Role Of Insulin In Regulation Research Articles

Insulin induces bioenergetic changes and alters mitochondrial dynamics in podocytes.

Diabetic nephropathy (DN) is one of the most frequent complications of diabetes. Early stages of DN are associated with hyperinsulinemia and progressive insulin resistance in insulin-sensitive cells, including podocytes. The diabetic environment induces pathological changes, especially in podocyte bioenergetics, which is tightly linked with mitochondrial dynamics. The regulatory role of insulin in mitochondrial morphology in podocytes has not been fully elucidated. Therefore, the main goal of the present study was to investigate effects of insulin on the regulation of mitochondrial dynamics and bioenergetics in human podocytes. Biochemical analyses were performed to assess oxidative phosphorylation efficiency by measuring the oxygen consumption rate (OCR) and glycolysis by measuring the extracellular acidification rate (ECAR). mRNA and protein expression were determined by real-time polymerase chain reaction and Western blot. The intracellular mitochondrial network was visualized by MitoTracker staining. All calculations were conducted using CellProfiler software. Short-term insulin exposure exerted inhibitory effects on various parameters of oxidative respiration and adenosine triphosphate production, and glycolysis flux was elevated. After a longer time of treating cells with insulin, an increase in mitochondrial size was observed, accompanied by a reduction of expression of the mitochondrial fission markers DRP1 and FIS1 and an increase in mitophagy. Overall, we identified a previously unknown role for insulin in the regulation of oxidative respiration and glycolysis and elucidated mitochondrial dynamics in human podocytes. The present results emphasize the importance of the duration of insulin stimulation for its metabolic and molecular effects, which should be considered in clinical and experimental studies of DN.

Investigating the Impact of ENPP1 Gene's K121Q (RS1044498) Polymorphism in Type 2 Diabetes via an Updated Meta-Analysis

Type 2 Diabetes Mellitus (T2DM) occurs due to a complex relationship of genetic, environmental, and physiological factors, encompassing insufficient pancreatic insulin synthesis, peripheral insulin resistance, and diverse molecular pathways. The transmembrane glycoprotein ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) plays a role in insulin regulation, with the K121Q (rs1044498) variant on the ENPP1 gene being a subject of extensive study due to its potential association with T2DM. To comprehensively evaluate this relationship, a meta-analysis was conducted, pooling data from 48 studies retrieved from databases such as PubMed, Google Scholar, Science Direct, and Medline. The analysis, performed using Review Manager Version 5.4.1 and Stata version 14.1, included a total of 24,979 T2DM cases and 33,005 controls. Employing fixed-effects or random-effects models, the combined Odds Ratio (OR) and 95% Confidence Intervals (CIs) were calculated to quantify the connection's magnitude. In the overall population, all genotypic models revealed a statistically noteworthy connotation between ENPP1 and T2DM (P < 0.05). Notably, the homozygous model exhibited an OR of 1.53 (95% CI = 1.23-1.90, P = 0.0001), while the heterozygous, dominant, recessive, and allelic models showed ORs of 1.22 (95% CI = 1.08-1.37, P = 0.001), 1.15 (95% CI = 1.11-1.41, P = 0.0003), 1.38 (95% CI = 1.17-1.64, P = 0.0002), and 1.22 (95% CI = 1.10-1.36, P = 0.0003), correspondingly. Subgroup analysis by population indicated no significant correlation between the K121Q polymorphism and T2DM in the African population, while a noteworthy association was detected in both Asian and Caucasian populations, with the heterozygous model lacking significance in the latter. Despite no evidence of publication bias, a notable amount of residual heterogeneity among studies was identified. Sensitivity analysis established the steadiness and dependability of the meta-analysis findings, underscoring the complex nature of the ENPP1 gene's involvement in T2DM across diverse populations.

1503-P: Insulin Regulates Expression and Secretion of Exosomal MicroRNAs in Adipocytes

Transfer of regulatory molecules, such as microRNAs (miRNAs), via small extracellular vesicles (sEVs) called exosomes represents a newly recognized mode of inter-organ communication that provides an important mechanism of metabolic regulation. We have previously shown that adipose tissue is a major contributor to the pool of circulating exosomal miRNAs and that these adipose-derived exosomal miRNAs can be taken up by target tissues and suppress gene expression. We have also shown that biogenesis, secretion, and exosomal cargo sorting is a highly regulated process and that miRNAs contain 4-8 nucleotide motifs which contribute to exosomal secretion. Several exosomal miRNAs have been linked to obesity and diabetes, yet their regulation by physiological stimuli such as insulin remains unknown. To identify the role of insulin in regulation of adipose miRNAs and their secretion in exosomes, we performed small RNA-seq on exosomes isolated from differentiated 3T3-L1 cells with and without insulin stimulation. We find that out of the 460 miRNAs detected, 39 were up- and 75 were down-regulated in exosomes in response to insulin, whereas only 14 were up- and 39 were down-regulated in cells. In some cases, exosomal secretion correlated with cellular expression of the miRNA. More interestingly, 103 of the 114 miRNAs (90.3%) that were regulated in exosomes had no significant change at the cellular level or even a change in the opposite direction, demonstrating insulin’s ability to regulate miRNA exosomal secretion independent of cellular level. These insulin-regulated exosomal miRNAs included members of the let-7 family, which have known functions in adipogenesis, as well miRNAs predicted to target mRNAs of molecules involved in fatty acid biosynthesis and insulin signaling. Thus, insulin can independently regulate miRNA expression and secretion in adipocyte-derived exosomes. These can serve as adipokines and create a novel mechanism for regulation of cell-cell and inter-organ communication. Disclosure M.Lino: None. H.Pan: None. J.Dreyfuss: None. C.Kahn: None. Funding National Institutes of Health (5R01DK082659)

Regulatory Role of Insulin on Endogenous L1 ORF1 and NEFM Gene Expression through PI3K Signaling Pathway Specifically in Neuroblastoma Cell Line.

One of the most important endogenous factors causing genomic instability in human cells is L1s retrotransposons. In this study, we assume that increased activity of L1 retrotransposons (specifically L1 expression) might be induced by hyperglycemia and hyperinsulinemia in neuroblastoma cell line. Two different cell lines (BE (2)-M17 and HEK293) were treated with insulin and its PI3K signaling pathway inhibitor under three conditional media including hyperglycemic and retinoic acid treatment in the department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran in 2018. The expression of L1 ORF1, as well as genes involved in insulin signaling pathway and neuronal stress and structure were measured at RNA level. Insulin could significantly down regulate the expression of L1 ORF1 and NEFM genes. Hyperglycemia result in severe decrease in expression of all candidate genes in control neuroblastoma but not HEK293 cells. Retinoic acid as the concentration used in this study cause increase stemness in neuroblastoma but not HEK293 cells. We could not find significant correlation between expression pattern of other genes tested in our study and L1 ORF1 expression. Total regulatory effect of insulin on L1 ORF1 RNA expression as well as NEFM markedly in BE (2)-M17 cell line. Although these results could not be interpreted as L1 retrotransposition, expression of L1 RNA during stress conditions might be considered following inhibition of the insulin pathway. The result of this study also confirms the impotence of insulin on human evolution.

Insulin regulates arginine-stimulated insulin secretion in humans

AimsInsulin potentiates glucose-stimulated insulin secretion. These effects are attenuated in beta cell–specific insulin receptor knockout mice and insulin resistant humans. This investigation examines whether short duration insulin exposure regulates beta cell responsiveness to arginine, a non-glucose secretagogue, in healthy humans. Materials and methodsArginine-stimulated insulin secretion was studied in 10 healthy humans. In each subject arginine was administered as a bolus followed by continuous infusion on two occasions one month apart, after sham/saline or hyperinsulinemic-isoglycemic clamp, respectively providing low and high insulin pre-exposure conditions. Arginine-stimulated insulin secretion was measured by C-peptide deconvolution, and by a selective immunogenic (DAKO) assay for direct measurement of endogenous but not exogenous insulin. ResultsPre-exposure to exogenous insulin augmented arginine-stimulated insulin secretion. The effect was seen acutely following arginine bolus (endogenous DAKO insulin incremental AUC240-255min 311.6 ± 208.1 (post-insulin exposure) versus 120.6 ± 42.2 μU/ml•min (sham/saline) (t-test P = 0.021)), as well as in response to continuous arginine infusion (DAKO insulin incremental AUC260-290min 1095.3 ± 592.1 (sham/saline) versus 564.8 ± 207.1 μU/ml•min (high insulin)(P = 0.009)). Findings were similar when beta cell response was assessed using C-peptide, insulin secretion rates by deconvolution, and the C-peptide to glucose ratio. ConclusionsWe demonstrate a physiologic role of insulin in regulation of the beta cell secretory response to arginine.

Mechanism of INSR clustering with insulin activation and resistance revealed by super-resolution imaging.

Insulin receptor (INSR) is a key protein in the INSR signaling pathway and plays a critical role in biological processes, especially in the regulation of glucose homeostasis. Many metabolic diseases are often accompanied by abnormal INSR signaling. However, the specific effector mechanisms regulating insulin resistance and the distribution patterns of INSR during cell membrane activation remain unclear. Here, we investigated the changes in the distribution of INSR during activation using super-resolution imaging. By observing the connection between INSR activation and its distribution, we found that insulin resistance inhibits its receptor clustering. More importantly, we found that INSR has a highly co-localized relationship with the skeletal protein βII-spectrin. Specific knockout of βII-spectrin inhibited the interaction of INSR with GLUT4 and affected the normal metabolism of glucose. Our work elucidates the effects of insulin activation and insulin resistance on INSR distribution and reveals a potential relationship between INSR and cytoskeleton at the single molecule level, which promotes a deeper understanding of the roles associated with insulin signaling and insulin resistance.

Factors responsible for low serum vitamin-D levels in type-2 diabetes mellitus females: a cross-sectional study

Background: Diabetes mellitus are a group of disorders as a result of reduced insulin secretion, insulin resistance, and increased glucose production and many more unknown etiologies. As compared to diabetic non-obese, diabetic obese individuals have an increased chance of 13.5 % in developing diabetic complications. Many diabetic patients are found to be vitamin-D deficient and its role in insulin regulation is being studied in many studies. Thus, diabetic complications and vitamin-D deficiency are likely to be developed in obese diabetic females.Methods: Type-2 diabetic females from 30 years to 60 years, attending in medicine as out-patient and female type-2 diabetes patients admitted in medical ward of RRMCH were assessed clinically. Parameters such as anthropometric measurements, weight, height, waist-hip ratio, tested for serum levels vitamin-D including other relevant investigations related to diabetes were done. Totally, 156 patients were assessed.Results: Assessment of 156 patients are as follows, 48.47±9.56years was the mean age of this study group. SD: 5.10±4.36 years is the mean diabetic duration of this study population. 0.98 was the mean waist-hip ratio. 98.93 cm was the mean waist circumference. 24.97 was the mean BMI. Mean fasting blood sugar (FBS) and post-prandial glucose test (PPBS) were: 202.73 mg/dl, 280.99 mg/dl respectively, 9.33% was the mean HbA1C. Majority of the females with type-2 diabetes (92.5%) had low levels of serum vitamin-D. 16.19 ng/ml was the mean serum vitamin-D levels. lower levels of serum vitamin-D were significantly associated with diabetic duration (p=0.082+), poor glycaemic control (p<0.001**) and increased BMI (p=0.011*).Conclusions: Majority of the females with type-2 diabetes mellitus were with waist-hip ratio more than 0.8 and Waist circumference more than 80 cm. Most of the study population had poor diabetic control. Lower levels of serum vitamin-D were found in almost all females with type-2 diabetes mellitus (92.5 %) and the most likely risk factor being obesity and poor glycaemic control as a conclusion of this study.

The regulatory role of insulin in energy metabolism and leukocyte functions.

Insulin is the hormone responsible for maintaining glucose homeostasis in the body, in addition to participating in lipid metabolism, protein synthesis, and the inhibition of gluconeogenesis. These functions are well characterized in the classic organ target cells that are responsible for general energy regulation: the liver, skeletal muscle, and adipose tissue. However, these actions are not restricted to these tissues because insulin has been shown to affect most cells in the body. This review describes the role of insulin in leukocyte signaling pathways, metabolism and functions, and how insulin resistance could affect this signaling and deteriorate leukocyte metabolism and function, in addition to showing evidence that suggests leukocytes may substantially contribute to the development of systemic insulin resistance.

Prevalence of Vitamin D3 insufficiency among rural ethnic population of Tripura and its association with type-2 diabetes mellitus

Background: Different studies suggest that Vitamin D3 has a role in regulation of insulin and its deficiency leads to the development of type 2 diabetes mellitus (DM). In a study by ICMR-INDIAB (NE), it was shown that the prevalence of type 2 DM is about 9% in Tripura. However, little is known regarding the blood level of Vitamin D3 among the rural ethnic population. Objectives: The objectives of this study were to assess the blood level of Vitamin D3 and its association with type 2 DM in rural ethnic population. Materials and Methods: This cross-sectional study was undertaken from February 2018 to January 2019 at Multidisciplinary Research Unit of Agartala Government Medical College to assess the blood Vitamin D3 level in the rural ethnic population and its association with Type 2 DM. Two hundred and eight subjects were recruited from 10 different health camps. Blood samples were collected for detecting different blood glycemic parameters. The data were statistically analyzed using the Statistical Package for the Social Sciences. Pearson’s correlation coefficient was used to evaluate the association of blood Vitamin D3 with glycemic parameters. Results: Of 208 ethnic subjects, 136 had insufficient while 72 had sufficient blood Vitamin D3 level. Overall, 65% prevalence of Vitamin D3 insufficiency has been observed in the study population. About 98.3% and 52.3% of insufficiency (

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice.

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria.

Insulin Attenuates Cardiac Myocyte Contractility via NADPH Oxidase: Implications for Diabetic Cardiomyopathy

Insulin is known to modulate metabolic responses in cardiac myocytes (CM), yet the role of insulin in regulation of CM contractility is less well understood. Type 2 diabetes mellitus (T2D) leads to diabetic cardiomyopathy, yet little is known about the role of insulin in CM function. Here we explore the role of insulin on CM contractility in adult mouse CM. Insulin had no effect on basal CM contractility, but inhibited the increase in contractility seen after stimulation of ß‐adrenergic receptors (ß‐AR) by isoproterenol (ISO; 40+/‐5% inhibition, p<0.01, n=115‐124 cells). The attenuation of ISO‐stimulated contractility by insulin was blocked by the NADPH oxidase inhibitor apocynin, implicating reactive oxygen species (ROS) in cardiac insulin responses. We found that insulin inhibited ß‐AR stimulation of CM Ca2+ transients (35+/‐4% inhibition, p<0.01, n=39‐41 cells); again, insulin inhibition of this ß‐AR response was blocked by apocynin. We analyzed CM contractile responses in CM isolated from mice fed a high‐fat diet, a model of T2D. We found that the insulin‐promoted decrease in contractility was blocked in CM isolated from diabetic mice. The phosphorylation of insulin‐dependent signaling proteins (insulin receptor, IRS‐1, Akt) was effectively abrogated in diabetic mice, consistent with myocyte insulin resistance. These data establish a central role for NADPH oxidase in insulin‐dependent physiological responses in CM, and show that these responses are deranged in mice with T2D. These observations suggest that alterations in ROS‐ and insulin‐dependent pathways play a role in the pathophysiology of diabetic cardiomyopathy. Research support: NIH and AHA.

Effects of insulin and its related signaling pathways on lipid metabolism in the yellow catfish Pelteobagrus fulvidraco.

The influence of insulin on hepatic metabolism in fish is not well understood. The present study was therefore conducted to investigate the effects of insulin on lipid metabolism, and the related signaling pathways, in the yellow catfish Pelteobagrus fulvidraco. Hepatic lipid and intracellular triglyceride (TG) content, the activity and expression levels of several enzymes and the mRNA expression of transcription factors (PPARα and PPARγ) involved in lipid metabolism were determined. Troglitazone, GW6471, fenofibrate and wortmannin were used to explore the signaling pathways by which insulin influences lipid metabolism. Insulin tended to increase hepatic lipid accumulation, the activity of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) and mRNA levels of FAS, G6PD, 6PGD, CPT IA and PPARγ, but down-regulated PPARα mRNA level. The insulin-induced effect could be stimulated by the specific PPARγ activator troglitazone or reversed by the PI3 kinase/Akt inhibitor wortmannin, demonstrating that signaling pathways of PPARγ and PI3 kinase/Akt were involved in the insulin-induced alteration of lipid metabolism. The specific PPARα pathway activator fenofibrate reduced insulin-induced TG accumulation, down-regulated the mRNA levels of FAS, G6PD and 6PGD, and up-regulated mRNA levels of CPT IA, PPARα and PPARγ. The specific PPARα pathway inhibitor GW6471 reduced insulin-induced changes in the expression of all the tested genes, indicating that PPARα mediated the insulin-induced changes of lipid metabolism. The present results contribute new knowledge on the regulatory role of insulin in hepatic metabolism in fish.

Amplification and Demultiplexing in Insulin-regulated Akt Protein Kinase Pathway in Adipocytes

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease.

Weight gain induced by an isocaloric pair-fed high fat diet: A nutriepigenetic study on FASN and NDUFB6 gene promoters

Experimental studies have demonstrated that dietary macronutrient distribution plays an important role in insulin regulation, a risk factor associated to obesity, diabetes and other metabolic disorders. To assess whether the macronutrient composition of the diet could be related to obesity onset by affecting the epigenetic regulation of gene expression, we investigated in rats the metabolic effects of two pair-fed isocaloric diets: control (rich in carbohydrates) and high fat diet (rich in fat; HFD). Compared to controls, HFD induced higher weight gain and adiposity and impaired glucose tolerance, which was accompanied by a slight increase in adiponectin levels and liver steatosis. Epididymal adipose tissue expression of the fatty acid synthase (FASN) gene and NADH dehydrogenase (ubiquinone) 1β-subcomplex 6 (NDUFB6) were significantly reduced in HFD group. These variations in mRNA levels were accompanied by changes in the methylation patterns of several CpG islands located in the promoter region of these genes. However, no correlations were found between gene expression and the methylation status. These results suggest that high fat intake produces overweighted rats independently of total energy intake. These diets could also induce some epigenetic changes in the promoters of key genes that could influence gene expression and may be behind metabolic alterations.

Prolonged Exposure to Insulin Suppresses Mitochondrial Production in Primary Hepatocytes

Insulin is the central regulator of metabolism and is necessary for storing energy as fat efficiently. Mitochondria are primary sites of energy consumption of most cells. Increased plasma insulin level and mitochondrial dysfunction are features of insulin resistance. The exact role of insulin in regulation of mitochondrial production and function remains unestablished. In this study, we observed that mitochondrial production in liver and skeletal muscle gastrocnemius was increased in mice with insulin deficiency (streptozotocin-induced type 1 diabetes). In contrast, prolonged exposure (24 h) of isolated hepatocytes to insulin decreased mitochondrial mass, mitochondrial DNA (mtDNA), intracellular ATP content, and cellular O(2) consumption. Transcript levels of genes associated with mitochondrial production and beta oxidation were decreased, whereas those of lipogenic genes were increased by the prolonged exposure to insulin. Insulin-induced changes in mtDNA, mitochondrial mass, intracellular ATP content, and transcripts of mitochondrion-associated genes were prevented by blockade of Akt activation with the phosphatidylinositol 3-kinase inhibitor LY294002. Conversely, levels of mtDNA, intracellular ATP content, and expression of mitochondrion-associated genes were decreased by overexpression of the constitutively active Akt. Finally, insulin suppression of mtDNA, ATP production, and expression of mitochondrion-related genes was largely prevented by inhibition of cyclic nucleotide phosphodiesterase with isobutylmethylxanthine. Together, our results show prolonged exposure of isolated hepatocytes to insulin suppresses mitochondrial production and function through the classical Akt-dependent insulin signaling pathway.

Effect of Permanent Hypoinsulinemia on Appetite, Performance, Carcass Composition, Blood Metabolites and Leptin Concentrations in Lambs

Insulin has crucial roles in energy metabolism in all mammals but has been less studied in ruminants. An experiment was conducted to investigate the effects of hypoinsulinemia induction on appetite, performance, carcass composition and blood metabolite levels in sheep. Treatments were intravenous injection of four doses of streptozotocin; 0, 25, 50 and 75 mg/kg BW named C, L, M and H, respectively. Twenty male lambs were divided into four treatment groups. Animals in group H could not continue the experiment because of abnormalities. The duration of the experiment was eight consecutive weeks, and injection was performed at the end of week 3. Feed and water intakes were measured weekly and weight changes of animals were recorded and used for calculation of other growth parameters. Blood samples were collected weekly via venipuncture at fasting and 2.5 h post-prandial and analyzed for hormones and blood metabolites. Results showed a marked hypoinsulinemia in group M with significant decrease in fasted and post-prandial insulin concentrations and also fasted leptin concentrations vs. the control group C (p 0.05). In conclusion, the results suggested a regulatory role of insulin in energy metabolism of ruminants by exerting two opposing effects: central catabolic and peripheral anabolic.

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats.

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (Vmax) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (Bmax) and affinity (Kd) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.

Insulin signalling in islets

Studies in transgenic animals, rodent insulin-secreting cell lines and rodent islets suggest that insulin acts in an autocrine manner to regulate beta-cell mass and gene expression. Very little is known about the in vitro roles played by insulin in human islets, and the regulatory role of insulin in protecting against beta-cell apoptosis. We have identified mRNAs encoding IRs (insulin receptors) and downstream signalling elements in dissociated human islet beta-cells by single-cell RT (reverse transcription)-PCR, and perifusion studies have indicated that insulin does not have an autocrine role to regulate insulin secretion from human islets, but activation of the closely related IGF-1 (insulin-like growth factor 1) receptors is linked to inhibition of insulin secretion. Knockdown of IR mRNA by siRNAs (small interfering RNAs) decreased IR protein expression without affecting IGF-1 receptor levels, and blocked glucose stimulation of preproinsulin gene expression. Similar results were obtained when human islet IRS (IR substrate)-2 was knocked down, whereas depletion of IRS-1 caused an increase in preproinsulin mRNA levels. Studies using the mouse MIN6 beta-cell line indicated that glucose protected beta-cells from undergoing apoptosis and that this was a consequence, at least in part, of insulin release in response to elevated glucose. IGF-1 also exerted anti-apoptotic effects. These data indicate that insulin can exert autocrine effects in human islets through receptors on beta-cells. It protects beta-cells against apoptosis and increases preproinsulin mRNA synthesis, but does not affect insulin secretion.

Differential Association of Basal and Postprandial Plasma Ghrelin With Leptin, Insulin, and Type 2 Diabetes

To gain further insight into the regulatory role of insulin and leptin on plasma ghrelin, 56 normal weight, 128 normoinsulinemic obese, 121 hyperinsulinemic obese, and 30 type 2 diabetic normoinsulinemic and 75 type 2 diabetic hyperinsulinemic obese patients were examined. In the obese subjects, basal hyperinsulinemia was associated with significantly lower ghrelin independent of BMI, age, and leptin. In normoinsulinemic (normal weight and normoinsulinemic obese) subjects, ghrelin was inversely related to stepwise increasing leptin. Multiple regression analysis and matching for insulin revealed a significant negative interaction of ghrelin with leptin but not insulin. In type 2 diabetic normoinsulinemic subjects, ghrelin was significantly lower compared with that in normoinsulinemic obese subjects. In type 2 diabetic hyperinsulinemic subjects, ghrelin was significantly lower than in normoinsulinemic subjects, whereas no further reduction was observed compared with hyperinsulinemic obese subjects. The postprandial decrease was significantly attenuated in normoinsulinemic obese and hyperinsulinemic obese subjects (-214.8 +/- 247 pg/ml [normal weight], -137.6 +/- 107 pg/ml [normoinsulinemic obese], -85.5 +/- 69 pg/ml [hyperinsulinemic obese], P < 0.001; mean +/- SD), whereas type 2 diabetes had no independent postprandial effect. In conclusion, the present data support the concept that leptin could be of importance for suppression of basal ghrelin during moderate weight gain in normoinsulinemic subjects, whereas hyperinsulinemia but not leptin is responsible in more severe obesity. Postprandial suppression of ghrelin is attenuated by as yet unknown mechanisms that are related to body weight but not to insulin or type 2 diabetes.

Regulation of Gene33 expression by insulin requires MEK–ERK activation

Gene33 and its human homologue, mitogen inducible gene-6/receptor-associated late transducer (mig-6, RALT), is a 53-kDa soluble protein that was identified as a hepatic gene regulated by glucocorticoids and insulin. Its mRNA is expressed in numerous tissues in addition to the liver. Mitogen inducibility of Gene33 mRNA has been described in several experimental systems. Recent reports have suggested a role for Gene33 in inhibition of proliferation induced by factors that bind to members of the ErbB family of receptors. In the present work, we examine the regulation of Gene33 protein by insulin in hepatoma cells of rat (H4IIE) and human (HepG2/Hep3B) origin. Inhibition of MEK1 significantly inhibited extracellularly regulated kinase (ERK)1/2 activation and insulin-regulated Gene33 transcription and protein levels in H4IIE cells. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity alone did not significantly alter transcription of Gene33. In Hep3B and HepG2 cells, insulin did not significantly induce either ERK1/2 activation or Gene33 expression. This work suggests that the MEK–ERK, but not the phosphatidylinositol 3-kinase (PI3-K), pathway plays a direct role in insulin regulation of Gene33 transcription and protein expression.