopt a stable helical conformation in a membrane-mimetic environment. All peptides stimulated release of insulin from BRIN-BD11 rat clonal β cells with phylloseptin-3.2TR being the most potent and effective and phylloseptin-2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure-activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin-3.3TR and plasticin-TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

The naturally occurring quassinoid compound brusatol improves the survival of insulin-producing cells when exposed to the proinflammatory cytokines IL-1β and IFN-γ in vitro. The aim of the present study was to investigate whether brusatol also promotes beneficial effects in mice fed a high-fat diet (HFD), and if so, to study the mechanisms by which brusatol acts. In vivo, we observed that the impaired glucose tolerance of HFD-fed male C57BL/6 mice was counteracted by a 2 wk treatment with brusatol. Brusatol treatment improved both β-cell function and peripheral insulin sensitivity of HFD-fed mice. In vitro, brusatol inhibited β-cell total protein and proinsulin biosynthesis, with an ED 50 of ∼40 nM. In line with this, brusatol blocked cytokine-induced iNOS protein expression via inhibition of iNOS mRNA translation. Brusatol may have affected protein synthesis, at least in part, via inhibition of eukaryotic initiation factor 5A (eIF5A) hypusination, as eIF5A spermidine association and hypusination in RIN-5AH cells was reduced in a dose- and time-dependent manner. The eIF5A hypusination inhibitor GC7 promoted a similar effect. Both brusatol and GC7 protected rat RIN-5AH cells against cytokine-induced cell death. Brusatol reduced eIF5A hypusination and cytokine-induced cell death in EndoC-βH1 cells as well. Finally, hypusinated eIF5A was reduced in vivo by brusatol in islet endocrine and endothelial islet cells of mice fed an HFD. The results of the present study suggest that brusatol improves glucose intolerance in mice fed an HFD, possibly by inhibiting protein biosynthesis and eIF5A hypusination.-Turpaev, K., Krizhanovskii, C., Wang, X., Sargsyan, E., Bergsten, P., Welsh, N. The protein synthesis inhibitor brusatol normalizes high-fat diet-induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination.

Designing strategies for the use of biopolymer-based nanoparticles as drug delivery carriers is a considerable challenge in pharmaceutical science. Present study reports synthesis of a novel glucose responsive and in-vitro pH triggered insulin delivery system comprised of glucose oxidase immobilized on acryloyl crosslinked dextran dialdehyde (ACDD) nanoparticles. Scanning electron microscopy, transmission electron microscopy and particle size analysis data revealed that these carriers possess nanosize which is an important parameter for drug delivery applications. In-vitro insulin release studies were performed under artificial gastric fluid (AGF, pH 1.2) and artificial intestinal fluid conditions (AIF, pH 7.4) at physiological temperature (37 °C). Insulin release profile showed controlled release of about 70% under AIF conditions for 24 h. Insulin release mechanism studied using different kinetic models revealed that Korsmeyer-Peppas model appropriately explained the mechanism as 'non-Fickian' diffusion release of insulin. These glucose responsive stimuli sensitive nanocarriers exhibited controlled release of about 90% under AIF conditions in the presence of glucose. These findings revealed that these nanoparticles are promising and reliable delivery systems to overcome problems related with subcutaneous insulin therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background/aims: In pancreatic β-cells, the intracellular Ca²⁺ homeostasis is an essential regulator of the cells major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca²⁺ store balances cellular Ca²⁺. In this study basal ER Ca²⁺ homeostasis was evaluated in order to reveal potential β-cell-specificity of ER Ca²⁺ handling and its consequences for mitochondrial Ca²⁺, ATP and respiration., Methods: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca²⁺ and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3β activity was measured with ELISA., Results: An atypical ER Ca²⁺ leak was observed exclusively in pancreatic islets and β-cells. This continuous ER Ca²⁺ efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3β-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3β. Expression of a presenlin-1 mutant that mimics GSK3β-mediated phosphorylation established a β-cell-like ER Ca²⁺ leak in HeLa and EA.hy926 cells. The ER Ca²⁺ loss in β-cells was compensated at steady state by Ca²⁺ entry that is linked to the activity of TRPC3., Conclusion: Pancreatic β-cells establish a cell-specific ER Ca²⁺ leak that is under the control of GSK3β and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca²⁺ handling for basal mitochondrial activity., Competing Interests: The authors declare no competing financial interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Key Points: A paternal high-fat diet/obesity before mating can negatively influence the metabolism of offspring. Exercise only early in life has a remarkable effect with respect to reprogramming adult rat offspring exposed to detrimental insults before conception. Exercise only early in life normalized adult whole body and muscle insulin resistance as a result of having a high-fat fed/obese father. Unlike the effects on the muscle, early exercise did not normalize the reduced adult pancreatic beta cell mass as a result of having a high-fat fed/obese father. Early-life exercise training may be able to reprogram an individual whose father was obese, inducing long-lasting beneficial effects on health., Abstract: A paternal high-fat diet (HFD) impairs female rat offspring glucose tolerance, pancreatic morphology and insulin secretion. We examined whether only 4 weeks of exercise early in life could reprogram these negative effects. Male Sprague-Dawley rats consumed a HFD for 10 weeks before mating with chow-fed dams. Female offspring remained sedentary or performed moderate intensity treadmill exercise (5 days week -1 , 60 min day -1 , 20 m min -1 ) from 5 to 9 weeks of age. Paternal HFD impaired (P < 0.05) adult offspring whole body insulin sensitivity (i.p. insulin sensitivity test), as well as skeletal muscle ex vivo insulin sensitivity and TBC1D4 phosphorylation. It also lowered β-cell mass and reduced in vivo insulin secretion in response to an i.p. glucose tolerance test. Early-life exercise in offspring reprogrammed the negative effects of a paternal HFD on whole body insulin sensitivity, skeletal muscle ex vivo insulin-stimulated glucose uptake and TBC1D4 phosphorylation and also increased glucose transporter 4 protein. However, early exercise did not normalize the reduced pancreatic β-cell mass or insulin secretion. In conclusion, only 4 weeks of exercise early in life in female rat offspring reprograms reductions in insulin sensitivity in adulthood caused by a paternal HFD without affecting pancreatic β-cell mass or insulin secretion., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Insulin entrapped alginate-gum tragacanth (ALG-GT) hydrogels at different ALG replacement ratios (100, 75, 50, 25) were prepared through an ionotropic gelation method, followed by chitosan (CH) polyelectrolyte complexation. A mild gelation process without the use of harsh chemicals was proposed to improve insulin efficiency. Retention of almost the full amount of entrapped insulin in a simulated gastric environment and sustained insulin release in simulated intestinal buffer indicated the pH sensitivity of the gels. Insulin release from hydrogels with different formulations showed significant differences ( p < 0.05). Time domain (TD) NMR relaxometry experiments also showed the differences for different formulations, and the presence of CH revealed that ALG-GT gel formulation could be used as an oral insulin carrier at optimum concentrations. The hydrogels formulated from biodegradable, biocompatible, and nontoxic natural polymers were seen as promising devices for potential oral insulin delivery.

Long-standing Type 2 diabetes is associated with loss of both β-cell function and β-cell mass. Peptides derived from the frog-skin host-defense peptide esculentin-1 have been shown to exhibit potent, broad-spectrum antimicrobial activity. The aim of the present study is to determine whether such peptides also show insulinotropic and β-cell protective activities. Esculentin-1a(1-21).NH 2 , esculentin-1b(1-18).NH 2 , and esculentin-1a(1-14).NH 2 produced concentration-dependent stimulations of insulin release from BRIN-BD11 rat clonal β-cells, 1.1B4 human-derived pancreatic β-cells, and isolated mouse islets with no cytotoxicity at concentrations of up to 3 μM. The mechanism of insulinotropic action involved membrane depolarization and an increase in intracellular Ca 2+ concentrations. The analogue [D-Lys 14 , D-Ser 17 ]esculentin-1a(1-21).NH 2 (Esc(1-21)-1c) was less potent in vitro than the all L-amino acid containing peptides and esculentin-1a(9-21) was inactive indicating that helicity is an important determinant of insulinotropic activity. However, intraperitoneal injection of Esc(1-21)-1c (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion, whereas administration of esculentin-1a(1-21).NH 2 , esculentin-1b(1-18).NH 2 , and esculentin-1a(1-14) was without significant effect on plasma glucose levels. Esc(1-21)-1c (1 µM) protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.01) and augmented proliferation of the cells (P < 0.01) to a similar extent as glucagon-like peptide-1. The data demonstrate that the multifunctional peptide Esc(1-21)-1c, as well as showing therapeutic potential as an anti-infective and wound-healing agent, may constitute a template for development of compounds for treatment of patients with Type 2 diabetes.

Insulin, as one of the most important hormones regulating energy metabolism, plays an essential role in maintaining glucose and lipid homeostasis in vivo. Failure or insufficiency of insulin secretion from pancreatic beta-cells increases glucose and free fatty acid level in circulation and subsequently contributes to the emergence of hyperglycaemia and dyslipidaemia. Therefore, stimulating the insulin release benefits the treatment of type 2 diabetes and obesity significantly. Frog skin peptides have been extensively studied for their biological functions, among which, Phylloseptin peptides discovered in Phyllomedusinae frogs have been found to exert antimicrobial, antiproliferative and insulinotropic activities, while the mechanism associated with Phylloseptin-induced insulin secretion remains elusive. In this study, we reported a novel peptide named Phylloseptin-PBu, isolated and identified from Phyllomedusa burmeisteri, exhibited dose-dependent insulinotropic property in rat pancreatic beta BRIN-BD11 cells without altering cell membrane integrity. Further mechanism investigations revealed that Phylloseptin-PBu-induced insulin output is predominantly modulated by K ATP -[K + ] channel depolarization triggered extracellular calcium influx and GLP-1 receptor initiated PKA signalling activation. Overall, our study highlighted that this novel Phylloseptin-PBu peptide has clear potential to be developed as a potent antidiabetic agent with established function-traced mechanism and low risk of cytotoxicity., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

One significant health issue that plagues contemporary society is that of Type 2 diabetes (T2D). This disease is characterised by higher-than-average blood glucose levels as a result of a combination of insulin resistance and insufficient insulin secretions from the β-cells of pancreatic islets of Langerhans. Previous developmental research into the pancreas has identified how early precursor genes of pancreatic β-cells, such as Cpal, Ngn3, NeuroD, Ptf1a, and cMyc, play an essential role in the differentiation of these cells. Furthermore, β-cell molecular characterization has also revealed the specific role of β-cell-markers, such as Glut2, MafA, Ins1, Ins2, and Pdx1 in insulin expression. The expression of these genes appears to be suppressed in the T2D β-cells, along with the reappearance of the early endocrine marker genes. Glucose transporters transport glucose into β-cells, thereby controlling insulin release during hyperglycaemia. This stimulates glycolysis through rises in intracellular calcium (a process enhanced by vitamin D) (Norman et al., 1980), activating 2 of 4 proteinases. The rise in calcium activates half of pancreatic β-cell proinsulinases, thus releasing free insulin from granules. The synthesis of ATP from glucose by glycolysis, Krebs cycle and oxidative phosphorylation plays a role in insulin release. Some studies have found that the β-cells contain high levels of the vitamin D receptor; however, the role that this plays in maintaining the maturity of the β-cells remains unknown. Further research is required to develop a more in-depth understanding of the role VDR plays in β-cell function and the processes by which the beta cell function is preserved., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Obesity is associated with an imbalance in the activity of the autonomic nervous system (ANS), specifically in the organs involved in energy metabolism. The pancreatic islets are richly innervated by the ANS, which tunes the insulin release due to changes in energy demand. Therefore, changes in the sympathetic input that reach the pancreas can lead to metabolic dysfunctions. To evaluate the role of the sympathetic ends that innervate the pancreas, 60-day-old male Wistar rats were subjected to sympathectomy (SYM) or were sham-operated (SO). At 120 day-old SYM rats exhibited an increase in body weight, fat pads and metabolic dysfunctions. Decreases in the HOMA-IR and reductions in insulin release were observed both in vivo and in vitro. Furthermore, the SYM rats exhibited altered pancreatic islet function in both muscarinic and adrenergic assays and exhibited high protein expression of the alpha-2 adrenergic receptor (α2AR). Because α2AR has been linked to type 2 diabetes, these findings demonstrate the clinical implications of this study., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Temporin A (FLPLIGRVLSGIL-NH 2 ), temporin F (FLPLIGKVLSGIL-NH 2 ), and temporin G (FFPVIGRILNGIL-NH 2 ), first identified in skin secretions of the frog Rana temporaria, produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal β-cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca 2+ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration-dependent stimulation of insulin release from 1.1B4 human-derived pancreatic β-cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon-like peptide-1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment., (Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.)

Background/aims: MicroRNAs (miRNAs) are a novel class of small RNAs that participate in a variety of biological processes. Although miRNAs have been linked to insulin synthesis and glucose homeostasis, their role in the targeting of mitochondrial uncoupling protein 2 (UCP2), a negative modulator of insulin secretion, remains unclear., Methods: miRNA levels were determined by real-time quantitative PCR analysis using TaqMan probes, and insulin secretion from isolated islets was quantified by ELISA. Effects of miRNAs on UCP2 expression were checked with a luciferase assay and western blotting analysis., Results: An overall change in a set of miRNAs was discovered, with miR-15a, miR-424, miR-497, and miR-185 coinciding with insulin levels in islets maintained under high-glucose conditions. Moreover, experiments in MIN6 cells illustrated that miR-15a, miR-424, miR-497, and miR-185 positively regulated insulin biosynthesis by co-inhibiting UCP2 expression. Furthermore, the four miRNAs were found to post-transcriptionally repress UCP2 expression by directly targeting the 3'UTR of UCP2 mRNA., Conclusions: Thus, our results shed further light on the regulatory network in β-cells consisting of miRNAs, UCP2, and insulin and provide novel therapeutic targets for diabetes., (© 2018 The Author(s). Published by S. Karger AG, Basel.)