Decreased Insulin Binding Research Articles

High-fat diet induced alterations in plasma membrane cholesterol content impairs insulin receptor binding and signalling in mouse liver but is ameliorated by atorvastatin

A high-fat diet (HFD) impairs insulin binding and signalling and may contribute to the development of insulin resistance. In addition, in vitro studies have shown that alterations in plasma membrane cholesterol influence ligand binding and downstream signalling for several receptor-tyrosine kinases (RTKs), including the insulin receptor. Using an ex vivo approach, we explored the effects of a HFD on insulin binding and signalling in mouse liver and relate these to observed changes in plasma membrane cholesterol. Mice fed a HFD demonstrated decreased insulin signalling compared to mice fed a normal chow diet (ND), indicated by a 3-fold decrease in insulin binding (P < 0.001) and a similar decrease in insulin receptor phosphorylation (~2.5-fold; P < 0.0001). Interestingly, we also observed a marked decrease in the cholesterol content of liver plasma membranes in the HFD fed mice (P < 0.0001). These effects of the HFD were found to be ameliorated by atorvastatin treatment (P < 0.0001). However, in ND mice, atorvastatin had no influence on membrane cholesterol content or insulin binding and signalling. The influence of membrane cholesterol on insulin binding and signalling was also corroborated in HepG2 cells. To the best of our knowledge, this is the first demonstration of the effects of a HFD and atorvastatin treatment on changes in plasma membrane cholesterol content and the consequent effects on insulin binding and signalling. Collectively, these findings suggest that changes in membrane cholesterol content could be an important underlying reason for the long-known effects of a HFD on the development of insulin resistance.

Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (KD: 2.3 ± 0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (KD: 2.1 ± 0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-β-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-β-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.

Insulin Receptor Levels Regulated by the Receptor- Associated Protein Progesterone Receptor Membrane Component 1 (PGRMC1)

As of 2014, 29.1 million Americans suffer from diabetes, creating a severe socioeconomic and medical burdenon society. Impaired insulin signaling is key to the development of type 2 diabetes, presenting a unique therapeutic challenge. Obese individuals demonstrate decreased insulin binding due to a reduction in IR levels, without an alteration in ligand-receptor binding affinity. The progesterone receptor membrane component 1 (PGRMC1) is an endosomal protein that promotes cellular signaling via altered receptor trafficking. A recent translational study determined that PGRMC1 was decreased in patients with insulin-resistant disease, suggesting a role in insulin signaling. In the present study, we hypothesized that PGRMC1 affects the levels of IR? (insulin receptor-? sub-unit) in adipocytes. Indeed, we show that treatment with PGRMC1 ligands significantly increase IR? protein levels in fully differentiated human subcutaneous adipocytes. Protein levels are likely affected through the direct interaction of PGRMC1 and IR?, as we demonstrate their co immunoprecipitation in differentiated 3T3-L1 cells. Notably, PGRMC1 ligand treatment significantly reduced IR? protein levels in two rodent model systems, indicating a pharmacological difference across species.

Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.

Periodic food shortages are a major challenge faced by organisms in natural habitats. Cave-dwelling animals must withstand long periods of nutrient deprivation, as-in the absence of photosynthesis-caves depend on external energy sources such as seasonal floods. Here we show that cave-adapted populations of the Mexican tetra, Astyanax mexicanus, have dysregulated blood glucose homeostasis and are insulin-resistant compared to river-adapted populations. We found that multiple cave populations carry a mutation in the insulin receptor that leads to decreased insulin binding in vitro and contributes to hyperglycaemia. Hybrid fish from surface-cave crosses carrying this mutation weigh more than non-carriers, and zebrafish genetically engineered to carry the mutation have increased body weight and insulin resistance. Higher body weight may be advantageous in caves as a strategy to cope with an infrequent food supply. In humans, the identical mutation in the insulin receptor leads to a severe form of insulin resistance and reduced lifespan. However, cavefish have a similar lifespan to surface fish and do not accumulate the advanced glycation end-products in the blood that are typically associated with the progression of diabetes-associated pathologies. Our findings suggest that diminished insulin signalling is beneficial in a nutrient-limited environment and that cavefish may have acquired compensatory mechanisms that enable them to circumvent the typical negative effects associated with failure to regulate blood glucose levels.

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata: Effects of osmotic stress

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG.

Rapid Non-Enzymatic Glycation of the Insulin Receptor under Hyperglycemic Conditions Inhibits Insulin Binding In Vitro: Implications for Insulin Resistance.

The causes of insulin resistance are not well-understood in either type 1 or type 2 diabetes. Insulin (INS) is known to undergo rapid non-enzymatic covalent conjugation to glucose or other sugars (glycation). Because the insulin receptor (IR) has INS-like regions associated with both glucose and INS binding, we hypothesize that hyperglycemic conditions may rapidly glycate the IR, chronically interfering with INS binding. IR peptides were synthesized spanning IR- associated INS-binding regions. Glycation rates of peptides under hyperglycemic conditions were followed over six days using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. INS conjugated to horse-radish peroxidase was used to determine INS binding to IR peptides in glycated and non-glycated forms. Several IR peptides were glycated up to 14% within days of exposure to 20–60 mM glucose. Rates of IR-peptide glycation were comparable to those of insulin. Glycation of four IR peptides significantly inhibits INS binding to them. Glycation of intact IR also decreases INS binding by about a third, although it was not possible to confirm the glycation sites on the intact IR. Glycation of the IR may therefore provide a mechanism by which INS resistance develops in diabetes. Demonstration of glycation of intact IR in vivo is needed.

Hepatocyte Ploidy Is a Diversity Factor for Liver Homeostasis.

Polyploidy, the existence of cells containing more than one pair of chromosomes, is a well-known feature of mammalian hepatocytes. Polyploid hepatocytes are found either as cells with a single polyploid nucleus or as multinucleated cells with diploid or even polyploid nuclei. In this study, we evaluate the degree of polyploidy in the murine liver by accounting both DNA content and number of nuclei per cell. We demonstrate that mouse hepatocytes with diploid nuclei have distinct metabolic characteristics compared to cells with polyploid nuclei. In addition to strong differential gene expression, comprising metabolic as well as signaling compounds, we found a strongly decreased insulin binding of nuclear polyploid cells. Our observations were associated with nuclear ploidy but not with total ploidy within a cell. We therefore suggest ploidy of the nuclei as an new diversity factor of hepatocytes and hypothesize that hepatocytes with polyploid nuclei may have distinct biological functions than mono-nuclear ones. This diversity is independent from the well-known heterogeneity related to the cells' position along the porto-central liver-axis.

Angiotensin II Inhibits Insulin Binding to Endothelial Cells

BackgroundInsulin-mediated glucose uptake in insulin target tissues is correlated with interstitial insulin concentration, rather than plasma insulin concentration. Therefore, insulin delivery to the interstitium of target tissues is very important, and the endothelium may also play an important role in the development of insulin resistance.MethodsAfter treating bovine aortic endothelial cells with angiotensin II (ATII), we observed the changes in insulin binding capacity and the amounts of insulin receptor (IR) on the cell membranes and in the cytosol.ResultsAfter treatment of 10-7M ATII, insulin binding was decreased progressively, up to 60% at 60 minutes (P<0.05). ATII receptor blocker (eprosartan) dose dependently improved the insulin binding capacity which was reduced by ATII (P<0.05). At 200 µM, eprosartan fully restored insulin binding capacity, althogh it resulted in only a 20% to 30% restoration at the therapeutic concentration. ATII did not affect the total amount of IR, but it did reduce the amount of IR on the plasma membrane and increased that in the cytosol.ConclusionATII decreased the insulin binding capacity of the tested cells. ATII did not affect the total amount of IR but did decrease the amount of IR on the plasma membrane. Our data indicate that ATII decreases insulin binding by translocating IR from the plasma membrane to the cytosol. The binding of insulin to IR is important for insulin-induced vasodilation and transendothelial insulin transport. Therefore, ATII may cause insulin resistance through this endothelium-based mechanism.

Insulin signaling pathways in a patient with insulin resistance of difficult management - a case report

Insulin signalling pathways were investigated in a 33 year-old woman with immunologic insulin resistance. Her past medical history was remarkable for intermittent use of insulin and allergic reactions to several drugs, and measure of plasma anti-insulin antibodies level corroborated the clinical suspicion of immune mediated insulin resistance (8074 nU/ml - RIA - Ref value: <60). Treatment with several immunosuppressive regimens was tried, however the results were disappointing. Possible subcellular mechanisms of insulin resistance were investigated by performing analysis of insulin receptor and post receptor signaling in skeletal muscle biopsy. The expression of insulin receptor (IR), insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) was evaluated in total extract from muscle tissue by Western blotting. Basal IR, IRS-1 and GLUT-4 expression was detected, however receptor autophosphorylation was not observed. A study of translocation of GLUT-4 to plasma membrane showed that tissue presented low levels of membrane-associated GLUT-4. When in vitro stimulation was undertaken, tissue was capable to be responsive to insulin. Our results suggest that even though IR expression was normally occurring, IR β-subunit tyrosine kinase activity in muscle was down-regulated leading to alterations in insulin post receptor signaling. Consistent with normal insulin receptor and post receptor signaling, our results were compatible with decreased insulin binding to IR probably due to neutralization by anti-insulin antibodies. In conclusion, this patient has immunologic insulin resistance and treatment should be based on immunosuppressive drugs as tolerated.

Insulin binding to erythrocyte receptors in acromegalic patients in relation to the activity of acromegaly and to concomitant diabetes mellitus.

Insulin binding to receptors on erythrocytes was studied in patients with acromegaly (n = 27) and in control subjects without any endocrine pathology, diabetes or obesity (n = 13). According to fasting serum concentration of growth hormone (GH), acromegalics were divided into two groups: A) GH less than 10 ng/ml (n = 16) and B) GH above 20 ng/ml (n = 11), in which patients were further divided into subgroups with regard to the presence or absence of diabetes. Insulin binding was decreased both in active and inactive acromegalics when compared with controls. A greater decrease was seen in active acromegaly coupled with diabetes. This was not the case of inactive hyperglycaemic acromegalics, where a compensatory increase in the affinity of "empty" receptors might account for a lack of a greater decrease in insulin binding.

P-26: Substitution of Arg 252 for cysteine in the insulin receptor leads to a conformational change of the α-subunit resulting in a decreased insulin binding

P-26: Substitution of Arg 252 for cysteine in the insulin receptor leads to a conformational change of the α-subunit resulting in a decreased insulin binding

Glucose binds to the insulin receptor affecting the mutual affinity of insulin and its receptor.

Insulin activity is sensitive to glucose concentration but the mechanisms are still unclear. An unexamined possibility is that the insulin receptor (IR) is sensitive to glucose concentration. We demonstrate here that insulin-like peptides derived from the IR bind glucose at low millimolar, and cytochalasin B at low micromolar, concentrations; several insulin-like IR peptides bind insulin at nanomolar Kd; and this binding is antagonized by increasing glucose concentrations. In addition, glucose and cytochalasin B bind to IR isolated from rat liver and increasing glucose decreases insulin binding to this IR preparation. The presence of GLUT 1 in our IR preparation suggests the possibility of additional glucose-mediated allosteric control. We propose a model in which glucose binds to insulin, the IR, and GLUT; insulin binds to the IR; and the IR binds to GLUT. This set of interactions produces an integrated system of insulin-dependent interactions that is highly sensitive to glucose concentration.

Role of the insulin receptor in IGF-system signaling and biology.

Abstract Abstract #4055 The role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in breast cancer has been extensively studied, and has led to the development of several IGF1R inhibitors currently in clinical trials. The closely related insulin receptor (IR) has been shown to contribute to IGF signaling through the formation of IGF1R/IR-hybrid receptors and through a homodimer of an alternatively spliced form of the IR (IR-A). Understanding the role of the IR in cancer biology will help determine which receptor(s) in the IGF system should be targeted to efficiently inhibit IGF signaling. Our previous work has shown that selective downregulation of IGF1R by siRNA resulted in enhanced sensitivity to insulin through increasing the numbers of insulin receptor homodimers. To determine how downregulation of IR affected IGF1R signaling, we used T47D cells, a breast cancer cell line expressing both the IR and IGF1R, as our model system. We transiently downregulated IR levels using siRNA and evaluated signaling. 5 nM of insulin activated downstream signaling molecules, such as insulin receptor substrate 1(IRS-1) and Akt in control cells, but this response was diminished in the IR-downregulated cells. In contrast, 5 nM of IGF-I did not result in differences in signaling between IR-downregulated cells and control cells. Using IR shRNA, we created stable clones with IR levels reduced by more than 72%. IR shRNA clones demonstrated decreased insulin binding, as measured by 125I-insulin binding assay. Additionally, clones showed decreased response to insulin signaling. 5 nM of insulin activated IRS-1 and Akt in parental T47D cells, but this response was attenuated in the IR-downregulated clones. Proliferation was also decreased in response to 5 nM insulin in the clones compared to parental cells. In contrast, IGF-I mediated signaling was not altered in clones when compared to vector control cells. Further, no change in proliferation was observed in response to IGF-I between wild-type and IR shRNA clones. We also generated stable IR shRNA clones using LCC6 cells. Insulin signaling was reduced in LCC6 clones when compared to wild-type LCC6 cells. However, in contrast to the T47D clones, IGF-I signaling was increased in LCC6 IR shRNA clones. Thus, the role of the insulin receptor in cancer biology may be dependent upon the ratio of IR, IGF1R, and the hybrid receptor. If cells contain mostly homodimers of IGF1R and IR, then downregulation of IR may affect only insulin response. However, if there are high levels of hybrid receptor, then downregulation of IR could affect, and even augment, IGF1R signaling. Our data suggest that the ratio and confirmation states of both IR and IGF1R should be understood to predict the effect of receptor downregulation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4055.

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.

Prolonged Treatment of Primary Hepatocytes with Oleate Induces Insulin Resistance through p38 Mitogen-activated Protein Kinase

Free fatty acid (FFA) is believed to be a major environmental factor linking obesity to Type II diabetes. We have recently reported that FFA can induce gluconeogenesis in hepatocytes through p38 mitogen-activated protein kinase (p38). In this study, we have investigated the role of p38 in oleate-induced hepatic insulin resistance. Our results show that a prolonged treatment of primary hepatocytes with oleate blunted insulin suppression of hepatic gluconeogenesis, and decreased insulin-induced phosphorylation of Akt in a p38-dependent manner. Reduction of the insulin-induced Akt phosphorylation by oleate correlated with activation of p38. In the presence of p38 inhibition, prolonged exposure of hepatocytes to oleate failed to reduce insulin-stimulated phosphorylation of Akt. An siRNA against p38alpha prevented oleate suppression of the insulin-induced phosphorylation of Akt. Furthermore, a prolonged exposure of hepatocytes to oleate decreased insulin-induced tyrosine phosphorylation of IRS1/2, while slightly increasing serine phosphorylation of IRS. The decrease of insulin-stimulated tyrosine phosphorylation of IRS1/2 in hepatocytes by oleate was reversed by the inhibition of p38. We further show that a prolonged exposure of primary hepatocytes to oleate elevated the protein level of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in a p38-dependent manner, but had no effect on the mRNA level of PTEN. Knocking down the PTEN gene prevented oleate to inhibit insulin activation of Akt and insulin suppression of gluconeogenesis. Together, results from this study demonstrate a critical role for p38 in oleate-induced hepatic insulin resistance.

Effect of N-benzoyl-d-phenylalanine and metformin on insulin receptors in neonatal streptozotocin-induced diabetic rats: Studies on insulin binding to erythrocytes

In the present study, we focused on the insulin-receptor binding in circulating erythrocytes of N-benzoyl-d-phenylalanine (NBDP) and metformin in neonatal streptozotocin (nSTZ)-induced male Wistar rats. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors in NBDP and metformin-treated diabetic rats. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (53.0 ± 3.1%) than in NBDP (62.0 ± 3.1%), metformin (66.0 ± 3.3%) and NBDP and metformin combination-treated (72.0 ± 4.2%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with NBDP and metformin-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from NBDP and metformin-treated diabetic rats having NBDP 2.0 ± 0.10×10−10 M−1 (Kd1); 12.0 ± 0.85×10−8 M−1 (Kd2), Metformin 2.1 ± 0.15×10−10 M−1 (Kd1); 15.0 ± 0.80×10−8 M−1 (Kd2), NBDP and metformin 2.7 ± 0.10×10−10 M−1 (Kd1); 20.0 ± 1.2×10−8 M−1 (Kd2) compared with 0.9 ± 0.06×10−10 M−1 (Kd1); 6.0 ± 0.30×10−8 M−1 (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in nSTZ induced diabetic control rats. Treatment with NBDP along with metformin significantly improved specific insulin binding, with receptor number and affinity binding reaching almost normal non-diabetic levels. The data presented here show that NBDP along with metformin increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.

Alterations in insulin receptor signalling in the rat epitrochlearis muscle upon cessation of voluntary exercise

The major purpose of this study was to elucidate mechanisms by which decreasing enhanced physical activity induces decreased insulin sensitivity in skeletal muscle. Rats with access to voluntary running wheels for 3 weeks had their wheels locked for 5 h (WL5), 29 h (WL29), or 53 h (WL53); a separate group of rats never had wheel access (sedentary, SED). Relative to WL5, submaximal insulin-stimulated 2-deoxyglucose uptake into the epitrochlearis muscle was lower in WL53 and SED. Insulin binding, insulin receptor beta-subunit (IRbeta) protein level, submaximal insulin-stimulated IRbeta tyrosine phosphorylation, and glucose transporter-4 protein level were each lower in both WL53 and SED than in WL5 and WL29. Akt/protein kinase B Ser(473) phosphorylation was lower in WL53 and SED than in WL5. Protein levels of protein tyrosine phosphatase-1B, Src homology phosphatase-2, and protein kinase C- did not vary among groups. The amount of protein tyrosine phosphatase-1B, Src homology phosphatase-2, and protein kinase C- associated with IRbeta in insulin-stimulated muscle also did not differ among the four groups. The mean of SED and WL53 had a significantly higher IRbeta-associated protein tyrosine phosphatase-1B than the mean of WL5 and WL29. The enclosure of multiple changes (decreases in insulin binding, IRbeta protein, IRbeta tyrosine phosphorylation, and glucose transporter-4 protein) in the epitrochlearis muscle within the 29th to 53rd hour after cessation of voluntary wheel running raises the possibility that a single regulatory event could be responsible for the coordinated decrease.

The effect of nitric oxide on glucose metabolism.

Nitric oxide (NO) is an important bioactive signaling molecule that mediates a variety of normal physiological functions, which, if altered, could contribute to the genesis of many pathological conditions, including diabetes. In this study, we examined the possible diabetogenicity of NO by noting differences in the cellular binding of insulin in dogs treated with the NO donor, S-nitrosoglutathione (GSNO) compared to captopril-treated controls. GSNO administration resulted in an abnormality in glucose metabolism which was attributed to decreased binding of insulin to its receptor on the cell membrane of mononuclear leucocytes, 11.60 ± 0.60% in GSNO-treated dogs compared with 18.10 ± 1.90% in captopril-treated control (p < 0.05). The decreased insulin binding was attributed to decreased insulin receptor sites per cell, 21.43 ± 2.51 × 10(4) in GSNO-treated dogs compared with 26.60 ± 1.57 × 10(4) in captopril-treated controls (p < 0.05). Average affinity analysis of the binding data demonstrated that this decrease in insulin binding was also due to a decrease in average affinity of the receptor on mononuclear leucocytes for insulin. This was evident by a decrease in empty and filled site affinities in GSNO-treated dogs compared with that of captopril-treated dogs (p < 0.05). It appears that GSNO is exerting its effect by decreasing the number of insulin receptor sites and/or decreasing the average receptor affinity. These results provide evidence for a novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus. (Mol Cell Biochem 263: 29-34, 2004).

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested.

The effects of protease inhibitors on basal and insulin-stimulated lipid metabolism, insulin binding, and signaling

The objective of our research was to investigate the effects of the protease inhibitors ritonavir, saquinavir, and indinavir on triglyceride synthesis, lipolysis, insulin binding, and signaling in differentiating 3T3 L1 pre-adipocytes. Saquinavir, ritonavir, and indinavir all stimulated triglyceride (TG) synthesis. Additionally, all concentrations of protease inhibitors employed (i.e., 0.1 micro M to 10 micro M) significantly decreased insulin-stimulated TG synthesis. No effects of any of the protease inhibitors were observed either on basal lipolysis or after stimulation of lipolysis with 100 nM noradrenaline. Specific (125)I-insulin binding was observed to be decreased by exposure to all the protease inhibitors throughout the period of adipocyte phenotype development. This was mediated by indinavir through a receptor decrease and had no effect on receptor affinity. During differentiation with ritonavir (i.e., 1-11 days post addition of differentiating cocktail), insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation was ascertained (day 11) and found to be decreased in the ritonavir exposed cells when compared with control cells. The results reported herein demonstrate protease inhibitor effects on basal TG synthesis while exhibiting decreased insulin-stimulated TG synthesis at physiological concentrations of protease inhibitors. These effects may be subsequent to decreased insulin binding and/or IRS-1 tyrosine phosphorylation.