Insulin Gene Expression Research Articles

Cloning, prokaryotic expression, purification, and functional verification of the insulin gene in black carp (Mylopharyngodon piceus)

In this paper, we studied the structure, expression and function of black carp insulin gene. The complete Mylopharyngodon piceus insulin (Mp-Ins) gene is 1,965 bp long and includes a 1,499 bp 5ʹ-untranslated region (UTR), a 139 bp 3′-UTR with a poly(A) tail, and an open reading frame (ORF) of 327 bp. The predicted molecular weight of the recombinant Mp-Ins(rMp-Ins) protein is 11.87 kDa. The mRNA expression of Mp-Ins is upregulated in the brain and liver. After the injection of rMp-Ins, Mp-Ins mRNA transcript abundance was significantly upregulated in the liver. The rMp-Ins protein could inhibit the concentration of glycogen phosphorylase (GP), growth hormone (GH), fatty acid synthase (FAS), and insulin-like growth factors-1 (IGF-1), and it also significantly increased the concentration of PI3K. Additionally, the injection of rMp-Ins did not have a significant impact on the glucose-6-phosphatase (G6Pase) content in blood. In situ hybridization results showed that the positive signal of the Mp-Ins gene was mainly concentrated in the cell nucleus of brain tissue and the cell membrane of liver tissue and muscle tissue. Together, these results demonstrated that Mp-Ins plays an important role in growth and metabolism in M. piceus.

Evidence from oyster suggests an ancient role for Pdx in regulating insulin gene expression in animals

Hox and ParaHox genes encode transcription factors with similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. Here, we report evidence indicating that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is a likely direct target of Pdx in Pacific oyster adults. We show that one insulin-related gene, cgILP, is co-expressed with cgPdx in oyster digestive tissue. Transcriptomic comparison suggests that this tissue plays a similar role to the vertebrate pancreas. Using ATAC-seq and ChIP, we identify an upstream regulatory element of the cgILP gene which shows binding interaction with cgPdx protein in oyster hepatopancreas and demonstrate, using a cell culture assay, that the oyster Pdx can act as a transcriptional activator through this site, possibly in synergy with NeuroD. These data argue that a classic homeodomain-target gene interaction dates back to the origin of Bilateria.

Optogenetic stimulation of entorhinal cortex reveals the implication of insulin signaling in adult rat’s hippocampal neurogenesis

Adult neurogenesis in the hippocampal dentate gyrus plays a critical role in learning and memory. Projections originating from entorhinal cortex, known as the perforant pathway, provide the main input to the dentate gyrus and promote neurogenesis. However, neuromodulators and molecular changes mediating neurogenic effects of this pathway are not yet fully understood. Here, by means of an optogenetic approach, we investigated neurogenesis and synaptic plasticity in the hippocampus of adult rats induced by stimulation of the perforant pathway. The lentiviruses carrying hChR2 (H134R)-mCherry gene under the control of the CaMKII promoter were injected into the medial entorhinal cortex region of adult rats. After 21 days, the entorhinal cortex region was exposed to the blue laser (473 nm) for five consecutive days (30 min/day). The expression of synaptic plasticity and neurogenesis markers in the hippocampus were evaluated using molecular and histological approaches. In parallel, the changes in the gene expression of insulin and its signaling pathway, trophic factors, and components of mitochondrial biogenesis were assessed. Our results showed that optogenetic stimulation of the entorhinal cortex promotes hippocampal neurogenesis and synaptic plasticity concomitant with the increased levels of insulin mRNA and its signaling markers, neurotrophic factors, and activation of mitochondrial biogenesis. These findings suggest that effects of perforant pathway stimulation on the hippocampus, at least in part, are mediated by insulin increase in the dentate gyrus and subsequently activation of its downstream signaling pathway.

Young Adult LEW.1WR1 Rats Develop Dysregulated Islet Function and Impaired Liver Insulin Responses

Obesity in humans can lead to metabolic problems such as glucose intolerance and insulin resistance, which may result from pancreatic islet dysregulation and reduced insulin sensitivity in the liver. LEW.1WR1 (1WR1) rats became more glucose intolerant than LEW/SsNHsd (SsNHsd) rats after 12 weeks on a moderate sucrose diet.1 We hypothesize that the 1WR1 rats develop decreased insulin sensitivity due to impaired islet function and liver responses to insulin. To test this hypothesis we measured blood hormone levels and islet and liver gene expression. The terminal blood insulin (14988+/- 4024 vs. 22703+/-5101 pg/mL; p=0.0085; n=7,7) and glucagon (127.3+/-73.31 vs. 188.6+/-46.87 pg/mL; p=0.0537; n=7,7) were higher in the the 1WR1 rats. Using qRT-PCR, we determined the islets of 1WR1 rats had 3 fold increased insulin (p<0.0001; n=3,3) and glucagon (p<0.0001; n=3,2) relative gene expression. Yet, the β-cell area (22.05+/-6.408 vs. 2.276 +/-1.284mm2; p=0.0016; n=3,4) was significantly reduced in 1WR1 rats. Islet Plin5 expression was upregulated in 1WR1 rats (5.388+/-0.3806 F.C.; p<0.0001; n=3,3) indicating increased lipid droplet production, while Cyclin D (0.5726+/-0.08797 F.C.; p=0.0035; n=3,2) was downregulated indicating decreased cell cycle proliferation. These results indicate that the islets of the 1WR1 rats were insensitive to insulin signaling, which may have been caused by increased lipid droplets and a decrease in compensatory islet area. We also measured the relative expression of insulin-sensitive genes in the liver tissue to determine if there were alterations in liver insulin signaling. Downregulation of Irs-2 (0.5840+/-0.001045 F.C.; p<0.0001; n=7,7) expression was likely caused by the upregulated fat10 gene in 1WR1 rats.2 Fat10 (2315+/-0.01380 F.C.; p<0.0001; n=4,6) expression in the liver was significantly increased. Foxo1 (2.644+/- 0.001211 F.C.; p<0.0001; n=7,7) expression, which is normally reduced by insulin, was upregulated which indicates reduced insulin sensitivity. Upregulated expression of Fgf21 (2.260+/-0.002376 F.C.; p<0.0001; n=6,7), which improves glucose homeostasis, in the liver is why the fasting blood glucose of 1WR1 rats were not significantly different from the SsNHsd rats.1 In conclusion, 1WR1 rats show increasingly impaired metabolism over time. These rats have increased insulin and glucagon levels coupled with liver fat10 overexpression leading to impaired gene regulation of insulin-responsive genes in the liver. These changes synergistically increase susceptibility to pathological obesity and metabolic disease.

Increased insulin and GLUT2 gene expression and elevated glucokinase activity in β-like cells of islets of langerhans differentiated from human haematopoietic stem cells on treatment with Costus igneus leaf extract.

In the quest to understand lost β-cells regeneration in the diabetic condition, we have demonstrated successful differentiation of human haematopoietic stem cells (HSCs) to functional β-like cells. Costus igneus (Ci) leaf extract is known to exhibit anti-diabetic properties by lowering the blood glucose level as demonstrated in mice models. To establish the anti-diabetic properties of Ci leaf extract on human subjects, we studied the effect of Ci on these differentiated β-like cells. Ci leaf extract showed its anti-diabetic property through elevated glucokinase activity which catalyzes the rate-limiting step of glucose catabolism in β-like cells and acts as a sensor for insulin production while decreasing the glucose-6-phosphatase activity. Upon increasing the concentrations of Ci leaf extract (25, 65, 105, 145, 185µg/ml) and glucose concentrations (5.5, 11.1, and 25mM) Ci leaf extract treated β-like cells showed enhanced glucokinase and decreased glucose-6-phosphatase activities and an exponential rise in gene expressions of INS and GLUT2 was observed. The present study shows enhanced INS and GLUT2 gene expression and elevated glucokinase activity in β-like cells differentiated from HSCs upon treatment with Ci leaf extract explain the anti-diabetic property of Ci leaf extract. This extract can be effectively used in the management of diabetes.

LEW.1WR1 Rats Have Decreased Normalized Beta Cell Area

Obesity in humans can lead to metabolic problems such as glucose intolerance and insulin resistance, which may result from pancreatic islet dysregulation and reduced insulin sensitivity in the liver. Increased fat10 expression is associated with adiposity, insulin resistance, and inflammation that increases with age as well as type 1 diabetes. The effect of increased fat10 expression on beta cell physiology, however, is not well understood. LEW.1WR1 (1WR1) rats possess a defect in the fat10 promoter which leads to sustained expression of diubiquitin. We suggested previously that fat10 may play a role in increased glucose intolerance, insulin sensitivity, age-related fat deposition, and changes in beta cell size and density in 1WR1 rats. In that study, we identified through semiquantitative analysis that the 1WR1 rats seemed to have fewer islets compared to the LEW/SsNHsd (SsNHsd) rats. The purpose of this project was to compare normalized beta cell area from these rodents and analyze this data to confirm that the perceived changes in islet number corresponded to differences in beta cell area. We hypothesized that, since the 1WR1 rats had a lower islet number, they would also have lower normalized beta cell area, dysregulated insulin signaling, and an altered cell cycle. Seven week old 1WR1 and SsNHsd rats were given a control diet for 12 weeks. Once the animals were sacrificed, the pancreases were harvested, fixed in formalin, and sent to Histowiz (New York, NY) for immunohistochemical staining against insulin. These stained slides were obtained digitally and opened in ImageJ, which was used to measure beta cell and pancreas area. Comparing these showed that the 1WR1 rats indeed have a lower average normalized beta cell area than SsNHsd rats on a moderate sucrose diet. Using qRT-PCR, we determined the islets of 1WR1 rats had 3 fold increased insulin and glucagon gene expression. Yet, the β-cell area (22.05+/-6.408 vs. 2.276 +/-1.284mm2; p=0.0016; n=3,4) was significantly reduced in 1WR1 rats. Islet Plin5 expression was upregulated in 1WR1 rats (5.388+/-0.3806 F.C.; p<0.0001; n=3,3) indicating increased lipid droplet production, while Cyclin D (0.5726+/-0.08797 F.C.; p=0.0035; n=3,2) was downregulated indicating decreased cell cycle proliferation. These results indicate that the islets of the 1WR1 rats were insensitive to insulin signaling, which may have been caused by increased lipid droplets and a decrease in compensatory islet area. This experiment increases our understanding of the characteristics of the pancreas of LEW.1WR1 rats, which may help us better understand the impact of unregulated fat10 expression on beta cell physiology.

Amino acid restriction alters survival mechanisms in pancreatic beta cells: possible role of the PI3K/Akt pathway

Background and aimsMalnutrition in the early stages of life may lead to changes in the glycemic metabolism during adulthood, such as pancreatic beta cells dysfunction and failure. Therefore, this study aimed to evaluate the effects of an in vitro amino acid restriction model on the function and viability of pancreatic beta cells.MethodsInsulin-producing cells (INS-1E) were maintained in control or amino acid restricted culture medium containing 1 × or 0.25 × of amino acids, respectively, for 48 h.ResultsAmino acid restricted group showed lower insulin secretion and insulin gene expression, reduced mitochondrial oxygen consumption rate and reactive oxygen species production. Besides, amino acid restricted group also showed higher levels of endoplasmic reticulum stress and apoptosis markers and enhanced Akt phosphorylation. However, even with higher levels of apoptosis markers, amino acid restricted group did not show higher levels of cell death unless the PI3K/Akt pathway was inhibited.ConclusionAmino acid restricted beta cell viability seems to be dependent on the PI3K/Akt pathway.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00394-021-02568-2.

The Role of Vascular Cells in Pancreatic Beta-Cell Function.

Insulin-producing β-cells constitute the majority of the cells in the pancreatic islets. Dysfunction of these cells is a key factor in the loss of glucose regulation that characterizes type 2 diabetes. The regulation of many of the functions of β-cells relies on their close interaction with the intra-islet microvasculature, comprised of endothelial cells and pericytes. In addition to providing islet blood supply, cells of the islet vasculature directly regulate β-cell activity through the secretion of growth factors and other molecules. These factors come from capillary mural pericytes and endothelial cells, and have been shown to promote insulin gene expression, insulin secretion, and β-cell proliferation. This review focuses on the intimate crosstalk of the vascular cells and β-cells and its role in glucose homeostasis and diabetes.

Resistance exercise training improves glucose homeostasis by enhancing insulin secretion in C57BL/6 mice

Resistance exercise exerts beneficial effects on glycemic control, which could be mediated by exercise-induced humoral factors released in the bloodstream. Here, we used C57Bl/6 healthy mice, submitted to resistance exercise training for 10 weeks. Trained mice presented higher muscle weight and maximum voluntary carrying capacity, combined with reduced body weight gain and fat deposition. Resistance training improved glucose tolerance and reduced glycemia, with no alterations in insulin sensitivity. In addition, trained mice displayed higher insulinemia in fed state, associated with increased glucose-stimulated insulin secretion. Islets from trained mice showed reduced expression of genes related to endoplasmic reticulum (ER) stress, associated with increased expression of Ins2. INS-1E beta-cells incubated with serum from trained mice displayed similar pattern of insulin secretion and gene expression than isolated islets from trained mice. When exposed to CPA (an ER stress inducer), the serum from trained mice partially preserved the secretory function of INS-1E cells, and prevented CPA-induced apoptosis. These data suggest that resistance training, in healthy mice, improves glucose homeostasis by enhancing insulin secretion, which could be driven, at least in part, by humoral factors.

Effect of oral nitrite administration on gene expression of SNARE proteins involved in insulin secretion from pancreatic islets of male type 2 diabetic rats

BackgroundNitrite stimulates insulin secretion from pancreatic β-cells; however, the underlying mechanisms have not been completely addressed. The aim of this study is to determine effect of nitrite on gene expression of SNARE proteins involved in insulin secretion from isolated pancreatic islets in Type 2 diabetic Wistar rats. MethodsThree groups of rats were studied (n = 10/group): Control, diabetes, and diabetes + nitrite, which treated with sodium nitrite (50 mg/L) for 8 weeks. Type 2 diabetes was induced using a low-dose of streptozotocin (25 mg/kg) combined with high-fat diet. At the end of the study, pancreatic islets were isolated and mRNA expressions of interested genes were measured; in addition, protein expression of proinsulin and C-peptide in pancreatic tissue was assessed using immunofluorescence staining. ResultsCompared with controls, in the isolated pancreatic islets of Type 2 diabetic rats, mRNA expression of glucokinase (59%), syntaxin1A (49%), SNAP25 (70%), Munc18b (48%), insulin1 (56%), and insulin2 (52%) as well as protein expression of proinsulin and C-peptide were lower. In diabetic rats, nitrite administration significantly increased gene expression of glucokinase, synaptotagmin III, syntaxin1A, SNAP25, Munc18b, and insulin genes as well as increased protein expression of proinsulin and C-peptide. ConclusionStimulatory effect of nitrite on insulin secretion in Type 2 diabetic rats is at least in part due to increased gene expression of molecules involved in glucose sensing (glucokinase), calcium sensing (synaptotagmin III), and exocytosis of insulin vesicles (syntaxin1A, SNAP25, and Munc18b) as well as increased expression of insulin genes.

Candidate plasticity gene 16 and jun dimerization protein 2 are involved in the suppression of insulin gene expression in rat pancreatic INS-1 β-cells

Chronic hyperglycemia causes pancreatic β-cell dysfunction, impaired insulin secretion and the suppression of insulin gene expression. This phenomenon is referred to as glucotoxicity, and is a critical component of the pathogenesis of type 2 diabetes. We previously reported that the expression of candidate plasticity gene 16 (CPG16) was higher in rat pancreatic INS-1 β-cells under glucotoxic conditions and CPG16 suppressed insulin promoter activity. However, the molecular mechanisms of the CPG16-mediated suppression of insulin gene expression are unclear. In this study, we found that CPG16 directly bound and phosphorylated jun dimerization protein 2 (JDP2), an AP-1 family transcription factor. CPG16 co-localized with JDP2 in the nucleus of INS-1 cells. JDP2 bound to the G1 element of the insulin promoter and up-regulated promoter activity. Finally, CPG16 suppressed the up-regulation of insulin promoter activity by JDP2 in a kinase activity-dependent manner. These results suggest that CPG16 suppresses insulin promoter activity by phosphorylating JDP2.

Perfluorooctane sulfonate continual exposure impairs glucose-stimulated insulin secretion via SIRT1-induced upregulation of UCP2 expression.

Per- and polyfluoroalkyl substances (PFASs) are environmentally and biologically persistent anthropogenic chemicals linked to adverse health outcomes. Epidemiological data have revealed association between exposure to specific PFAS and disruption of insulin level in bodies. However, the effect of PFASs on insulin secretion and the responsible molecular mechanism are poorly understood. In the present study, we used perfluorooctane sulfonate (PFOS) as a representative PFAS family member to investigate its effect on the insulin secretion in mouse pancreatic β cells (β-TC-6). Our results showed that exposure to PFOS inhibited silent information regulator 1 (SIRT1) activity, and molecular simulation showed PFOS could fit into the pocket overlapped with the nicotinamide adenine dinucleotide (NAD+) binding cavity in SIRT1. PFOS exposure upregulated uncoupling protein 2 (UCP2) expression, and this upregulation was blunted in the presence of Ex-527, a SIRT1 specific inhibitor. The mitochondria membrane potential (ΔΨm), as well as the glucose-induced ATP production and Ca2+ influx decreased under PFOS treatment. PFOS continual exposure (48h) impaired glucose stimulated insulin secretion (GSIS), while the gene expression of insulin was not significantly altered. Importantly, the SIRT1 activator and UCP2 inhibitor could partly reverse the PFOS-induced impairment of GSIS. Taken together, the results suggested that PFOS continual exposure could inhibit SIRT1 activity, and the SIRT1-UCP2 pathway mediated, at least partially, the PFOS induced GSIS impairment.

Relationship between insulin sensitivity and gene expression in human skeletal muscle

BackgroundInsulin resistance (IR) in skeletal muscle is a key feature of the pre-diabetic state, hypertension, dyslipidemia, cardiovascular diseases and also predicts type 2 diabetes. However, the underlying molecular mechanisms are still poorly understood.MethodsTo explore these mechanisms, we related global skeletal muscle gene expression profiling of 38 non-diabetic men to a surrogate measure of insulin sensitivity, i.e. homeostatic model assessment of insulin resistance (HOMA-IR).ResultsWe identified 70 genes positively and 110 genes inversely correlated with insulin sensitivity in human skeletal muscle, identifying autophagy-related genes as positively correlated with insulin sensitivity. Replication in an independent study of 9 non-diabetic men resulted in 10 overlapping genes that strongly correlated with insulin sensitivity, including SIRT2, involved in lipid metabolism, and FBXW5 that regulates mammalian target-of-rapamycin (mTOR) and autophagy. The expressions of SIRT2 and FBXW5 were also positively correlated with the expression of key genes promoting the phenotype of an insulin sensitive myocyte e.g.PPARGC1A.ConclusionsThe muscle expression of 180 genes were correlated with insulin sensitivity. These data suggest that activation of genes involved in lipid metabolism, e.g.SIRT2, and genes regulating autophagy and mTOR signaling, e.g.FBXW5, are associated with increased insulin sensitivity in human skeletal muscle, reflecting a highly flexible nutrient sensing.

Preparation and characterisation of a new form of silymarin as a potential antidiabetic agent in the adult male rat

Silymarin is used for a wide variety of biological applications including, antidiabetic activities. However, the effectiveness of Silymarin is affected by its poor aqueous solubility and low systemic bioavailability after oral administration. The present study aimed to formulate a new, simple, and inexpensive form of silymarin solution. A new form of silymarin solution (NFSM) characterised by small particle size (227.5 nm), high entrapment efficiency (>82%), and appropriate zeta potential(–24.7mv). Moreover, the antidiabetic effects of NESM were evaluated relative to native Silymarin (SM). Oral administration of NFSM for 14 days in diabetic rats significantly decreased fasting blood glucose, oxidative stress levels, and improved lipid profile compared with SM. Also, NFSM significantly increased serum insulin levels, the gene expression of insulin and Pdx1, restored and improved the structure of the liver, and pancreas histologically. Our results concluded that NFSM may be an efficient carrier for oral delivery of silymarin for the management of diabetes and aggravated antioxidant status.

Mitochondrial gene expression in single cells shape pancreatic beta cells' sub-populations and explain variation in insulin pathway

Mitochondrial gene expression is pivotal to cell metabolism. Nevertheless, it is unknown whether it diverges within a given cell type. Here, we analysed single-cell RNA-seq experiments from human pancreatic alpha (N = 3471) and beta cells (N = 1989), as well as mouse beta cells (N = 1094). Cluster analysis revealed two distinct human beta cells populations, which diverged by mitochondrial (mtDNA) and nuclear DNA (nDNA)-encoded oxidative phosphorylation (OXPHOS) gene expression in healthy and diabetic individuals, and in newborn but not in adult mice. Insulin gene expression was elevated in beta cells with higher mtDNA gene expression in humans and in young mice. Such human beta cell populations also diverged in mitochondrial RNA mutational repertoire, and in their selective signature, thus implying the existence of two previously overlooked distinct and conserved beta cell populations. While applying our approach to human alpha cells, two sub-populations of cells were identified which diverged in mtDNA gene expression, yet these cellular populations did not consistently diverge in nDNA OXPHOS genes expression, nor did they correlate with the expression of glucagon, the hallmark of alpha cells. Thus, pancreatic beta cells within an individual are divided into distinct groups with unique metabolic-mitochondrial signature.

Potential Antidiabetic and Anti-Genotoxic Activities of Silver Nanoparticles of Alkaloid Extract of Rhazya stricta in Rat Animal Model

In most of Arabian courtiers Rhazya stricta is extensively utilized in public medicine for several diseases treatment. In this study, crude alkaloid extract of R. stricta (CAERS) coated with silver nanoparticles (CAERS-AgNPs) as potential treatment against diabetes in DM animal model was evaluated. Swiss albino male rats (n = 80) were injected with STZ to induce Diabetes Mellitus type-2 (DM). DM-rats were injected different doses of CAERS or CAERS-nanoparticles (CAERS-NPs) for 2 months. The results exhibited that mRNA expression of insulin and insulin receptor was down-regulated, activity levels of antioxidant enzymes were decreased, generation of ROS mediated DNA adducts and apoptosis in DM-rats was increased significantly than those in negative control rats. In contrast, the expression of insulin and insulin receptor genes was up-regulated, activity levels of antioxidant enzymes, ROS generation, DNA adducts and apoptosis incidence in DM-rats supplemented with high dose of CAERS and all doses of CAERS-NPs were improved. In conclusion: R. stricta nanoparticles improved the anti-diabetic effect of the plant much more than the powder form of the extract. This action could be attributed to modification of the chemical and physical properties of the plant materials. The properties modification might be improved the ability of plant compounds to penetrate the cell membrane which facilitating release of plant materials into the target cells.

Evaluation of Plasma Total Antioxidant Capacity Levels and Osteocalcin in Prediabetes and Healthy Subjects

Prediabetes can also be termed as intermediate or borderline hyperglycaemia. These individuals were defined as having impaired fasting glucose IFG. Prediabetes is associated with oxidative stress predisposing the patients to an increased risk of cardiovascular disorders. Osteocalcin OCN is a protein that is secreted in large amounts in the bone extracellular matrix. OCN regulates Beta cell proliferation insulin gene expression and secretion suggesting a potential role in glucose metabolism and in the pathogenesis of glucose alterations in Type2 Diabetes mellitus T2DM physiopathology. Oxidative stress has an inhibitory role on osteoblast. Hence this study was carried out with a total of 80 male subjects within age group of 30-70 years containing 40 controls and 40 cases. Total antioxidant capacity TAC and OCN were evaluated in prediabetic and healthy subjects and were correlated among the study groups. This may lay a step mainly in early diagnosis of diabetes and prevent its complications.

Novel signaling pathways that regulate suppression of insulin gene expression in type 2 diabetes

Novel signaling pathways that regulate suppression of insulin gene expression in type 2 diabetes

Making, Cloning, and the Expression of Human Insulin Genes in Bacteria: The Path to Humulin.

In the mid- to late 1970s, recombinant deoxyribonucleic acid methods for cloning and expressing genes in E. coli were under intense development. The important question had become: Can humans design and chemically synthesize novel genes that function in bacteria? This question was answered in 1978 and in 1979 with the successful expression in E. coli of 2 mammalian hormones, first somatostatin and then human insulin. The successful production of human insulin in bacteria provided, for the first time, a practical, scalable source of human insulin and resulted in the approval, in 1982, of human insulin for the treatment of diabetics. In this short review, I give my personal view of how the making, cloning, and expressing of human insulin genes was accomplished by a team of scientists led by Keiichi Itakura, Herbert W. Boyer, and myself.

The Role of Estrogens in Insulin Secretion. Implications for Aromatase Inhibitor Treatment

Introduction The main hormonal treatment of estrogen sensitive breast cancer includes the use of selective estrogen receptor modulators, aromatase inhibitors and GnRH-Analogs. It has been observed that administering aromatase inhibitor to breast cancer patients not only impairs their glucose metabolism but it can even cause frank diabetes mellitus. Moreover, it has been hypothesized that aromatase inhibitors may have an impact on glucose metabolism by their effect on estrogen levels. Therefore, we designed an experiment in order to assess the effect of estrogens on insulin secretion from rat beta pancreatic insulinoma INS-1 cells. Methods Experiments were conducted on an INS-1 cell line, a rodent beta cell line derived from a rat insulinoma induced by X-ray irradiation, which displays high insulin content, production of both proinsulin I and II and responsiveness to glucose and hormones. INS-1 cells were routinely cultured in 75cm2 flasks (T75) containing 10ml of appropriate culture media and then were transferred into 6-well plates and treated with 17β-E2. Proliferation, as well as insulin expression in the 17β-E2 treated cells in mRNA and protein level was then investigated. Cell cultures were treated with 12.5mM, 25mM, 50mM, 100mM and 200mM estradiol. After 24 hours, RNA as well as protein extraction was carried out. Gene expression was examined in mRNA and protein level, by real time quantitative reverse transcriptase PCR and by western blotting, respectively. Extraction of total RNA was achieved with the use of NucleoZOL (Macherey-Nagel, Duren, Germany). Protein concentration of samples was measured by using the bicinchoninic acid (BCA) assay and bovine serum albumin (BSA) as the standard (Thermo Scientific TM Pierce TM BCA Protein Assay Kit, USA). Results Real time PCR showed a dose-dependent up regulation of insulin gene expression by estradiol. The findings were consistent with the results from western blot, although the estradiol dose-dependent increase in gene expression was not so clear. Finally, both protein and mRNA expression of insulin increased in a dose dependent manner, with mRNA reaching a plateau at 100nM estradiol treatment. Conclusion The experimental data suggest that there might be a direct effect of estrogen on beta cells and insulin secretion.