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Every young researcher dreams of making a great discovery, but few achieve it. If they do, success does not guarantee happiness. There is little satisfaction in discovering something if others get the credit, and those who achieve fame must face the 'winner's curse' of living up to their reputation. Few discoveries have been more dramatic than the isolation of insulin which, as Michael Bliss said, resembled a secular miracle. And yet, as he also pointed out, this great discovery brought little happiness to those who made it. Some were sidelined, and Banting and Best were saddled with the winner's curse. Here, we look at the ways in which a great discovery can haunt its discoverers., (© 2021 Diabetes UK.)

There has been 100 years of research detailing the role of insulin in glucose, protein and free fatty acid metabolism. We explore the learnings though evolution and changes in management with an understanding of how it has impacted the care of people with diabetes. The discrimination endured is described and recent advances to empower and counter this are highlighted., (© 2021 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)

Background: D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. Methods: Using a novel stereospecific bioluminescent assay, chiral chromatography, enzyme kinetics and a transgenic mouse model we identify endogenous D-cysteine. We elucidate its function in two mice models of type 1 diabetes (STZ and NOD), and in tests of Glucose Stimulated Insulin Secretion in isolated mouse and human islets and INS-1 832/13 cell line. Results and Discussion: D-cysteine is synthesized by serine racemase (SR) and SR−/− mice produce 6–10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR−/− mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR−/− mice. Conclusions: Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

Introduction: The incidence of diabetes is rising in the South Asian region including Sri Lanka. Insulin is one of the therapeutic options which has a significant impact in optimal control of diabetes but lack of knowledge and negative attitudes among patients pose a significant barrier to proper adherence to insulin therapy. This study aims to assess the knowledge and attitudes towards insulin therapy among patients treated with insulin for diabetes attending National Hospital, Kandy and to assess the association of demographic parameters with knowledge and attitude scores. Methods: An institution-based, descriptive, cross-sectional study was conducted which included 298 randomly selected patients with diabetes who are on insulin attending National Hospital, Kandy. They were assessed with a structured interviewer-administered questionnaire. The data was analysed using SPSS version 22. Results: The knowledge scores of participants were 70%, 26.8% and 3% for good, moderate and inadequate, respectively. The majority (74%) had a favourable attitude towards insulin therapy. The mean knowledge and attitudes scores were 81.9% (95% CI: 79.5% to 84.3%) and 78.3% (95% CI: 73.5% to 83.1%), respectively. Being retired (p= .012) and being on insulin for more than 5 years (p = .028) were associated with a good knowledge score. Conclusions: Majority of the patients with diabetes assessed in the study had good knowledge and a favourable attitude regarding insulin therapy. It is therefore essential to maintain and reinforce the educational and training modalities made available for patients with diabetes.

The review article describes the involvement of epigenetic factors in type 1 diabetes mellitus (T1DM) etiopathogenesis. The disease is characterized by changes in expression of microRNAs that affect the transcription of genes involved in autoimmune reactions, destruction of beta cells and insulin production. However, the cause of the observed epigenetic changes is still unclear. In evolution, the sources of microRNA genes are transposable elements, which occupy up to 45 % of the entire human DNA sequence and are drivers of epigenetic regulation in ontogenesis. They are sources of transcription factor sequences and binding sites for them. Features of the genome distribution of transposable elements can cause changes in the number of 5’VNTR (variable number of tandem repeats) — repeats of insulin promoter region and HERV insertions into HLA genes, which affects their expression. Therefore, I assume that the cause of the development of type 1 diabetes mellitus may be an imbalance in transcription activation of transposons, which contributes to changes in the expression of specific microRNAs and protein-coding genes, and also contributes to autoimmune response development. Triggers for this may be individual features of genome distribution of transposons, viral infections and stress. An analysis of the scientific literature confirms my proposed mechanisms for T1DM development, since the global role of retroelements in hormonal regulation, the sensitivity of transposable elements to exogenous viral infections and stress, and HERV-W expression of the majority of patients with T1DM with activation of the autoimmune response have been proven. Analysis of the MDTE DB (miRNAs derived from transposable elements database) database showed the transposon origin of 12 T1DM-associated microRNAs (miR-192, miR-224, miR-31, miR-320c, miR-326, miR-340, miR-342, miR-44661, miR-548c, miR-652, miR-95), the use of which can become the basis for targeted therapy for T1DM.

Type 2 diabetes mellitus (T2D) is a metabolic disease reaching pandemic levels worldwide. In parallel, Alzheimer’s disease (AD) and vascular dementia (VaD) are the two leading causes of dementia in an increasingly long-living Western society. Numerous epidemiological studies support the role of T2D as a risk factor for the development of dementia. However, few basic science studies have focused on the possible mechanisms involved in this relationship. On the other hand, this review of the literature also aims to explore the relationship between T2D, AD and VaD. The data found show that there are several alterations in the central nervous system that may be promoting the development of T2D. In addition, there are some mechanisms by which T2D may contribute to the development of neurodegenerative diseases such as AD or VaD.

Abstract Objective Prader‐Willi syndrome (PWS) is the most frequently diagnosed genetic cause of early childhood obesity. Individuals with PWS typically progress through 7 different nutritional phases during their lifetime. The main objective of this study was to assess potential factors, particularly insulin, that may be responsible for the weight gains in sub‐phase 2a and their role in the subsequent increase in fat mass and obesity in sub‐phase 2b and insatiable appetite in phase 3. Methods Fasting plasma insulin levels were measured in children with PWS between the ages of 0–12 years and in age‐matched non‐PWS participants with early‐onset major (clinically severe) obesity (EMO) and in healthy‐weight sibling controls (SC). Results Participants with PWS in nutritional phases 1a and 1b had plasma insulin levels comparable to SC. However, the transition from phase 1b up to phase 3 in the PWS group was accompanied by significant increases in insulin, coinciding in weight gains, obesity, and hyperphagia. Only individuals with PWS in phase 3 had comparable insulin levels to the EMO group who were higher than the SC group at any age. Conclusions Elevated insulin signaling is a probable trigger for weight gain and onset of hyperphagia in children with Prader‐Willi syndrome. Regulating insulin levels early in childhood before the onset of the early weight gain may be key in modulating the onset and severity of obesity and hyperphagia in individuals with PWS, as well as in other young children with non‐PWS early‐onset obesity. Preventing or reversing elevated insulin levels in PWS with pharmacological agents and/or through diet restrictions such as a combined low carbohydrate, low glycemic‐load diet may be a viable therapeutic strategy in combating obesity in children with PWS and others with early childhood obesity.

Objective: Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM), requiring insulin therapy similar to T1D. While the negative effects on insulin processing and secretion are known, how dominant insulin mutations result in a continued decline of beta cell function after birth is not well understood. Methods: We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations using patient-derived iPSCs and mutated hESCs. Results: we detected accumulation of misfolded proinsulin and impaired proinsulin processing in vitro, and a dominant-negative effect of these mutations on beta-cell mass and function after transplantation into mice. In addition to anticipated ER stress, we found evidence of beta-cell dedifferentiation, characterized by an increase of cells expressing both Nkx6.1 and ALDH1A3, but negative for insulin and glucagon. Conclusions: These results highlight a novel mechanism, the loss of beta cell identity, contributing to the loss and functional failure of human beta cells with specific insulin gene mutations.

Objective: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time. Methods: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs’ effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain. Results: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure. Conclusions: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP’ binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.

Objective: Endothelin receptor B (ETB) together with ETA mediates cellular effects of endothelin 1 (ET-1), an autocrine and endocrine peptide produced by the endothelium and other cells. It regulates vascular tone and controls kidney function. Metabolic syndrome is due to high caloric intake and is characterized by insulin resistance, dyslipidemia, and white adipose tissue (WAT) accumulation. ETA/ETB antagonism has been demonstrated to favorably influence insulin resistance. Our study explored the role of ETB in metabolic syndrome. Methods: Wild type (etb+/+) and rescued ETB-deficient (etb−/−) mice were fed a high-fat diet, and energy, glucose, and insulin metabolism were analyzed, and hormones and lipids measured in serum and tissues. Cell culture experiments were performed in HepG2 cells. Results: Compared to etb+/+ mice, etb−/− mice exhibited better glucose tolerance and insulin sensitivity, less WAT accumulation, lower serum triglycerides, and higher energy expenditure. Protection from metabolic syndrome was paralleled by higher hepatic production of fibroblast growth factor 21 (FGF21) and higher serum levels of free thyroxine (fT4), stimulators of energy expenditure. Conclusions: ETB deficiency confers protection from metabolic syndrome by counteracting glucose intolerance, dyslipidemia, and WAT accumulation due to enhanced energy expenditure, effects at least in part dependent on enhanced production of thyroid hormone/FGF21. ETB antagonism may therefore be a novel therapeutic approach in metabolic syndrome.

In recent years, the incidence of diabetes mellitus and hepatocellular carcinoma (HCC) has been increasing worldwide, in the context of an increasing prevalence of non-alcoholic fatty liver disease (NAFLD). In patients with diabetes mellitus, exogenous insulin is commonly prescribed and used in long-term settings. Recent studies suggest that insulin use may elevate the risk of HCC. A substantial body of work seeks to unpack the association between insulin use and the risk of developing HCC, although there may be conflicting evidence. Further validation is necessary to clarify the true relationship between insulin mechanisms and its hepatocarcinogenic effect. Given the burden of diabetic patients developing HCC, diabetologists and hepatologists must collaborate, particularly regarding the prevention and surveillance of HCC in diabetic patients.

Objective: The consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. Methods: To model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) β cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. Results: Interestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-β cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-β cell proliferation but had no impact on the expansion of SC-α cells. Conclusions: Using iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.

Objective: Insulin acts on the liver via changes in gene expression to maintain glucose and lipid homeostasis. This study aimed to the Forkhead box protein K1 (FOXK1) associated gene regulatory network as a transcriptional regulator of hepatic insulin action and to determine its role versus FoxO1 and possible actions of the insulin receptor at the DNA level. Methods: Genome-wide analysis of FoxK1 binding were studied by chromatin immunoprecipitation sequencing and compared to those for IR and FoxO1. These were validated by knockdown experiments and gene expression analysis. Results: Chromatin immunoprecipitation (ChIP) sequencing shows that FoxK1 binds to the proximal promoters and enhancers of over 4000 genes, and insulin enhances this interaction for about 75% of them. These include genes involved in cell cycle, senescence, steroid biosynthesis, autophagy, and metabolic regulation, including glucose metabolism and mitochondrial function and are enriched in a TGTTTAC consensus motif. Some of these genes are also bound by FoxO1. Comparing this FoxK1 ChIP-seq data to that of the insulin receptor (IR) reveals that FoxK1 may act as the transcription factor partner for some of the previously reported roles of IR in gene regulation, including for LARS1 and TIMM22, which are involved in rRNA processing and cell cycle. Conclusion: These data demonstrate that FoxK1 is an important regulator of gene expression in response to insulin in liver and may act in concert with FoxO1 and IR in regulation of genes in metabolism and other important biological pathways.

Pregnancy complicated by gestational diabetes mellitus (GDM) is accompanied by a higher risk of serious obstetric and perinatal complications. The results of the HAPO (Hyperglycemia and Adverse Pregnancy Outcomes) study showed that the previously used criteria for the diagnosis of GDM require revision. Universal standards for the diagnosis and treatment of carbohydrate metabolism disorders during pregnancy are required. GDM is a risk factor for the development of obesity, type 2 diabetes and cardiovascular diseases in the mother and offspring in the future. GDM treatment is based on modification of diet and lifestyle, self-monitoring of glycemia, as well as timely initiated insulin therapy. However, the adherence to glucose self-control among pregnant women with GDM remains quite low, as well as the correctness of filling out handwritten diaries. The wider use of glucose meters with the setting of an individual target range of glycemia, the function of wireless transmission of blood glucose data to an endocrinologist with the formation of reports, as well as the ability to enter information about nutrition, physical activity, insulin dose into a mobile application allows to increase satisfaction with treatment and reduce poor obstetric and perinatal outcomes in GDM.

Background and Aim Music therapy is an established health profession where music is used to address cognitive, emotional, social, and physical needs of individuals. Stress is a reaction of an organism to stimulus that changes psychophysiological equilibrium with harmful consequences, by affecting the concentration of hormones related to homeostasis. The current study aimed to assess the possible effect of enhancing the quality of environment to alleviate stressful environmental conditions. Materials and methods A total of 45 adult male albino rats were chosen for this study. They were divided into five groups as follows: group 1, uncrowded control rats; group 2, rats exposed to crowding; group 3, rats exposed to crowding and listening to instrumental music; group 4, rats exposed to crowding and listening to soft songs; and group 5, rats exposed to crowding and listening to rallying songs. After 90 days, all rat groups were anesthetized, and a blood sample was collected for measurement of glucagon-like peptide 1 level, insulin level, and leptin level for these groups. Results Music had no significant (P

This study aimed to test the hypothesis that short-term supplementation with a high-energy diet promotes embryo production following ovum pick-up (OPU) in Japanese Black cows. After a period of adaptation to the maintenance diet, a 200% maintenance diet was fed to the high-energy diet group (HD group, n = 6) for four weeks, and a maintenance diet was fed to the other group (MD group, n = 6). OPU-in vitro fertilization (IVF) procedures were performed on days 14, 21, and 28; follicles and oocytes were counted and morphologically graded, and cultivable oocytes were cultured for in vitro maturation, fertilization, and culture. The mean plasma insulin concentrations on days 14 and 21 were significantly higher in the HD group than in the MD group (P < 0.05). The number of follicles observed at OPU, recovered oocytes, cultivable (Grades 1 to 4) oocytes, and the rate of degenerated (Grade 6) oocytes in the HD group were significantly higher than those in the MD group (P < 0.05). The proportion of cleaved oocytes was lower in the HD group than in the MD group (P < 0.05); consequently, there was no significant difference in the number of blastocysts obtained between the HD and MD groups. The present findings suggest that high-energy diets can promote follicular growth in parallel with an increase in plasma concentrations of insulin, but have a detrimental effect on the quality of oocytes with the OPU-IVF procedure in Japanese Black cows.

Introduction: Chronic kidney disease (CKD) poses a financial burden on most patients from low/ middle income countries. Glycaemic control with affordable hypoglycemic agents may influence on the prognosis of diabetic nephropathy. Objectives: We aimed to compare the rates of CKD progression and proteinuria in the type 2 diabetic population in response to the use of various hypoglycemic agents. Patients and Methods: A retrospective cross-sectional study of a total of 250 patients of Afro-Caribbean descent at the University hospital of the West Indies between 2018 and 2019 was conducted. The use of hypoglycaemic agents and changes in albuminuria were calculated as odds ratios with a 95% confidence interval (CI). A P value

Objective: The insulin/insulin-like growth factor 1 (IGF1) pathway is emerging as a crucial component of prostate cancer progression. Therefore, we investigated the role of the novel insulin/IGF1 signaling modulator inceptor in prostate cancer. Methods: We analyzed the expression of inceptor in human samples of benign prostate epithelium and prostate cancer. Further, we performed signaling and functional assays using prostate cancer cell lines. Results: We found that inceptor was expressed in human benign and malignant prostate tissue and its expression positively correlated with various genes of interest, including genes involved in androgen signaling. In vitro, total levels of inceptor were increased upon androgen deprivation and correlated with high levels of androgen receptor in the nucleus. Inceptor overexpression was associated with increased cell migration, altered IGF1R trafficking and higher IGF1R activation. Conclusions: Our in vitro results showed that inceptor expression was associated with androgen status, increased migration, and IGF1R signaling. In human samples, inceptor expression was significantly correlated with markers of prostate cancer progression. Taken together, these data provide a basis for investigation of inceptor in the context of prostate cancer.

The authors conclude that in patients with DM2, more advanced CKD was associated with greater insulin use, proposing different explanations of this relationship, however, we consider that in addition to these findings, within the study there are no patients using Dipeptidyl Peptidase-4 Inhibitors (DPP4i), probably by that time they were not being used yet. Therefore, patients with advanced CKD only had insulin as the only option, which explains a greater frequency of its use in this group of patients.

Background: Hypoglycemia is frequently associated with insulin therapy in diabetic patients; it leads to many short and long-term complications and even death if not addressed in time. This study was undertaken to observe the circadian propensity of hypoglycemia and its recovery time based on type 2 diabetes mellitus patients’ clinical parameters. Methods: We included type 2 diabetes mellitus (DM) hospitalized patients with the exclusion of patients suffering from critical illness. Data were collected for a period of three months (September to November 2019). Results: A total of 120 patients were included, comprising 60% males and 40% females. Approximately 55% of patients had hypoglycemic episodes at around 12 am– 6 am. The most common comorbidity present in these patients was hypertension (43.3%, P=0.931). Anemia (OR-3.765, CI-1.350-5.500, P=0.011), retinopathy (OR 6.066, CI-2.031-8.113, P=0.001), and duration of DM (OR-6.266, CI-2.209-7.774, P=0.001) were significantly associated with the recovery time of hypoglycemia, around 50±14.14min in the elderly population of age 60-70. People with BMI 22.5- 27.5 Kg/m2 took around 45.66 ± 19.37 min to recover after treatment. Conclusion: Time taken to recover from hypoglycemic episodes vary with age and BMI. Associated comorbidities such as anemia, retinopathy, and DM duration had a significant bearing on the time taken to recover from hypoglycemia. Recovery time was directly proportional to BMI, a new finding that needs further molecular level evaluation. Circadian propensity of hypoglycemia in these patients have been identified in the early morning hours of the day.

Abnormalities that characterize the pathophysiology of type 2 diabetes (T2D) include deficiencies of β-cells and the expansion of α-cells in pancreatic islets, manifested by lower insulin release and glucagon oversecretion. The molecular mechanisms that determine intra-islet interactions between pancreatic α- and β-cells are still not fully understood. The present study showed that stearoyl-coenzyme A (CoA) desaturase 1 (SCD1), an enzyme that is implicated in fatty acid metabolism, serves as a checkpoint in the control of endocrine cell equilibrium in pancreatic islets. Our data showed that SCD1 activity is essential for proper α-cell and β-cell lineage determination during morphogenesis of the pancreas and the maintenance of mature β-cell identity. The inhibition of SCD1 expression/activity led to both a decrease in the expression of β-cell signature genes (e.g., Pdx1, Nkx6.1, MafA, and Neurod1, among others) and induction of the expression of the dedifferentiation marker Sox9 in mature pancreatic islets. The transcriptional repression of Pdx1 and MafA in SCD1-deficient β-cells was related to the excessive methylation of promoter regions of these transcription factors. In contrast, SCD1 ablation favored the formation of α-cells over β-cells throughout pancreas organogenesis and did not compromise α-cell identity in adult pancreatic islets. Such molecular changes that were caused by SCD1 downregulation resulted in the mislocalization of α-cells within the core of islets and increased the ratio of pancreatic α- to β-cell mass. This was followed by islet dysfunction, including impairments in glucose-stimulated insulin release, simultaneously with elevations of basal glucagon secretion. Altogether, these findings provide additional mechanistic insights into the role of SCD1 in the pathogenesis of T2D.

Objective: Insulin is a principal metabolic hormone. It regulates a plethora of metabolic pathways in peripheral tissues. The highly homologous insulin-like growth factor 1 (IGF-1), on the other hand, is important for development and growth. Recent studies have shown that insulin and IGF-1 signaling plays fundamental roles in the brain. Loss of insulin or IGF-1 receptors in astrocytes leads to altered glucose handling, mitochondrial metabolism, neurovascular coupling, and behavioral abnormalities in mice. Here, we aim to investigate molecular mechanisms by which insulin and IGF-1 signaling regulates astrocyte functions. Methods: IR-flox and IRKO primary astrocytes were treated with 100 nM insulin or IGF-1 for 6 h, and their transcriptomes were analyzed. Astrocytes with either IR deletion, IGF1R deletion or both were used to examine receptor-dependent transcriptional regulations using qPCR. Additional immunoblotting and confocal imaging studies were performed to functionally validate pathways involved in protein homeostasis. Results: Using next-generation RNA sequencing, we show that insulin significantly regulates the expression of over 1,200 genes involved in multiple functional processes in primary astrocytes. Insulin-like growth factor 1 (IGF-1) triggers a similar robust transcriptional regulation in astrocytes. Thus, over 50% of the differentially expressed genes are regulated by both ligands. As expected, these commonly regulated genes are highly enriched in pathways involved in lipid and cholesterol biosynthesis. Additionally, insulin and IGF-1 induce the expression of genes involved in ribosomal biogenesis, while suppressing the expression of genes involved in autophagy, indicating a common role of insulin and IGF-1 on protein homeostasis in astrocytes. Insulin-dependent suppression of autophagy genes, including p62, Ulk1/2, and several Atg genes, is blunted only when both IR and IGF1R are deleted. Conclusions: In summary, insulin and IGF-1 potently suppress autophagy in astrocytes through transcriptional regulation. Both IR and IGF1R can elicit ligand-dependent transcriptional suppression of autophagy. These results demonstrate an important role of astrocytic insulin/IGF-1 signaling on proteostasis. Impairment of this regulation in insulin resistance and diabetes may contribute to neurological complications related to diabetes.

Objectives: Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive β-like cells by targeted inhibition of FOXO1. We have previously shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes. Methods: We profiled two novel FOXO1 inhibitors in reporter gene assays, and hepatocyte gene expression studies, and in vivo pyruvate tolerance test (PTT) for their activity and specificity. We evaluated their glucose-lowering effect in mice rendered insulin-deficient by administration of streptozotocin. Results: We provide evidence that two novel FOXO1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut β-like cell-inducing properties in mice. FBT432 is also highly effective in combination with a Notch inhibitor in this model. Conclusion: The data add to a growing body of evidence suggesting that FOXO1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.

Objective: Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive β-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate β-like gut cells. Methods: We used Ngn3-or Villin-driven FoxO1 ablation to capture the distinctive developmental effects of FoxO1 on EEC pool. We combined FoxO1 ablation with Notch inhibition to enhance the expansion of EEC pool. We tested the ability of an orally available small molecule of FoxO1 inhibitor, Cpd10, to phenocopy genetic ablation of FoxO1. We evaluated the therapeutic impact of genetic ablation or chemical inhibition of FoxO1 on insulin-deficient diabetes in Ins2Akita/+ mice. Results: Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced β-like cells, and improved glucose tolerance in Ins2Akita/+ mice. This genetic effect was phenocopied by Cpd10. Cpd10 induced β-like cells that released insulin in response to glucose in gut organoids, and this effect was enhanced by the Notch inhibitor, DBZ. In Ins2Akita/+ mice, a five-day course of either Cpd10 or DBZ induced intestinal insulin-immunoreactive β-like cells, lowered glycemia, and increased plasma insulin levels without apparent adverse effects. Conclusion: These results provide proof of principle of gut cell conversion into β-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications.

Objective: Pancreatic insulin was discovered a century ago, and this discovery led to the first lifesaving treatment for diabetes. While still controversial, nearly one hundred published reports suggest that insulin is also produced in the brain, with most focusing on hypothalamic or cortical insulin-producing cells. However, specific function for insulin produced within the brain remains poorly understood. Here we identify insulin expression in the hindbrain's dorsal vagal complex (DVC), and determine the role of this source of insulin in feeding and metabolism, as well as its response to diet-induced obesity in mice. Methods: To determine the contribution of Ins2-producing neurons to feeding behavior in mice, we used the cross of transgenic RipHER-cre mouse and channelrhodopsin-2 expressing animals, which allowed us to optogenetically stimulate neurons expressing Ins2 in vivo. To confirm the presence of insulin expression in Rip-labeled DVC cells, in situ hybridization was used. To ascertain the specific role of insulin in effects discovered via optogenetic stimulation a selective, CNS applied, insulin receptor antagonist was used. To understand the physiological contribution of insulin made in the hindbrain a virogenetic knockdown strategy was used. Results: Insulin gene expression and presence of insulin-promoter driven fluorescence in rat insulin promoter (Rip)-transgenic mice were detected in the hypothalamus, but also in the DVC. Insulin mRNA was present in nearly all fluorescently labeled cells in DVC. Diet-induced obesity in mice altered brain insulin gene expression, in a neuroanatomically divergent manner; while in the hypothalamus the expected obesity-induced reduction was found, in the DVC diet-induced obesity resulted in increased expression of the insulin gene. This led us to hypothesize a potentially divergent energy balance role of insulin in these two brain areas. To determine the acute impact of activating insulin-producing neurons in the DVC, optic stimulation of light-sensitive channelrhodopsin 2 in Rip-transgenic mice was utilized. Optogenetic photoactivation induced hyperphagia after acute activation of the DVC insulin neurons. This hyperphagia was blocked by central application of the insulin receptor antagonist S961, suggesting the feeding response was driven by insulin. To determine whether DVC insulin has a necessary contribution to feeding and metabolism, virogenetic insulin gene knockdown (KD) strategy, which allows for site-specific reduction of insulin gene expression in adult mice, was used. While chow-fed mice failed to reveal any changes of feeding or thermogenesis in response to the KD, mice challenged with a high-fat diet consumed less food. No changes in body weight were identified, possibly resulting from compensatory reduction in thermogenesis. Conclusions: Together, our data suggest an important role for hindbrain insulin and insulin-producing cells in energy homeostasis.

Objective: Identifying the transcripts which mediate genetic association signals for type 2 diabetes (T2D) is critical to understand disease mechanisms. Studies in pancreatic islets support the transcription factor ZMIZ1 as a transcript underlying a T2D GWAS signal, but how it influences T2D risk is unknown. Methods: β-Cell-specific Zmiz1 knockout (Zmiz1βKO) mice were generated and phenotypically characterised. Glucose homeostasis was assessed in Zmiz1βKO mice and their control littermates on chow diet (CD) and high fat diet (HFD). Islet morphology and function were examined by immunohistochemistry and in vitro islet function was assessed by dynamic insulin secretion assay. Transcript and protein expression were assessed by RNA sequencing and Western blotting. In islets isolated from genotyped human donors, we assessed glucose-dependent insulin secretion and islet insulin content by static incubation assay. Results: Male and female Zmiz1βKO mice were glucose intolerant with impaired insulin secretion, compared with control littermates. Transcriptomic profiling of Zmiz1βKO islets identified over 500 differentially expressed genes including those involved in β-cell function and maturity, which we confirmed at the protein level. Upon HFD, Zmiz1βKO mice fail to expand β-cell mass and become severely diabetic. Human islets from carriers of the ZMIZ1-linked T2D-risk alleles have reduced islet insulin content and glucose-stimulated insulin secretion. Conclusions: β-Cell Zmiz1 is required for normal glucose homeostasis. Genetic variation at the ZMIZ1 locus may influence T2D-risk by reducing islet mass expansion upon metabolic stress and the ability to maintain a mature β-cell state.

Objective: Maternal obesity increases the incidence of excess adiposity in newborns, resulting in lifelong diabetes risk. Elevated intrauterine fetal adiposity has been attributed to maternal hyperglycemia; however, this hypothesis does not account for the increased adiposity seen in newborns of mothers with obesity who have euglycemia. We aimed to explore the placental response to maternal hyperinsulinemia and the effect of insulin-like growth factor 2 (IGF-2) in promoting fetal adiposity by increasing storage and availability of nutrients to the fetus. Methods: We used placental villous explants and isolated trophoblasts from normal weight and obese women to assess the effect of insulin and IGF-2 on triglyceride content and insulin receptor signaling. Stable isotope tracer methods were used ex vivo to determine effect of hormone treatment on de novo lipogenesis (DNL), fatty acid uptake, fatty acid oxidation, and esterification in the placenta. Results: Here we show that placentae from euglycemic women with normal weight and obesity both have abundant insulin receptor. Placental depth and triglyceride were greater in women with obesity compared with normal weight women. In syncytialized placental trophoblasts and villous explants, insulin and IGF-2 activate insulin receptor, induce expression of lipogenic transcription factor SREBP-1 (sterol regulatory element-binding protein 1), and stimulate triglyceride accumulation. We demonstrate elevated triglyceride is attributable to increased esterification of fatty acids, without contribution from DNL and without an acceleration of fatty acid uptake. Conclusions: Our work reveals that obesity-driven aberrations in maternal metabolism, such as hyperinsulinemia, alter placental metabolism in euglycemic conditions, and may explain the higher prevalence of excess adiposity in the newborns of obese women.

Background: Combination of insulin and GLP-1RAs have shown reductions in the HbA1c, body weight, and the risk of hypoglycemia. To date, there are conflicting data regarding the effect of GLP-1RAs on insulin dosage(s). Objective: The objective of this study was to evaluate adjustments of insulin doses upon initiation of GLP-1RAs. Methods: This was a retrospective chart review of patients on insulin therapy initiated on GLP-1RAs at NYU Langone Health. Patients were included in the study if they were at least 18 years of age, history of type 2 diabetes, and were on concurrent basal or mixed insulin therapy. 45 patients met inclusion criteria and were included in the study analysis. The primary endpoint was the median change in overall basal insulin doses. Secondary endpoints included median changes in total basal, mixed, and bolus insulin doses, oral antidiabetic medications and GLP-1RA doses, HbA1c, body weight, fasting glucose, and creatinine clearance. Safety results included any adverse reactions to insulin and/or GLP-1RA. Results: In the per-protocol analysis, there was a significant reduction in overall total basal insulin doses from baseline to week 24 (50 units vs. 44 units, p < 0.05). There was a median reduction in patients receiving glargine (50 units vs. 44 units) and detemir (29 units vs. 21.5 units). Conclusions: Use of GLP-1RAs after 24 weeks resulted in a statistically significant reduction in overall total basal insulin dosages from baseline. The median HbA1C in our patient population was >8%. Consider a ≥10% reduction in the overall basal insulin dose upon initiation of GLP-1RA in patients with a HbA1C >8%. [ABSTRACT FROM AUTHOR]

Macrophages, the central mediators of innate immune responses, being in the first-line of defense, they have to readily respond to pathogenic or tissue damage signals to initiate the inflammatory cascade. Such rapid responses require energy to support orchestrated production of pro-inflammatory mediators and activation of phagocytosis. Being a cell type that is present in diverse environments and conditions, macrophages have to adapt to different nutritional resources. Thus, macrophages have developed plasticity and are capable of utilizing energy at both normoxic and hypoxic conditions and in the presence of varying concentrations of glucose or other nutrients. Such adaptation is reflected on changes in signaling pathways that modulate responses, accounting for the different activation phenotypes observed. Macrophage metabolism has been tightly associated with distinct activation phenotypes within the range of M1-like and M2-like types. In the context of diseases, systemic changes also affect macrophage metabolism, as in diabetes and insulin resistance, which results in altered metabolism and distinct activation phenotypes in the adipose tissue or in the periphery. In the context of solid tumors, tumor-associated macrophages adapt in the hypoxic environment, which results in metabolic changes that are reflected on an activation phenotype that supports tumor growth. Coordination of environmental and pathogenic signals determines macrophage metabolism, which in turn shapes the type and magnitude of the response. Therefore, modulating macrophage metabolism provides a potential therapeutic approach for inflammatory diseases and cancer.

Objective: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. Methods: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. Results: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. Conclusions: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.

Introduction. Insulin injection refusal is a challenge when initiating insulin therapy. This study aimed to investigate the effects of an actual insulin injection demonstration on insulin acceptance in patients with type 2 diabetes mellitus (T2DM).

In recent decades, type 2 diabetes is increasingly recorded in young people, adolescents and children. The most significant risk factors for type 2 diabetes in young patients are family history of the disease, overweight and obesity, and the presence of insulin resistance. The main diagnostic criteria for type 2 diabetes mellitus in young patients, in addition to the traditional ones (hyperglycemia and glycosylated hemoglobin), are a high level of C-peptide and insulin resistance in the absence of antibodies to ICA, insulin, GAD. In connection with the projected increase in the incidence of type 2 diabetes at a young age, it is necessary to pay attention to the implementation of preventive measures and timely clinical examination among groups of children and adolescents with the presence of abdominal obesity, burdened with a family history of obesity and type 2 diabetes, in order to prevent or delay the early development of disorders carbohydrate metabolism among this group at an earlier age. Given the predominance of insulin resistance in the pathogenesis of type 2 diabetes in young people, it is necessary to give priority to drugs from the metformin group when prescribing treatment. Thus, the global growth of type 2 diabetes, the tendency to "rejuvenate" diabetes, insufficient information on the prevalence, risk factors and clinical manifestations of the disease at a young age emphasize the urgency of studying this problem.

The aim of the present work was to investigate the impact of maternal obesity on DNA methylation in ovulated oocytes, and to compare the response of in vitro-developing preimplantation embryos originating from control and obese mice to insulin. An intergenerational, diet-induced obesity model was used to produce outbred mice with an increased body weight and body fat. Two-cell and eight-cell embryos recovered from obese and control mice were cultured in a medium supplemented with 1 or 10 ng/ml insulin until blastocyst formation. In the derived blastocysts, cell proliferation, differentiation, and death rates were determined. The results of immunochemical visualization of 5-methylcytosine indicated a slightly higher DNA methylation in ovulated metaphase II oocytes recovered from obese females; however, the difference between groups did not reach statistical significance. Expanded blastocysts developed from embryos provided by control dams showed increased mean cell numbers (two and eight-cell embryos exposed to 10 ng/ml), an increased inner-cell-mass/trophectoderm ratio (two-cell embryos exposed to 1 ng/ml and eight-cell embryos exposed to 10 ng/ml), and a reduced level of apoptosis (two and eight-cell embryos exposed to 10 ng/ml). In contrast, embryos originating from obese mice were significantly less sensitive to insulin; indeed, no difference was recorded in any tested variable between the embryos exposed to insulin and those cultured in insulin-free medium. Real-time RT-PCR analysis showed a significant increase in the amount of insulin receptor transcripts in blastocysts recovered from obese dams. These results suggest that maternal obesity might modulate the mitogenic and antiapoptotic responses of preimplantation embryos to insulin.

Objective: The glucose tolerance test (GTT) is widely used in preclinical research to investigate glucose metabolism, but there is no standardised way to administer glucose. The aim of this study was to directly compare the effect of the route of glucose administration on glucose and insulin kinetics during a GTT in mice. Methods: A GTT was performed in lean male and female mice and obese male mice and glucose was administered via the oral or intraperitoneal (I.P.) route. Samples were collected frequently during the GTT to provide a full time-course of the insulin and glucose excursions. In another cohort of lean male mice, plasma concentrations of insulin, c-peptide, and incretin hormones were measured at early time points after glucose administration. A stable-isotope labelled GTT (SiGTT) was then performed to delineate the contribution of exogenous and endogenous glucose to glycemia during the GTT, comparing both methods of glucose administration. Finally, we present a method to easily measure insulin from small volumes of blood during a GTT by directly assaying whole-blood insulin using ELISA and show a good concordance between whole-blood and plasma insulin measurements. Results: We report that I.P. glucose administration results in an elevated blood glucose excursion and a largely absent elevation in blood insulin and plasma incretin hormones when compared to oral administration. Utilising stable-isotope labelled glucose, we demonstrate that the difference in glucose excursion between the two routes of administration is mainly due to the lack of suppression of glucose production in I.P. injected mice. Additionally, rates of exogenous glucose appearance into circulation were different between lean and obese mice after I.P., but not after oral glucose administration. Conclusion: Reflecting on these data, we suggest that careful consideration be given to the route of glucose administration when planning a GTT procedure in mice and that in most circumstances the oral route of glucose administration should be preferred over the I.P. route to avoid possible artifacts originating from a non-physiological route.

Objective: Age is a risk factor for type 2 diabetes (T2D). We aimed to elucidate whether β-cell glucose metabolism is altered with aging and contributes to T2D. Methods: We used senescence-accelerated mice (SAM), C57BL/6J (B6) mice, and ob/ob mice as aging models. As a diabetes model, we used db/db mice. The glucose responsiveness of insulin secretion and the [U-13C]-glucose metabolic flux were examined in isolated islets. We analyzed the expression of β-cell-specific genes in isolated islets and pancreatic sections as molecular signatures of β-cell identity. β cells defective in the malate-aspartate (MA) shuttle were previously generated from MIN6-K8 cells by the knockout of Got1, a component of the shuttle. We analyzed Got1 KO β cells as a model of increased glycolysis. Results: We identified hyperresponsiveness to glucose and compromised cellular identity as dysfunctional phenotypes shared in common between aged and diabetic mouse β cells. We also observed a metabolic commonality between aged and diabetic β cells: hyperactive glycolysis through the increased expression of nicotinamide mononucleotide adenylyl transferase 2 (Nmnat2), a cytosolic nicotinamide adenine dinucleotide (NAD)-synthesizing enzyme. Got1 KO β cells showed increased glycolysis, β-cell dysfunction, and impaired cellular identity, phenocopying aging and diabetes. Using Got1 KO β cells, we show that attenuation of glycolysis or Nmnat2 activity can restore β-cell function and identity. Conclusions: Our study demonstrates that hyperactive glycolysis is a metabolic signature of aged and diabetic β cells, which may underlie age-related β-cell dysfunction and loss of cellular identity. We suggest Nmnat2 suppression as an approach to counteract age-related T2D.

Objective: Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to play a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. Methods: We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. Results: Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP and the PDI family member PDIA4, but without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa, and Slc2a2, along with increased expression of α-cell markers, Mafb, and glucagon was observed in adult mice, suggesting loss of β-cell identity. Conclusions: The results demonstrate that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.

Objective: β-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We previously showed that increased miR-7 levels trigger β-cell dedifferentiation and diabetes. We used β-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of β-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes. Methods: We performed bulk and single-cell RNA sequencing (RNA-seq) in islets obtained from β-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-bH1 cell lines. We analysed previously published mouse and human T2D data sets. Results: Bulk RNA-seq revealed that β-cell dedifferentiation is associated with the induction of genes associated with epithelial-to-mesenchymal transition (EMT) in prediabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in β-cells. These molecular changes are associated with a weakening of β-cell: β-cell contacts, increased extracellular matrix (ECM) deposition, and TGFβ-dependent islet fibrosis. We found that the mesenchymal reprogramming of β-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in β-cells. Notable among genes transactivated by Pdx1 is Ovol2, which encodes a transcriptional repressor of the EMT transcription factor Zeb2. Following compromised β-cell identity, the reduction in Pdx1 gene expression causes a decrease in Ovol2 protein, triggering mesenchymal reprogramming of β-cells through the induction of Zeb2. We provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islets in T2D subjects. Conclusions: Our study indicates that miR-7-mediated β-cell dedifferentiation induces EMT signalling and a chronic response to tissue injury, which alters the islet microenvironment and predisposes to fibrosis. This research suggests that regulators of EMT signalling may represent novel therapeutic targets for treating β-cell dysfunction and fibrosis in T2D.

Objective: Genetic and acquired abnormalities contribute to pancreatic β-cell failure in diabetes. Transcription factors Hnf4α (MODY1) and FoxO1 are respective examples of these two components and act through β-cell-specific enhancers. However, their relationship is unclear. Methods: In this report, we show by genome-wide interrogation of chromatin modifications that ablation of FoxO1 in mature β-cells enriches active Hnf4α enhancers according to a HOMER analysis. Results: To model the functional significance of this predicted unusual enhancer utilization, we generated single and compound knockouts of FoxO1 and Hnf4α in β-cells. Single knockout of either gene impaired insulin secretion in mechanistically distinct fashions as indicated by their responses to sulfonylurea and calcium fluxes. Surprisingly, the defective β-cell secretory function of either single mutant in hyperglycemic clamps and isolated islets treated with various secretagogues was completely reversed in double mutants lacking FoxO1 and Hnf4α. Gene expression analyses revealed distinct epistatic modalities by which the two transcription factors regulate networks associated with reversal of β-cell dysfunction. An antagonistic network regulating glycolysis, including β-cell “disallowed” genes, and a synergistic network regulating protocadherins emerged as likely mediators of the functional restoration of insulin secretion. Conclusions: The findings provide evidence of antagonistic epistasis as a model of gene/environment interactions in the pathogenesis of β-cell dysfunction.

Objective: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism. Methods: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor, rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice. Results: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver, in a hepatocyte-intrinsic manner by insulin in response to feeding. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of nonessential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. Conclusions: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in the liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

Objective: Natural sources of molecular diversity remain of utmost importance as a reservoir of proteins and peptides with unique biological functions. We recently identified such a family of viral insulin-like peptides (VILPs). We sought to advance the chemical methods in synthesis to explore the structure-function relationship within these VILPs, and the molecular basis for differential biological activities relative to human IGF-1 and insulin. Methods: Optimized chemical methods in synthesis were established for a set of VILPs and related analogs. These modified forms included the substitution of select VILP chains with those derived from human insulin and IGF-1. Each peptide was assessed in vitro for agonism and antagonism at the human insulin and the human insulin-like growth factor 1 receptor (IGF-1R). Results: We report here that one of these VILPs, lymphocystis disease virus-1 (LCDV1)-VILP, has the unique property to be a potent and full antagonist of the IGF-1R. We demonstrate the coordinated importance of the B- and C-chains of the VILP in regulating this activity. Moreover, mutation of the glycine following the first cysteine in the B-chain of IGF-1 to serine, in concert with substitution to the connecting peptide of LCDV1-VILP, converted native IGF-1 to a high potency antagonist. Conclusions: The results reveal novel aspects in ligand–receptor interactions at the IGF-1 receptor and identify a set of antagonists of potential medicinal importance.

Background: Since its discovery 100 years ago, insulin, as the ‘cure’ for type 1 diabetes, has rescued the lives of countless individuals. As the century unfolded and the autoimmune nature of type 1 diabetes was recognised, a darker side of insulin emerged. Autoimmunity to insulin was found to be an early marker of risk for type 1 diabetes in young children. In humans, it remains unclear if autoimmunity to insulin is primarily due to a defect in the beta cell itself or to dysregulated immune activation. Conversely, it may be secondary to beta-cell damage from an environmental agent (e.g., virus). Nevertheless, direct, interventional studies in non-obese diabetic (NOD) mouse models of type 1 diabetes point to a critical role for (pro)insulin as a primary autoantigen that drives beta cell pathology. Scope of review: Modelled on Koch's postulates for the pathogenicity of an infectious agent, evidence for a pathogenic role of (pro)insulin as an autoantigen in type 1 diabetes, particularly applicable to the NOD mouse model, is reviewed. Evidence in humans remains circumstantial. Additionally, as (pro)insulin is a target of autoimmunity in type 1 diabetes, its application as a therapeutic tool to elicit antigen-specific immune tolerance is assessed. Major conclusions: Paradoxically, insulin is both a ‘cure’ and a potential ‘cause’ of type 1 diabetes, actively participating as an autoantigen to drive autoimmune destruction of beta cells - the instrument of its own destruction.

Background: The 100th anniversary of the discovery of insulin in Toronto in 1921 is an important moment in medical and scientific history. The demonstration that an extract of dog pancreas reproducibly lowered blood glucose, initially in diabetic dogs and then in humans with type 1 diabetes, was a medical breakthrough that changed the course of what was until then a largely fatal disease. The discovery of the “activity”, soon named “insulin”, was widely celebrated, garnering a Nobel Prize for Banting and McLeod in 1923. Over the ensuing 100 years, research on insulin has advanced on many fronts, producing insights that have transformed our understanding of diabetes and our approach to its treatment. Scope of Review: This paper will review research on insulin that had another consequence of far broader scientific significance, by serving as a pacesetter and catalyst to bioscience research across many fields. Some of this was directly insulin-related and was also recognized by the Nobel Prize. Equally important, however, was research stimulated by the discovery of insulin that has profoundly influenced biomedical research, sometimes also recognized by the Nobel Prize and sometimes without this recognition. Major Conclusions: By reviewing some of the most notable examples of both insulin-related and insulin-stimulated research, it becomes apparent that insulin had an enormous and frequently under-appreciated impact on the course of modern bioscience.

The discovery of insulin 100 years ago and its application to the treatment of human disease in the years since have marked a major turning point in the history of medicine. The availability of purified insulin allowed for the establishment of its physiological role in the regulation of blood glucose and ketones, the determination of its amino acid sequence, and the solving of its structure. Over the last 50 years, the function of insulin has been applied into the discovery of the insulin receptor and its signaling cascade to reveal the role of impaired insulin signaling—or resistance—in the progression of type 2 diabetes. It has also become clear that insulin signaling can impact not only classical insulin-sensitive tissues, but all tissues of the body, and that in many of these tissues the insulin signaling cascade regulates unexpected physiological functions. Despite these remarkable advances, much remains to be learned about both insulin signaling and how to use this molecular knowledge to advance the treatment of type 2 diabetes and other insulin-resistant states.

Background: Insulin's discovery 100 years ago and its ongoing use since that time to treat diabetes belies the molecular complexity of its structure and that of its receptor. Advances in single-particle cryo-electron microscopy have over the past three years revolutionized our understanding of the atomic detail of insulin-receptor interactions. Scope of review: This review describes the three-dimensional structure of insulin and its receptor and details on how they interact. This review also highlights the current gaps in our structural understanding of the system. Major conclusions: A near-complete picture has been obtained of the hormone receptor interactions, providing new insights into the kinetics of the interactions and necessitating a revision of the extant two-site cross-linking model of hormone receptor engagement. How insulin initially engages the receptor and the receptor's traversed trajectory as it undergoes conformational changes associated with activation remain areas for future investigation.

Background: The brain was once thought of as an insulin-insensitive organ. We now know that the insulin receptor is present throughout the brain and serves important functions in whole-body metabolism and brain function. Brain insulin signaling is involved not only in brain homeostatic processes but also neuropathological processes such as cognitive decline and Alzheimer's disease. Scope of review: In this review, we provide an overview of insulin signaling within the brain and the metabolic impact of brain insulin resistance and discuss Alzheimer's disease, one of the neurologic diseases most closely associated with brain insulin resistance. Major conclusions: While brain insulin signaling plays only a small role in central nervous system glucose regulation, it has a significant impact on the brain's metabolic health. Normal insulin signaling is important for mitochondrial functioning and normal food intake. Brain insulin resistance contributes to obesity and may also play an important role in neurodegeneration.

Background: Diabetes is a metabolic disorder with a highly complex, multifaceted and intricate etiologies and thus may require management options that proffers multimodal mechanism of action. This present study evaluated the antidiabetic and antioxidant potential of the methanolic extract/fractions of leaves of Ocimum canum. Methods: The antidiabetic potential was evaluated and using STZ-induced diabetic Wistar rat model (in vivo) and inhibition of α-amylase and α-glucosidase activity (in vitro). Antioxidant activity was assessed in vitro by free radical scavenging and reducing power assays and in vivo via monitoring SOD and CAT activities; GSH and MDA levels. Results: The total phenolic content (221.0±3.0 mg catechol/g of sample) and tannins (146.0±4.0 mg tannic acid/g of sample) of the crude extract; and flavonoid of the aqueous-methanol fraction (216.0.0±1.0 mg of rutin/g of sample) were found to be significantly higher relative to others. The crude extract and the aqueous-methanol fraction exhibited a significantly (p

Objective: Maintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (α, β, and δ cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting δ cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. Methods: Glucose tolerance tests were performed in mice after administration of selective GPR120 agonist Compound A. Insulin, glucagon, and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous (ω-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knock-out of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in α, β, and δ cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. Results: Acute exposure to Compound A increased glucose tolerance, circulating insulin, and glucagon levels in vivo. Endogenous and/or pharmacological GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in δ cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in δ cells but increased these signals in α and β cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. Conclusions: Inhibitory GPR120 signaling in δ cells contributes to both insulin and glucagon secretion in part by mitigating somatostatin release.

Objective: Members of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time. Methods: The dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. Results: GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT. Conclusions: Our results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.

Introduction: Atorvastatin hinders cardiovascular disease by reducing cholesterol levels. Proprotein convertase subtilisin/kexin type 9 (PCSK9) enhances the secretion of insulin by binding to LDL receptor. Sortilin is committed in the transfer of intracellular proteins through the plasma membrane. Objectives: The purpose of this research was to determine the effect of atorvastatin consumption on alterations in the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R), PCSK9 and sortilin in diabetic patients and pre-diabetics. Patients and Methods: This study was carried out on 80 individuals including normal subjects, diabetic patients and pre-diabetics. The participated individuals were divided as control group (i) (healthy individuals without diabetes mellitus), diabetic group receiving statin (ii), diabetic group not receiving statin (iii), pre-diabetic group receiving statin (iv) and pre-diabetic group not receiving statin (v). Levels of HMG-COA-R, PCSK9 and sortilin were determined by ELISA method. Results: In diabetics and pre-diabetics taking atorvastatin, the level of HMG-COA-R was not altered significantly compared to diabetics and pre-diabetics not taking atorvastatin, respectively (P> 0.05). The serum PCSK9 level in diabetics and pre-diabetics was significantly higher than the healthy individuals (P= 0.001). Additionally, the serum PCSK9 level in diabetics and pre-diabetics receiving atorvastatin was significantly higher than diabetics and pre-diabetics not receiving atorvastatin, respectively (P=0.001). The serum sortilin level in diabetics and pre-diabetics was significantly higher than the healthy individuals (P=0.001). In addition, the serum sortilin level in pre-diabetics receiving atorvastatin was significantly higher than pre-diabetics not receiving atorvastatin (P=0.001). Conclusion: Atorvastatin improved insulin secretion and sensitivity by increasing serum sortilin and PCSK9 levels. Thereby, it prevented the development of diabetes in diabetics and the progression of pre-diabetes to diabetes in pre-diabetics.