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Objective To explore the pharmacological mechanism of active saponin compounds of Tuchao Baibiandouren (Lablab Semen Album fried with earth, TCBBDR) in treating type 2 diabetes (T2DM) using UHPLC-Q-Exactive Orbitrap MS and network pharmacology. Methods UHPLC-Q-Exactive Orbitrap MS was used for a qualitative analysis of saponin compounds in TCBBDR. PharmMapper and CTD were used to screen drug active compounds and disease targets, and an active compound-target network was constructed via Cytoscape 3.8.0. Molecular docking was applied with the drug active compounds and key targets using AutoDock Vina 1.1.2, and a trajectory for the molecular dynamics simulation was completed by GROMACS 2019-3. Results Sixteen saponin compounds were identified from TCBBDR, along with 292 saponin compoud targets and 792 T2DM targets. Through Venn analysis of target saponin constituents and T2DM related targets, a total of 91 intersection targets were screened out in the treatment of T2DM with saponin. The mean values of degree, betweenness centrality and closeness centrality were taken as the thresholds to screen out 22 key genes, among which 4 key proteins namely MAPK1, IGF1 EGFR, PIK3R1 were selected in the top 10 key genes. On this basis, the saponin active constituent-target-signaling pathway network was established. The Gene Ontology (GO) enrichment analysis showed that the related biological modules included activity of steroid hormone receptor, steroid binding, and insulin receptor binding, etc.; the related signaling pathways were EGFR, PI3K-Akt and MAPK, etc.; regulating signaling pathways like MAPK could induce the proliferation, inhibition and apoptosis of pancreatic β cells, increase the quantity of pancreatic β cells, improve the functions of pancreatic β cells and stimulate the insulin secretion. Docking experiment analysis showed that all selected saponin compounds could enter the active sites of targets and form 3 – 14 hydrogen bonds with residues of the active sites. Moreover, van der Waals forces were present between chemical compounds and active sites. By combining the docking binding energy, we determined that the chemical compounds showed strong binding energy to the targets. Conclusion TCBBDR exerts therapeutic effects on diabetes through multi-compound and multi-target collaboration. Specifically, saponin components mediate pathways including inflammatory reaction and signal transduction to treat T2DM by regulating several key proteins that interact with EGFR and a series of signaling pathways related to disease development.

Introduction: The C allele of the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP-1) rs1044498 polymorphism has previously been associated with increased binding of ENPP-1 to the insulin receptor (IR), resulting in decreased IR signalling and enhanced insulin resistance. It has also been associated with reduced kidney function in participants with diabetes of predominantly European and Asian descent. The association of this polymorphism with kidney disease in healthy Black South African participants has yet to be ascertained. Objective: This study, therefore, aimed to determine whether the K121Q polymorphism is associated with estimated glomerular filtration rate (eGFR) in a Black South African cohort. Methods: Black South African participants (n = 348) from an existing cohort with known eGFR levels were genotyped for the K121Q polymorphism using PCR-RFLP and assessed for any statistical association between genotype and kidney function. Results: Individuals with the A allele had significantly lower eGFR levels than individuals with the CC genotype (86.52 ± 18.95 vs. 93.29 ± 23.55 mL/min; p = 0.022). The association of the A allele with lower eGFR levels remained after controlling for sex, blood pressure, insulin resistance, age, smoking, thyroid-stimulating hormone, insulin-like growth factor-1, and BMI (R2 = 0.030, p < 0.001). Conclusion: The rs1044498 A allele was significantly associated with lower eGFR levels in a cohort of apparently healthy Black South Africans, through an unknown mechanism that was independent of insulin resistance. It is possible that the rs1044498 polymorphism affects kidney function by altering the role of ENPP-1 in endothelial wound healing, podocyte signalling, or oxidative stress. Thus, the presence of this polymorphism may predispose individuals to a greater risk of CKD even in the absence of diabetes.

Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal levels of insulin and allows for once-daily administration. MYL-1501D is a biosimilar insulin glargine designed to offer greater access of insulin glargine to patients, with comparable efficacy and safety to the marketed reference product. We conducted a comprehensive panel of studies based on a formal analysis of critical quality attributes to characterize the structural and functional properties of MYL-1501D and reference insulin glargine products available in the United States and European Union. MYL-1501D was comprehensively shown to have high similarity to the reference products in terms of protein structure, metabolic activity (both in vitro cell-based assays and in vivo rabbit bioassays), and in vitro cell-based assays for mitogenic activity. The structural analyses demonstrated that the primary protein sequence was identical, and secondary and tertiary structures are similar between the proposed biosimilar and the reference products. Insulin receptor binding affinity and phosphorylation studies also established analytical similarity. MYL-1501D demonstrated high similarity in different metabolic assays of glucose uptake, adipogenesis activity, and inhibition of stimulated lipolysis. Rabbit bioassay studies showed MYL-1501D and EU-approved insulin glargine are highly similar to US-licensed insulin glargine. These product quality studies show high similarity between MYL-1501D and licensed or approved insulin glargine products and suggest the potential of MYL-1501D as an alternative cost-effective treatment option for patients and clinicians.

Introduction CNS insulin levels are decreased and insulin receptor signalling is dampened in Alzheimer's disease (AD). Increasing CNS insulin levels through a variety of methods has been shown to improve memory. Indeed, medications routinely used to improve insulin resistance in type 2 diabetes are now being repurposed for memory enhancement. CNS insulin is primarily derived from the circulation, by an active transport system at the blood‐brain barrier (BBB). The goal of this study was to determine whether rosiglitazone (RSG), a drug used to improve insulin sensitivity in type 2 diabetes, could enhance insulin transport at the BBB, as a potential therapeutic for improving memory. Methods Using radioactively labelled insulin and the multiple‐time regression analysis technique, we measured the rate of insulin BBB transport and level of vascular binding in mice pretreated with vehicle or 10 µg RSG in the presence or absence of an insulin receptor inhibitor. Results Although we found acute RSG administration does not affect insulin transport at the BBB, it does restore BBB vascular binding of insulin in an insulin receptor–resistant state. Conclusions Acute RSG treatment does not alter insulin BBB transport in healthy mice but can restore insulin receptor binding at the BBB in an insulin‐resistant state., Here, the impact of rosiglitazone (RSG) on insulin transport across the murine BBB, the primary means for CNS access to insulin, is investigated. While RSG did not affect the transport rate of insulin across the BBB, it did improve vascular binding under conditions in which the insulin receptor was acutely inhibited.

Insulin analogue X10 has a higher mitogenic potency than native human insulin in vitro and supra-pharmacological doses of insulin X10 increased the incidence of mammary tumours in rats. Compared to native human insulin, insulin X10 has increased binding affinity to the insulin receptor and the IGF-1 receptor, but it is not known whether either or both characteristics are important for stimulation of cell proliferation in vivo. The aim of this study was to explore how increased binding affinity to the insulin receptor or the IGF-1 receptor contributes to stimulation of cell proliferation in vivo. A mouse xenograft model was established with rat L6 myoblast cells transfected with the human insulin receptor (L6hIR cells) and effects of supra-pharmacological doses of native human insulin, insulin X10 or novel insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor were examined. Treatment with insulin X10 and insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor increased growth of L6hIR cell xenografts significantly compared to native human insulin. Thus, increased binding affinity to the insulin receptor and the IGF-1 receptor are each independently linked to increased growth of L6hIR cell xenografts in vivo.

SUMMARY Although the widely used anticoagulant drug heparin has been shown to have many other biological functions independent of its anticoagulant role, its effects on energy homeostasis are unknown. Here, we demonstrate that heparin level is negatively associated with nutritional states and that heparin treatment increases food intake and body weight gain. By using electrophysiological, pharmacological, molecular biological, and chemogenetic approaches, we provide evidence that heparin increases food intake by stimulating AgRP neurons and increasing AgRP release. Our results support a model whereby heparin competes with insulin for insulin receptor binding on AgRP neurons, and by doing so it inhibits FoxO1 activity to promote AgRP release and feeding. Heparin may be a potential drug target for food intake regulation and body weight control., Graphical Abstract, In Brief Zhu et al. demonstrate that heparin competes with insulin for insulin receptor binding on AgRP neurons, and by doing so it inhibits FoxO1 activity to promote AgRP release and feeding. Heparin is identified as a potential drug target for food intake regulation and body weight control.

The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.

Manufacturers of insulin products for diabetes therapy have long sought ways to modify the absorption rate of exogenously administered insulins in an effort to better reproduce the naturally occurring pharmacokinetics of endogenous insulin secretion. Several mechanisms of protraction have been used in pursuit of a basal insulin, for which a low injection frequency would provide tolerable and reproducible glucose control; these mechanisms have met with varying degrees of success. Before the advent of recombinant DNA technology, development focused on modifications to the formulation that increased insulin self-association, such as supplementation with zinc or the development of preformed precipitates using protamine. Indeed, NPH insulin remains widely used today despite a frequent need for a twice-daily dosing and a relatively high incidence of hypoglycaemia. The early insulin analogues used post-injection precipitation (insulin glargine U100) or dimerization and albumin binding (insulin detemir) as methods of increasing therapeutic duration. These products approached a 24-hour glucose-lowering effect with decreased variability in insulin action. Newer basal insulin analogues have used up-concentration in addition to precipitation (insulin glargine U300), and multihexamer formation in addition to albumin binding (insulin degludec), to further increase duration of action and/or decrease the day-to-day variability of the glucose-lowering profile. Clinically, the major advantage of these recent analogues has been a reduction in hypoglycaemia with similar glycated haemoglobin control when compared with earlier products. Future therapies may bring clinical benefits through hepato-preferential insulin receptor binding or very long durations of action, perhaps enabling once-weekly administration and the potential for further clinical benefits.

Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site-specific incorporation of stable (13) C=(18) O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis-via a key ester insulin intermediate-of 97 % enriched [(1-(13) C=(18) O)Phe(B24) ] human insulin: stable-isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1-(13) C=(18) O)Phe(B24) ] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red-shifted amide I carbonyl band peak at 1595 cm(-1) resulting from the (1-(13) C=(18) O)Phe(B24) backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.

The world’s population is living much longer than in the past. It is crucial to find as many pathological factors that deteriorate the health condition and well-being of elderly people as possible. Loss of activity and functions over time is typical for elderly people. Aging affects brain function, metabolism and structure in different ways, and these effects have multiple etiologies. Cognitive impairment, impaired neurotransmitter activity and reduction of brain volume are observed in the elderly population. The process of brain aging is associated with a decrease of central insulin concentration as well as impairment of insulin receptor binding ability, resulting in deterioration of glucose homeostasis in the brain. Peripheral insulin resistance is a typical feature of older age. Data from the literature suggest that high circulating insulin and insulin resistance are important contributors to progressive cognitive impairment and neurodegenerative processes. The maintenance of insulin sensitivity and proper insulin signaling may lead to preserved cognition that results in well-being of elderly people.

Optimal conditions for a high cell-density fermentation of Escherichia coli strain harboring a recombinant anti-thrombosis insulin variant (named rAT-INS) were investigated by using fed-batch culture employing pH-stat method. The optimized main medium composition were glycerol 10 g/L, yeast extract 30 g/L, trypton 10 g/L, NaCl 5 g/L. The late-stage induction with 0.05 mM isopropyl-β- d - thiogalactopyranoside showed the highest productivity after 28 h of the fed-batch fermentation. This optimized process yielded about 150 mg of purified rAT-INS from 1 L of wet cell mass with high-homogeneity. The amino acid compositions and mass data of the purified rAT-INS were in good agreement with those as expected. Purified rAT-INS exhibited potent inhibitory activity of platelet aggregation. The in vivo assay showed that rAT-INS had a higher activity in prolonging the bleeding time in mice than native-insulin. The purified rAT-INS had almost no insulin receptor binding activity. Our study demonstrates the promise for mass production of novel recombinant antiplatelet agents.

James Raphael Gavin III, MD, PhD, represents the quintessential scientist, educator, trailblazer, and type of leader most people aspire to become. The breadth and depth of his contributions to the scientific and medical community in the area of diabetes are unparalleled, while his passion has pushed him to unprecedented heights in American medicine. His original research as a young investigator at the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health (NIH) has greatly contributed to our understanding of insulin receptor binding and function. This factor advanced the understanding of the mechanism of insulin resistance and the role of hyperinsulinemia in obesity and type 2 diabetes. These observations have become the pathophysiologic hallmarks of both diseases. James R. Gavin III was born 23 November 1945 in Mobile, Alabama, the fourth of James R. Gavin II and Bessie Smoke Gavin’s seven children. Gavin’s father worked for the shipbuilding company in Mobile doing welding and sandblasting, and he eventually became a self-made chemist. As a child Gavin played sports and “went to church a lot.” Because he was a precocious child and easily bored with kindergarten, he started public school at an early age, attending segregated public schools (in Mobile). “[I] had the most fantastic teachers and educational experience at all levels, including opportunities to pursue a number of extracurricular activities,” said Gavin. One particular highlight was meeting Rev. Dr. Martin Luther King Jr. As a high school freshman, he was selected to give the Gettysburg Address at the annual Emancipation Proclamation celebration in Mobile, where Dr. King was the keynote speaker. Gavin said, “I sat mesmerized behind him as he spoke for almost 90 minutes in spellbinding fashion, without a single note! I will always remember that event and always cherish that he leaned over …

Insulin resistance is a key feature of obesity and type 2 diabetes mellitus (T2DM). Interaction of insulin with the insulin receptor (IR) leads to both its auto-phosphorylation and phosphorylation of tyrosine residues on the IR substrate (IRS) proteins, initiating the activation of intracellular signaling cascades. The metabolic effects of IRS are known to be mediated through pathways involving phosphatidyl-inositol 3-kinase (PI-3K), which result in the activation of Akt signaling. The C-terminal region of the IR ectodomain is required to facilitate the conformational changes that are required for high-affinity binding to insulin. Furthermore, the CH2 and CH3 domains in the Fc fragments of immunoglobulins are responsible for their binding to the Fc receptor, which triggers transcytosis. In this study, we created a fusion peptide of the C-terminal end of the human IR ectodomain with the IgG4 Fc fragment, including an intervening polyG fragment to ensure enough space for insulin binding. We named this new peptide "Yiminsu", meaning an insulin sensitizer. The results of our analyses show that Yiminsu significantly facilitates insulin signaling via the activation of Akt in hepatocytes in a dose- and time-dependent manner. Further studies are required to determine whether Yiminsu can act as an insulin sensitizer.

Insulin resistance is associated with endothelial dysfunction and ensuing cardiovascular diseases in type 2 diabetes mellitus (T2DM) patients. ENPP1 is a key modulator of insulin signaling and its polymorphism, K121Q, increases the potency to competitively inhibit insulin receptor binding. We investigated the association of ENPP1 121Q variant with coronary artery disease (CAD) in patients with and without T2DM in South Indian population. Our study was conducted in 913 subjects: 198 patients with CAD, 284 patients in whom T2DM and CAD co-exists, 160 patients with T2DM and no CAD history, and 271 healthy volunteers. Genotyping was performed using PCR-RFLP and PCR-DNA sequencing. Genotype frequency of ENPP1 121Q was higher in disease groups compared to healthy subjects (p 0.05). T2DM patients who carried polymorphic AC/CC genotypes were at 12.8-fold enhanced risk to CAD (95% CI 4.97-37.18, p 0.01). Moreover we observed that 121Q, both in heterozygous and homozygous polymorphic states, was a risk factor for CAD without diabetes (OR 4.15, p 0.01). 121Q variant was associated with T2DM patients with no CAD history too, but the risk was statistically insignificant after multivariate logistic regression analysis (OR 2.32, p 0.05). We conclude that ENPP1 121Q variant is associated with increased risk for CAD in patients with T2DM in South Indian population. We also report that 121Q variant of ENPP1 was an independent risk factor for CAD irrespective of diabetic milieu. Factors which enhance insulin resistance increase the risk for onset and progression of coronary atherosclerosis irrespective of a diabetic background.

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is a most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones at isolated rat hepatocytes, the internalization time course of 125I-insulin and 125I-IGF-I are traced at 37 and 12°C. There are established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37°C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. But essential differences in the internalization course of these two related peptides were obvious at the temperature of 12°C. The internalization level of insulin receptors at 12°C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocyte plasma membrane. At 12°C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12°C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar “inhibition mechanism” of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to action of cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.

Although interaction of human insulin with its receptor is studied to considerable extent such studies are currently lacking with recombinant insulin in-spite of its rampant clinical use. It is known that at molecular level the interaction of recombinant insulin with insulin receptor is similar to human insulin but not exactly same. With the increasing incidence of diabetes throughout the globe use of recombinant insulin is also increasing at a considerable rate. Therefore it is need of the hour to explore the recombinant insulin- insulin receptor interaction by all possible means. In this paper we have studied insulin receptor binding of lispro and glargine; the two commonly used recombinant insulins using tools of computational biology. We have observed that the binding pattern of insulin receptor (L1-CR-L2 ectodomain) with lispro and glargine is different when compared with human insulin. Analyzing the ligand receptor interactions we have hypothesized that the tail region of insulin beyond B26 is a critical regulator of insulin insulin receptor interactions detail of which cannot be understandable from docking studies due to lack of consideration of the flexibility of the tail region while docking studies. We have recommended experimental validation of our study. However, our docking procedure may also be explored for preliminary screening of novel anti-diabetic peptide.

Aim: Non-alcoholic fatty liver disease (NAFLD) is an insulin resistance disease that can progress to cirrhosis and liver failure. We hypothesized that in NAFLD, insulin resistance dysregulates lipid metabolism, increasing production of cytotoxic lipids including ceramides, which exacerbate hepatic insulin resistance and injury. Methods: Long Evans rats were pair-fed low (LFD) or high (HFD) fat diets for 8 weeks. Livers were used to measure lipids, gene expression, insulin receptor binding, integrity of insulin signaling, and pro-inflammatory cytokines. In vitro experiments characterized effects of ceramides on Huh7 cell viability, mitochondrial function, and insulin signaling. Results: High fat diet feeding caused NAFLD with peripheral and hepatic insulin resistance, increased hepatic expression of pro-ceramide genes, sphingomyelinase activity, and lipid peroxidation, and increased serum ceramide. Ceramide treatment impaired Huh7 cell viability, mitochondrial function, and insulin signaling. Conclusions: Increased hepatic ceramide generation and release may mediate both hepatic and peripheral insulin resistance in NAFLD.

Scope: Carnitine improves protein accretion, muscle mass, and protein:fat accretion in piglets. The underlying mechanisms, however, are largely unknown. Methods and results: To gain insight into mechanisms through which carnitine exerts these effects, we fed piglets either a control or a carnitine-supplemented diet, and analyzed the transcriptome in skeletal muscle. Carnitine concentrations in plasma and muscle were about four-fold higher in the carnitine group when compared to the control group. Transcript profiling revealed 211 genes to be differentially expressed in muscle by carnitine supplementation. The identified genes were mainly involved in molecular processes such as cytoskeletal protein binding, insulin-like growth factor (IGF) binding, transcription factor activity, and insulin receptor binding. Identified genes with the molecular function transcription factor activity encoded primarily transcription factors, most of which were down-regulated by carnitine, including pro-apoptotic transcription factors such as proto-oncogene c-fos, proto-oncogene c-jun and activating transcription factor 3. Furthermore, atrophy-related genes such as atrogin-1, MuRF1, and DRE1 were significantly down-regulated by carnitine. IGF signalling and insulin signalling were identified as significantly up-regulated regulatory pathways in the carnitine group. Conclusion: Carnitine may have beneficial effects on skeletal muscle mass through stimulating the anabolic IGF-1 pathway and suppressing pro-apoptotic and atrophy-related genes, which are involved in apoptosis of muscle fibers and proteolysis of muscle proteins, respectively.

Numerous analogues of insulin have been prepared over the past three decades for use in diabetic therapy. However, only two long-acting insulins have been approved for clinical use. One is Levemir (Novo Nordisk) and the other is Lantus (Sanofi-Aventis). Glargine (commercial name: Lantus) is characterized by a substitution of Gly in place of Asn at the C terminus of the A-chain and addition of two Arg residues to the C terminus of the B-chain. Despite the clinical advantages of glargine, it is not without concern that its increased affinity for the IGF-1 receptor may correlate with increased mitogenic activity. Recently, a systematic study of modified analogues of glargine showed that placement of an extra Arg residue at the N terminus of the A-chain conferred improved insulin:IGF-1 receptor selectivity without significant loss of pharmacological profile. However, as it is difficult to prepare such an analogue in high yield by recombinant DNA methods, we undertook its chemical assembly by our refined solid phase synthesis method. We describe herein its chemical preparation and biological activity in both insulin receptor binding assays and DNA synthesis assays. The synthetic analogue, A0:R glargine, showed slightly reduced affinity for IR-B (twofold) compared to native insulin. In stimulating DNA synthesis, A0:R glargine was slightly less potent compared to insulin or glargine. This result ultimately confirms the previous report that A0:R glargine has a lower potency in mitogenic assays compared to glargine. This glargine analogue thus could be a potential lead compound for drug design and development for the treatment of diabetes.

A 5-hour insulin clamp was performed in 7 normal subjects (N) and 6 type-II-diabetics. After a 10-min-priming insulin infusion, a constant infusion of 1.813 micrograms/m2 s.a./min was given to all subjects. Glycemia was kept at fasting levels by a variable glucose infusion. Under these conditions the amount of metabolized glucose (M) has been calculated and served as a measure of insulin-stimulated glucose disposal. M differed markedly between N (35.6 +/- 3.11 mumol glucose/kg b.w./min and diabetics (17.8 +/- 1.17 mumol glucose/kg b.w./min; p less than 0.01) indicating a diminished insulin sensitivity in the latter group. However, M increased slightly but significantly until the end of the study in both groups. Under fasting conditions the mean percent 125I-insulin specifically bound to 3.5 X 10(9) erythrocytes/ml at tracer concentrations was 12 +/- 1.2% and 8.9 +/- 0.9% in N and diabetics, respectively. During insulin infusion specific insulin binding decreased significantly in both groups by 34% and 41%. Thus, the downregulation of insulin binding was similar in both groups and was due to changes in receptor affinity. Assuming that insulin binding to red blood cells mimic that to target cells we conclude that the cause of reduced glucose utilization in type-II-diabetes lies mainly in changes of postreceptor events rather than in receptor binding.

Glucagon-like peptide-1 (GLP-1) is the major incretin hormone from the distal small intestine which stimulates basal and glucose-induced insulin secretion. Using the rat insulinoma cell line RINm5F (Gazdar et al. 1980) we investigated the effects of GLP-1 on insulin secretion, insulin content, and insulin receptor binding. During a 1 hour incubation, GLP-1 [1 nM] stimulated insulin secretion 2-fold (p < 0.01 vs controls). Incubating RINm5F for 24 h with GLP-1 [1 nM], a 1.6-fold higher cellular insulin content was observed (p < 0.01 vs controls). Moreover, GLP-1 induced a 2-fold higher capacity and a 15-fold higher affinity of 125I-insulin binding on the cell surface (p < 0.01 vs controls). Glucagon, known as a potent stimulator of insulin secretion, yielded a similar effect only in 1,000-fold higher concentrations, whereas the intracellular insulin content as well as insulin receptor binding was not increased. Taken together, in RINm5F insulinoma cells GLP-1 potently stimulates insulin secretion and insulin content, and improves insulin receptor binding.

Background/Aims Chronic ethanol exposure impairs liver regeneration due to inhibition of insulin signaling and oxidative injury. PPAR agonists function as insulin sensitizers and anti-inflammatory agents. We investigated whether treatment with a PPARδ agonist could restore hepatic insulin sensitivity, survival signaling, and regenerative responses vis-a-vis chronic ethanol feeding. Methods Adult rats were fed isocaloric liquid diets containing 0% or 37% ethanol, and administered a PPARδ agonist by i.p. injection. We used liver tissue to examine histopathology, gene expression, oxidative stress, insulin signaling, and regenerative responses to 2/3 hepatectomy. Results Chronic ethanol feeding caused insulin resistance, increased oxidative stress, lipid peroxidation, DNA damage, and hepatocellular injury in liver. These effects were associated with reduced insulin receptor binding and affinity, impaired survival signaling through PI3K/Akt/GSK3β, and reduced expression of insulin responsive genes mediating energy metabolism and tissue remodeling. PPARδ agonist treatment reduced ethanol-mediated hepatic injury, oxidative stress, lipid peroxidation, and insulin resistance, increased signaling through PI3K/Akt/GSK3β, and enhanced the regenerative response to partial hepatectomy. Conclusions PPARδ agonist administration may attenuate the severity of chronic ethanol-induced liver injury and ethanol's adverse effects on the hepatic repair by restoring insulin responsiveness, even in the context of continued high-level ethanol consumption.

Single chain insulins (SCIs) are single polypeptide chains in which the insulin B-chain links contiguously with the insulin A-chain via an uncleaved connecting peptide. While direct linkage of insulin Band A-chains produces SCIs with little insulin receptor binding, biologists have been interested in bioengineering linker peptides that form a flexible reverse turn, allowing SCIs to activate insulin receptors. In this report, we have investigated a series of cDNAs intended to explore the significance of linker length, cleavability, and the impact of certain site-dependent residues for the bioactivity of recombinant SCIs on insulin receptors. SCI concentration is readily measured by radioimmunoassay with a (proinsulin insulin)-specific polyclonal antibody. While dibasic flanking residues may result in potential endoproteolytic susceptibility, a linker with -Gln-Argflanking sequences resisted cleavage even in secretory granules, ensuring single-chain behavior. Effective SCIs exhibit favorable and specific binding with insulin receptors. SCIs with linkers bearing an Arg residue immediately preceding the A-chain were most bioactive, although efficient receptor interaction was inhibited as SCI linker length increased, approaching that observed for proinsulin. SCIs activate downstream metabolic signaling — stimulating glucose uptake into adipocytes and suppressing gluconeogenic enzyme biosynthesis in hepatocytes — with only limited cross-reactivity on IGF1 receptors. SCIs might theoretically have utility either in immunotherapy or gene therapy in insulin-deficient diabetes. The MOR-1 Opioid Receptor Regulates Glucose Homeostasis by Modulating Insulin Secretion Ting Wen, Bonnie Peng, and John E. Pintar (Mol Endocrinol, published February 12, 2009, 10.1210/me.2008-0345) ABSTRACT In addition to producing analgesia, opioids have also been proposed to regulate glucose homeostasis by altering insulin secretion. A considerable controversy exists, however, regarding the contribution of the opioid receptor (MOR-1) to insulin secretion dynamics. We employed congenic C57Bl/6J MOR-1 KO mice to clarify the role of MOR in glucose homeostasis. We first found that both sexes of MOR-1 KO mice weigh more than WT mice throughout postnatal life and that this increase includes preferentially increased fat deposition. We also found that MOR-1 KO mice exhibit enhanced glucose tolerance that results from insulin hypersecretion that reflects increased beta-cell mass and increased secretory dynamics in the MOR-1 mutant mice compared to WT. Analysis of the isolated islets indicated that islet insulin hypersecretion is mediated directly by MOR expressed on islet cells via a mechanism downstream of ATP-sensitive K channel activation by glucose. These findings indicate that MOR-1 regulates body weight by a mechanism that involves insulin secretion and thus may represent a novel target for new diabetes therapies. T R A N S L A T I O N A L H I G H L I G H T S F R O M M O L E C U L A R E N D O C R I N O L O G YIn addition to producing analgesia, opioids have also been proposed to regulate glucose homeostasis by altering insulin secretion. A considerable controversy exists, however, regarding the contribution of the opioid receptor (MOR-1) to insulin secretion dynamics. We employed congenic C57Bl/6J MOR-1 KO mice to clarify the role of MOR in glucose homeostasis. We first found that both sexes of MOR-1 KO mice weigh more than WT mice throughout postnatal life and that this increase includes preferentially increased fat deposition. We also found that MOR-1 KO mice exhibit enhanced glucose tolerance that results from insulin hypersecretion that reflects increased beta-cell mass and increased secretory dynamics in the MOR-1 mutant mice compared to WT. Analysis of the isolated islets indicated that islet insulin hypersecretion is mediated directly by MOR expressed on islet cells via a mechanism downstream of ATP-sensitive K channel activation by glucose. These findings indicate that MOR-1 regulates body weight by a mechanism that involves insulin secretion and thus may represent a novel target for new diabetes therapies. T R A N S L A T I O N A L H I G H L I G H T S F R O M M O L E C U L A R E N D O C R I N O L O G Y

Insulin-induced antilipolysis was investigated in fat cells obtained after an overnight fast and 60 min after glucose ingestion in seven non-obese patients with non-insulin-dependent diabetes mellitus (NIDDM). The study was performed before and after long-term therapy with diet and glibenclamide. After treatment, the antilipolytic potency of insulin in fat cells was threefold enhanced (p less than 0.05) in the fasting state and remained unaltered after glucose ingestion. In untreated NIDDM oral glucose induced a significant (p less than 0.01) increase in insulin sensitivity. In consequence, in the glucose-fed state insulin sensitivity was similar before and after therapy. Adipocyte insulin receptor binding was comparable before and after therapy, both in the fasting state and following glucose intake. In untreated NIDDM, despite relative hypoinsulinemia, plasma glycerol was markedly reduced after oral glucose. After therapy, plasma glycerol was significantly reduced both in the fasting state and following glucose ingestion. At the same time, fasting and glucose-stimulated circulating insulin were significantly (p less than 0.01) increased. It is concluded that conventional antidiabetes therapy in NIDDM mediates a suppression of adipose tissue lipolysis. This seems to be due to an improvement in insulin secretion in combination with a potentiation of the antilipolytic effectiveness of insulin in fat cells in the fasting state, the latter being secondary to post-binding alterations in insulin action.

The human [12-Asparagine-B] insulin [Asn12-B] insulin) which differs from the parent molecule in that the valine residue at position B12 is substituted by an asparagine residue, has been synthesized by the procedures developed in this laboratory. In stimulating glucose oxidation and lipogenesis the analogue exhibited potencies of 0.19% and 0.14%, respectively, as compared to insulin. In insulin receptor binding [Asn12-B] insulin was found to possess a potency of ca. 0.29% compared to the natural hormone. At high concentrations this analogue is shown to have the same maximal activity in the in vitro assays as the natural hormone. This indicates that despite the low affinity for of the analogue for the insulin receptor, the analogue-receptor complex is fully capable of initiating the series of chemical events that leads to the biological response. It is concluded that the B12 valine contributes greatly in the maintenance of a structure possessing the proper receptor-binding characteristics, but does not have any role in modulating the activity of the hormone-receptor complex. The potency of this analogue by radioimmunoassay is at least 100-fold higher than the in vitro biological assays, indicating that the immunological determinants of insulin can be essentially unrelated to the biological activity of the molecule.

Many food bioactive peptides with diverse functions have been discovered by studying plant proteins. We have previously identified a 68-residue insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia that exhibits hypoglycemic effects in mice via interaction with IR. By in vitro digestion, we found that mcIRBP-19, spanning residues 50-68 of mcIRBP, enhanced the binding of insulin to IR, stimulated the phosphorylation of PDK1 and Akt, induced the expression of glucose transporter 4, and stimulated both the uptake of glucose in cells and the clearance of glucose in diabetic mice. Furthermore, mcIRBP-19 homologs were present in various plants and shared similar β-hairpin structures and IR kinase-activating abilities to mcIRBP-19. In conclusion, our findings suggested that mcIRBP-19 is a blood glucose-lowering bioactive peptide that exhibits IR-binding potentials. Moreover, we newly identified novel IR-binding bioactive peptides in various plants which belonged to different taxonomic families.

Display Omitted A novel design of peptide inhibitor competitive insulin receptor is proposed.The peptide inhibitor developed based on the binding site of insulin-IR.Modification of peptide performs to obtain the promising peptide inhibitor.The complex of modified peptide-IR is more stable than the commercially IR blocker. A designing peptide as agent for inducing diabetes mellitus type 2 (T2DM) in an animal model is challenging. The computational approach provides a sophisticated tool to design a functional peptide that may block the insulin receptor activity. The peptide that able to inhibit the binding between insulin and insulin receptor is a warrant for inducing T2DM. Therefore, we designed a potential peptide inhibitor of insulin receptor as an agent to generate T2DM animal model by bioinformatics approach. The peptide has been developed based on the structure of insulin receptor binding site of insulin and then modified it to obtain the best properties of half life, hydrophobicity, antigenicity, and stability binding into insulin receptor. The results showed that the modified peptide has characteristics 100h half-life, high-affinity -95.1ź20, and high stability 28.17 in complex with the insulin receptor. Moreover, the modified peptide has molecular weight 4420.8g/Mol and has no antigenic regions. Based on the molecular dynamic simulation, the complex of modified peptide-insulin receptor is more stable than the commercial insulin receptor blocker. This study suggested that the modified peptide has the promising performance to block the insulin receptor activity that potentially induce diabetes mellitus type 2 in mice.

Enhanced skeletal muscle and whole body insulin sensitivity can persist for up to 24–48 h after one exercise session. This review focuses on potential mechanisms for greater postexercise and insulin-stimulated glucose uptake (ISGU) by muscle in individuals with normal or reduced insulin sensitivity. A model is proposed for the processes underlying this improvement; i.e., triggers initiate events that activate subsequent memory elements, which store information that is relayed to mediators, which translate memory into action by controlling an end effector that directly executes increased insulin-stimulated glucose transport. Several candidates are potential triggers or memory elements, but none have been conclusively verified. Regarding potential mediators in both normal and insulin-resistant individuals, elevated postexercise ISGU with a physiological insulin dose coincides with greater Akt substrate of 160 kDa (AS160) phosphorylation without improved proximal insulin signaling at steps from insulin receptor binding to Akt activity. Causality remains to be established between greater AS160 phosphorylation and improved ISGU. The end effector for normal individuals is increased GLUT4 translocation, but this remains untested for insulin-resistant individuals postexercise. Following exercise, insulin-resistant individuals can attain ISGU values similar to nonexercising healthy controls, but after a comparable exercise protocol performed by both groups, ISGU for the insulin-resistant group has been consistently reported to be below postexercise values for the healthy group. Further research is required to fully understand the mechanisms underlying the improved postexercise ISGU in individuals with normal or subnormal insulin sensitivity and to explain the disparity between these groups after similar exercise.

Introduction Imprinted genes play an important role in mammalian fetoplacental growth and development. We have evaluated whether the placental expression of two imprinted genes, growth factor receptor-binding protein 10 ( GRB10 ) and pleckstrin homology-like domain, family A, member 2 ( PHLDA2 ) correlate with human fetoplacental growth parameters. Methods Placentae (n = 77) were collected from small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies (n = 36 SGA and 41 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of GRB10 and PHLDA2 normalized to a panel of reference genes. Results Placental GRB10 transcript abundance associated positively with placental weight (r = 0.307, P = 0.007), birth weight (r = 0.267, P = 0.019) and neonatal head circumference (r = 0.280, P = 0.014). Placental GRB10 transcript levels were significantly lower in male SGA placentae compared to the male AGA placentae. Placental PHLDA2 transcript abundance did not show any associations with maternal, placental or neonatal parameters. Discussion Placental GRB10 expression was found to be associated positively with placental weight, birth weight, and neonatal head circumference, especially in males. Hence, we speculate that placental GRB10 plays a role in regulating fetoplacental growth and thereby in the pathophysiology of fetal growth restriction in the context of fetal gender.

Functional disturbance in the novel adenylyl cyclase signaling mechanism (ACSM) of insulin and relaxin action in rat streptozotocin (STZ) type I diabetes was studied on the basis of the authors’ conception of molecular defects in hormonal signaling systems as the main causes of endocrine diseases. Studying the functional state of molecular components of the ACSM and the mechanism as a whole, the following changes were found in the skeletal muscles of diabetic rats compared with control animals: 1) increase of insulin receptor binding due to an increase in the number of insulin binding sites with high and low affinity; 2) increase of the basal adenylyl cyclase (AC) activity and the reduction of AC-activating effect of non-hormonal agents (guanine nucleotides, sodium fluoride, forskolin); 3) reduction of ACSM response to stimulatory action of insulin and relaxin; 4) decrease of the insulin-activating effect on the key enzymes of carbohydrate metabolism, glycogen synthase and glucose-6-phosphate dehydrogenase. Hence, the functional activity of GTP-binding protein of stimulatory type, AC and their functional coupling are decreased during experimental type 1 diabetes that leads to the impairment of the transduction of insulin and relaxin signals via ACSM.

In fetal alcohol syndrome (FAS), cerebellar hypoplasia is associated with impaired insulin-stimulated survival signaling. This study characterizes ethanol dose-effects on cerebellar development, expression of genes required for insulin and insulin-like growth factor (IGF) signaling, and the upstream mechanisms and downstream consequences of impaired signaling in relation to acetylcholine (ACh) homeostasis. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0%, 2%, 4.5%, 6.5%, or 9.25% ethanol from gestation day 6. Ethanol caused dose-dependent increases in severity of cerebellar hypoplasia, neuronal loss, proliferation of astrocytes and microglia, and DNA damage. Ethanol also reduced insulin, IGF-I, and IGF-II receptor binding, insulin and IGF-I receptor tyrosine kinase activities, ATP, membrane cholesterol, and choline acetyltransferase (ChAT) expression. In vitro studies linked membrane cholesterol depletion to impaired insulin receptor binding and insulin-stimulated ChAT. In conclusion, cerebellar hypoplasia in FAS is mediated by insulin/IGF resistance with attendant impairments in energy production and ACh homeostasis.

An eight amino acid sequence, CAKGDWNC, from disintegrin barbourin, was introduced into an inactive human proinsulin molecule between the B28 and A2 sites to construct a chimeric, anti-thrombosis recombinant protein. The constructed Lys-Gly-Asp (KGD)-proinsulin gene was expressed in Escherichia coli and then purified. The KGD-proinsulin chimera protein inhibits human platelet aggregation, induced by ADP, with an IC50 value (molar concentration causing 50% inhibition of platelet aggregation) of 830 nM: and demonstrates also specific affinity to glycoprotein IIb/IIIa receptor. Its insulin receptor binding activity remains as low as 0.04% with native insulin as a control.

New insulin analogues have been created by amino-acid exchange to provide an improved pharmacokinetic profile. However, safety issues have been raised regarding their use, as amino-acid exchange of insulin may induce altered metabolic and mitogenic effects. For example, the insulin analogue Asp(B10) causes breast cancer in rodents. The aim of this study was to compare two new insulin analogues HMR1964 (Lys[B3],Glu[B29]) (insulin glulisine) and HMR1423 (Gly[A21],His[B31],His[B32]) with regular insulin and the mitogenic analogue Asp(B10).We analysed insulin receptor binding characteristics and dissociation kinetics, as well as insulin-induced receptor auto- and dephosphorylation kinetics, in rat-1 fibroblasts overexpressing the human insulin receptor isoform B. Mitogenic activity was tested in the non-malignant cell line MCF10.Regular insulin, HMR1964 and HMR1423 showed no significant differences in receptor association, dissociation and receptor binding affinity, while Asp(B10) displayed markedly increased insulin receptor affinity. All of the analogues induced rapid insulin receptor autophosphorylation, reaching a maximum 10 min after stimulation (10(-9) mmol/l insulin). In contrast, Asp(B10) induced a prolonged phosphorylation and dephosphorylation state of the 95 kDa insulin receptor beta-subunit. With respect to [3H]thymidine incorporation, the new analogues had similar (HMR1423) or even lower (HMR1964) effects than regular insulin in the mammary epithelial cell line MCF10, while Asp(B10) showed increased [3H]thymidine incorporation.HMR1964 and HMR1423 displayed the same association, dissociation and insulin receptor affinity kinetics as regular insulin, and might therefore be useful for the treatment of diabetes.

Nitric oxide, a messenger molecule has been reported as having various antineoplastic properties. The activation of insulin-activated nitric oxide synthase (IANOS) was found to be related to the production of NO as a result of the binding of insulin to its receptor through the activation of tyrosine kinase in erythrocyte membrane. As nitric oxide is reported to be a systemic anticancer agent, studies were carried out to determine the role of insulin receptor binding that lead to the activation of tyrosine kinase and IANOS in erythrocytes in breast cancer. Blood samples were collected from female breast cancer patients who, at the time of participation in the study, had not undergone any therapeutic intervention but had opted for surgery. The binding of insulin to its receptor in erythrocyte membrane and the activation of both receptor tyrosine kinase and IANOS due to the hormone binding were determined and compared with the appropriate control. It was found that the impaired NO synthesis in erythrocyte membrane in breast cancer was related to the marked decrease of insulin binding sites of the high-affinity hormone receptor population. This impaired insulin binding to high-affinity receptors resulted in the impairment of both reaction velocity (Vmax) of the IANOS and receptor tyrosine kinase activation. These results indicated that the impairment of interaction between insulin and its high-affinity receptors in erythrocyte membrane might be a critical pathophysiological event in the development of breast cancer.

Insulin receptors and renal sodium handling in hypertensive fructose-fed rats. Background Insulin resistance and hypertension are present in Sprague-Dawley rats fed a fructose-enriched diet. In these rats, insulin might elevate blood pressure via an antinatriuretic action. Methods To investigate the sodium-insulin interaction in fructose-fed rats, we compared insulin sensitivity, insulin receptor binding, and insulin receptor mRNA levels in the kidney and skeletal muscle of rats that were fed standard rat chow or a fructose-enriched diet (66%) with either low (0.07%), normal (0.3%), or high (7.5%) NaCl concentrations for 3weeks. Results Systolic blood pressure increased in the fructose-fed rats receiving the normal and high-salt diet, but not the low-salt diet. When the rats were fed the low-salt diet, the rate of glucose infusion required to maintain euglycemia during a hyperinsulinemic clamp and insulin receptor number and mRNA levels in skeletal muscle were lower in fructose-fed than control rats. High-salt diet decreased significantly the rate of glucose disposal during the clamp and muscular insulin receptor number and mRNA levels in control, but not fructose-fed rats. During the low-salt diet, renal insulin receptor number and mRNA levels were comparable in fructose-fed and control rats and hyperinsulinemia had comparable acute antinatriuretic effects in the two groups; when the rats were maintained on the high-salt diet, the expected decrease in renal insulin receptor number and mRNA levels occurred in control but not fructose-fed rats and, consistent with this finding, the antinatriuretic response to hyperinsulinemia was blunted only in controls. An inverse relationship between dietary NaCl content and renal insulin receptor mRNA levels was observed in control but not fructose-fed rats. Conclusion Fructose-fed rats appear to have lost the feedback mechanism that limits insulin-induced sodium retention through a down-regulation of the renal insulin receptor when the dietary NaCl content is increased. This abnormality might possibly contribute to the elevation of blood pressure in these rats.

我々は、低脂防食およびトレッドミル訓練がヒトの肥満に類似した高脂肪飼料誘発肥満ラットの体脂肪率、摂食効率及びインスリン作用に及ぼす影響について研究した。高脂肪飼料誘発肥満ラットにおいて、摂食効率(体重増加量/摂取エネルギー)の上昇、高インスリン血症、肝細胞膜インスリン受容体結合能の低下が認められた。トレッドミル訓練および低脂防食+トレッドミル訓練は肥満ラットの肝細胞膜インスリン受容体の結合能を高め、高インスリン血症を抑制する働きがあった。とりわけ、低脂防食+トレッドミル訓練は肥満ラットの体重増加を抑制し体脂肪率を減少させた。これらの代謝変化が高脂肪飼料誘発肥満ラットにおいてダイエット効果を発揮していると考えられる。, Diet-induced obesity (DIO) was produced in 1-month-old male Sprague-Dawley rats during a 10-week period who were fed a high-fat (20%), high-calorie (7.6 kcal/g) DIO diet. Effects of three weight-reducing methods (treadmill training, low-fat diet, treadmill training plus low-fat diet) on body weight, body fat rate, feed efficiency, total cholesterol and triglyceride, plasma insulin and insulin receptor binding capacity on the hepatic cell membrane in the obese rats were investigated. The results indicated that feed efficiency increased, the insulin receptor binding capacity on the hepatic cell membrane decreased and hyperinsulinemia developed in the obese rats. A low-fat diet did not affect body fat rate, feed efficiency, insulin receptor binding capacity on the hepatic cell membrane or hyperinsulinemia. With unrestricted food intake, treadmill training in DIO rats depressed hyperinsulinemia significantly and reduced body fat. Furthermore, treadmill training plus low-fat diet was most effective in inhibiting the body weight gain in the obese rats; it also reduced body fat and feed efficiency to the non-obese control levels. The results indicate that DIO is related to the increase in feed efficiency, the decrease in insulin receptor binding capacity on the hepatic cell membrane and hyperinsulinemia.