Glutathione reductase is expressed at high levels in pancreatic islet cells

Reactive oxygen species are, at least partly, involved in the diabetogenic agent-induced dysfunction of pancreatic beta-cells because the expression of antioxidative and redox proteins is low. We examined the levels of antioxidant/redox proteins, peroxiredoxins-1, -4, and -6 and glutathione reductase (GR), by immunohistochemistry and found that the expression of GR was very high in pancreatic islet cells compared to exocrine cells. When diabetes was induced by an intravenous injection of streptozotocin, the pre-administration of 1,3-bis[2-chloroethyl]-1-nitrosourea, an irreversible inhibitor of GR, made islet cells more vulnerable to streptozotocin. These data point to a pivotal role of the glutathione redox system in pancreatic islet cells against diabetogenic stress.     

Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIP-mHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin.     

Expression of a glutathione reductase from <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> enhanced cellular redox homeostasis by modulating antioxidant proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H₂O₂, menadione, and heavy metal (CdCl₂, ZnCl₂ and AlCl₂)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H₂O₂ stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.     

IFN-gamma induces islet cell MHC antigens and enhances autoimmune, streptozotocin-induced diabetes in the mouse

To explore the role of the lymphokine, IFN-gamma, in the development of autoimmune-mediated insulin-dependent diabetes, we examined the effects of systemically administered IFN-gamma on the clinical features and pancreatic immunohistology of CBA mice made diabetic with multiple low doses of the pancreatic islet beta-cell toxin, streptozotocin. Mice given streptozotocin and IFN-gamma were significantly more hyperglycemic than those given streptozotocin alone and had significantly decreased body weight. Mice given IFN-gamma alone did not differ in glycemia or weight from vehicle-injected mice. On day 11, Ia proteins were detected on islet cells from mice given streptozotocin and their expression was potentiated by IFN-gamma; they could not be detected on islet cells from mice given IFN-gamma alone or vehicle. H-2K protein expression was increased on islet cells from mice given streptozotocin and was potentiated by IFN-gamma. IFN-gamma alone also increased H-2K protein expression on islet cells compared with vehicle-treated mice. These findings show that IFN-gamma enhances the severity of diabetes in mice given multiple-low doses of streptozotocin, in association with enhanced expression of Ia and H-2K proteins on islet cells. They indicate an important role for IFN-gamma in amplifying the autoimmune process leading to beta-cell destruction in diabetes. The ability of IFN-gamma to worsen autoimmune disease has implications for its use in man.     

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

Effect of glutathione reductase knockdown in response to UVB-induced oxidative stress in human lung adenocarcinoma

Background

Glutathione reductase (GR) plays a critical role in the maintenance of physiological redox status in cells. However, the comprehensive investigations of GR-modulated oxidative stress have not been reported.

Methods

In the present study, we cultured a human lung adenocarcinoma line CL1-0 and its GR-knockdown derivative CL1-0ΔGR to evaluate their differential responses to UVB-irradiation.

Results

We identified 18 proteins that showed significant changes under UVB-irradiation in CL1-0ΔGR cells rather than in CL1-0 cells. Several proteins involving protein folding, metabolism, protein biosynthesis and redox regulation showed significant changes in expression.

Conclusions

In summary, the current study used a comprehensive lung adenocarcinoma-based proteomic approach for the identification of GR-modulated protein expression in response to UVB-irradiation. To our knowledge, this is the first global proteomic analysis to investigate the role of GR under UVB-irradiation in mammalian cell model.     

Glutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells

BACKGROUNDCellular metabolism regulates stemness in health and disease.  A reduced redox state is essential for self-renewal of normal and cancer stem cells (CSCs). However, while stem cells rely on glycolysis, different CSCs, including pancreatic CSCs, favor mitochondrial metabolism as their dominant energy-producing pathway. This suggests that powerful antioxidant networks must be in place to detoxify mitochondrial reactive oxygen species (ROS) and maintain stemness in oxidative CSCs. Since glutathione metabolism is critical for normal stem cell function and CSCs from breast, liver and gastric cancer show increased glutathione content, we hypothesized that pancreatic CSCs also rely on this pathway for ROS detoxification.AIMTo investigate the role of glutathione metabolism in pancreatic CSCs.METHODSPrimary pancreatic cancer cells of patient-derived xenografts (PDXs) were cultured in adherent or CSC-enriching sphere conditions to determine the role of glutathione metabolism in stemness. Real-time polymerase chain reaction (PCR) was used to validate RNAseq results involving glutathione metabolism genes in adherent vs spheres, as well as the expression of pluripotency-related genes following treatment. Public TCGA and GTEx RNAseq data from pancreatic cancer vs normal tissue samples were analyzed using the webserver GEPIA2. The glutathione-sensitive fluorescent probe monochlorobimane was used to determine glutathione content by fluorimetry or flow cytometry. Pharmacological inhibitors of glutathione synthesis and recycling [buthionine-sulfoximine (BSO) and 6-Aminonicotinamide (6-AN), respectively] were used to investigate the impact of glutathione depletion on CSC-enriched cultures. Staining with propidium iodide (cell cycle), Annexin-V (apoptosis) and CD133 (CSC content) were determined by flow cytometry. Self-renewal was assessed by sphere formation assay and response to gemcitabine treatment was used as a readout for chemoresistance.RESULTSAnalysis of our previously published RNAseq dataset E-MTAB-3808 revealed up-regulation of genes involved in the KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Glutathione Metabolism in CSC-enriched cultures compared to their differentiated counterparts. Consistently, in pancreatic cancer patient samples the expression of most of these up-regulated genes positively correlated with a stemness signature defined by NANOG, KLF4, SOX2 and OCT4 expression (P < 10^-5). Moreover, 3 of the upregulated genes (MGST1, GPX8, GCCT) were associated with reduced disease-free survival in patients [Hazard ratio (HR) 2.2-2.5; P = 0.03-0.0054], suggesting a critical role for this pathway in pancreatic cancer progression. CSC-enriched sphere cultures also showed increased expression of different glutathione metabolism-related genes, as well as enhanced glutathione content in its reduced form (GSH). Glutathione depletion with BSO induced cell cycle arrest and apoptosis in spheres, and diminished the expression of stemness genes. Moreover, treatment with either BSO or the glutathione recycling inhibitor 6-AN inhibited self-renewal and the expression of the CSC marker CD133. GSH content in spheres positively correlated with intrinsic resistance to gemcitabine treatment in different PDXs r = 0.96, P = 5.8 × 10¹¹). Additionally, CD133⁺ cells accumulated GSH in response to gemcitabine, which was abrogated by BSO treatment (P < 0.05). Combined treatment with BSO and gemcitabine-induced apoptosis in CD133⁺ cells to levels comparable to CD133^- cells and significantly diminished self-renewal (P < 0.05), suggesting that chemoresistance of CSCs is partially dependent on GSH metabolism.CONCLUSIONOur data suggest that pancreatic CSCs depend on glutathione metabolism. Pharmacological targeting of this pathway showed that high GSH content is essential to maintain CSC functionality in terms of self-renewal and chemoresistance.     

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD⁺ and NADPH/NADP⁺. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].     

Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes

BackgroundProtection of pancreatic islet cells against dysfunction or death by regulating autophagy is considered to be an effective method for treatment of type 2 diabetes mellitus (T2DM). Morus alba leaves (mulberry leaves), a popular herbal medicine, have been used for prevention of T2DM since ancient times.PurposeThis study aimed to clarify whether Morus alba leaves ethanol extract (MLE) could protect islet cells in vivo and in vitro by regulating autophagy in T2DM, and explore the possible mechanism of action.MethodsThe main chemical constituents in MLE were analyzed by HPLC. The T2DM rat model was induced via high-fat diet combined with peritoneal injection of low-dose streptozotocin, and MLE was administered by oral gavage. Fasting blood glucose (FBG) and plasma insulin were measured, and homeostatic model assessment of β cell function (HOMA-β) and insulin resistance (HOMA-IR) were determined. The histomorphology of pancreas islets was evaluated by haematoxylin and eosin staining. In palmitic acid (PA)-stressed INS-1 rat insulinoma cells, cell viability was assayed by an MTT method. Expression of the autophagy-related proteins LC3 I/II, p62, p-AMPK and p-mTOR in islet tissues and INS-1 cells was evaluated by western blotting or immunohistochemistry analysis.ResultsThe four main chemical constituents in MLE were identified as chlorogenic acid, rutin, isoquercitrin and quercitrin. MLE ameliorated hyperglycemia, insulin resistance and dyslipidemia of T2DM rats with prominent therapeutic effect. Further study indicated that MLE observably improved islet function, alleviated islet injury of T2DM rats, and inhibited PA-induced INS-1 cell death. On the other hand, MLE significantly induced autophagy in islet cells both in vivo and in vitro, and autophagy inhibitors abolished its therapeutic effect on T2DM rats and protective effect on islet cells. Apart from this, MLE markedly activated the AMPK/mTOR pathway in INS-1 cells, and the AMPK inhibitor prevented the autophagy induction ability of MLE.ConclusionTogether, MLE could protect islet cells against dysfunction and death by inducing AMPK/mTOR-mediated autophagy in T2DM, and these findings provide a new perspective for understanding the treatment mechanism of Morus alba leaves against T2DM.     

Protective effects of polyamine depletion in mouse models of type 1 diabetes: implications for therapy

The underlying pathophysiology of type 1 diabetes involves autoimmune-mediated islet inflammation, leading to dysfunction and death of insulin-secreting islet β cells. Recent studies have shown that polyamines, which are essential for mRNA translation, cellular replication, and the formation of the hypusine modification of eIF5A may play an important role in the progression of cellular inflammation. To test a role for polyamines in type 1 diabetes pathogenesis, we administered the ornithine decarboxylase inhibitor difluoromethylornithine to two mouse models—the low-dose streptozotocin model and the NOD model—to deplete intracellular polyamines, and administered streptozotocin to a third model, which was haploinsufficient for the gene encoding the hypusination enzyme deoxyhypusine synthase. Subsequent development of diabetes and/or glucose intolerance was monitored. In the low-dose streptozotocin mouse model, continuous difluoromethylornithine administration dose-dependently reduced the incidence of hyperglycemia and led to the preservation of β cell area, whereas in the NOD mouse model of autoimmune diabetes difluoromethylornithine reduced diabetes incidence by 50 %, preserved β cell area and insulin secretion, led to reductions in both islet inflammation and potentially diabetogenic Th17 cells in pancreatic lymph nodes. Difluoromethylornithine treatment reduced hypusinated eIF5A levels in both immune cells and islets. Animals haploinsufficient for the gene encoding deoxyhypusine synthase were partially protected from hyperglycemia induced by streptozotocin. Collectively, these studies suggest that interventions that interfere with polyamine biosynthesis and/or eIF5A hypusination may represent viable approaches in the treatment of diabetes.     

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse

Summary This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2K^b-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products α-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.     

Controlled aggregation of primary human pancreatic islet cells leads to glucose‐responsive pseudoislets comparable to native islets

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three‐dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose‐responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re‐associated human islet cells showed an a‐typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C‐peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.     

单核细胞增多性李斯特菌谷胱甘肽还原酶GR的生物学特性

[目的]本文旨在构建单核细胞增多性李斯特菌谷胱甘肽还原酶(glutathione reductase,GR)基因lmo1433(gr)缺失株,并研究GR在细菌生长和运动过程中发挥的作用及与谷氧还蛋白(glutaredoxin,Grx)系统间的调控关系,探究GR参与细菌抗氧化应激和致病力的生物学功能,为阐明氧化还原蛋白介...     

Type 1 diabetes cadaveric human pancreata exhibit a unique exocrine tissue proteomic profile

Type 1 diabetes (T1D) is an autoimmune disorder resulting from a self‐destruction of pancreatic islet beta cells. The complete proteome of the human pancreas, where both the dysfunctional beta cells and their proximal environment co‐exist, remains unknown. Here, we used TMT10‐based isobaric labeling and multidimensional LC‐MS/MS to quantitatively profile the differences between pancreatic head region tissues from T1D (N = 5) and healthy subjects (N = 5). Among the 5357 (1% false discovery rate) confidently identified proteins, 145 showed statistically significant dysregulation between T1D and healthy subjects. The differentially expressed pancreatic proteome supports the growing notion of a potential role for exocrine pancreas involvement in T1D. This study also demonstrates the utility for this approach to analyze dysregulated proteins as a means to investigate islet biology, pancreatic pathology and T1D pathogenesis.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Apoptosis signal regulating kinase-1 and NADPH oxidase mediate human amylin evoked redox stress and apoptosis in pancreatic beta-cells

Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet β-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to β-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic β-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. β-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in β-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic β-cells.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.