Postreceptor Insulin Resistance Research Articles

FRI114 PUFAs Implicated In Platelet Activation Differ In Receptor Mediated Vs Post-receptor Insulin Resistance

Abstract Disclosure: R. Klein: None. S.T. Chung: None. R.J. Brown: None. Background: Severe insulin resistance (IR) can be classified as receptor mediated, in which all downstream signaling is blocked, or post receptor IR where some downstream signaling pathways are decreased and others are increased. Receptor-mediated IR occurs in rare mutations of the insulin receptor (INSR), whereas in most forms of IR, including type 2 diabetes (T2DM) and lipodystrophy (LD), IR is post-receptor. LD is a group of syndromes characterized by deficiency of fat depots leading to metabolic dysfunction similar to obesity. Patients with post-receptor IR have increased atherosclerotic cardiovascular disease (ASCVD) risk. Thrombosis underlies pathology of ASCVD and is a result of platelet (PLT) activation. Polyunsaturated fatty acids (PUFAs) are embedded in the PLT membrane and play a key role in thrombosis. During PLT activation, cytosolic phospholipase A2 (cPLA2) hydrolyzes membrane lipids, generating free PUFAs, leading to synthesis of mediators of blood vessel constriction, inflammation, and thrombosis. Methods: This was a cross-sectional study of subjects with INSR (N=30) and LD (N=30) matched for age, sex, HbA1c and pubertal stage. Untargeted metabolomic (848 compounds) and lipidomic (984 compounds) analysis was completed on serum after an 8-12 hour fast. Analyses were conducted by t-test and Mann-Whitney with adjustment for multiplicity. Bonferroni corrected P<0.05 was considered significant. Results: Of 19 long chain PUFAs (ω=3, ω=6) measured, 14 were significantly elevated in LD vs INSR, including arachidonate, docosahexaenoate, docosapentaenoate, stearidonate, and adrenate. There was no difference in linoleate, dihomo-linoleate, tetradecadienoate, linolenate, or docosadienoate levels between LD vs INSR. Among 8 long chain saturated FAs, only 3 were significantly higher in LD, 4 were comparable, and 1 was higher in INSR. All long and medium chain monounsaturated FAs were similar in LD vs INSR. cPLA2 was not different between LD and INSR. Conclusions: Our study shows that liberated PUFAs were higher in the patients with LD compared to INSR. A similar trend was not seen for other FFA such as saturated and monounsaturated FFA, suggesting that higher PUFAs in LD are unlikely to be caused by global differences in FFA metabolism, such as different peripheral uptake of FFA via lipoprotein lipase activity or diet. Instead, our data suggest an increase in PLT membrane ability to store and release PUFAs. These data suggest increased ASCVD risk in post-receptor insulin resistance may be partially mediated through differences in platelet activation. Presentation: Friday, June 16, 2023

FRI117 Major Adverse Cardiovascular Events In Severe Insulin Resistance Syndromes: Lipodystrophy Compared To Pathogenic Variants Of The Insulin Receptor

Abstract Disclosure: M. Lightbourne: None. R.J. Brown: None. Patients with insulin resistance (IR) associated with obesity and/or type 2 diabetes have increased atherosclerotic cardiovascular disease (ASCVD) risk. Rare, syndromic forms of severe IR (SIR) exist, in which ASCVD risk is not established. SIR can be classified into post-receptor IR, where insulin signaling is increased in some signaling pathways, and decreased in others (i.e. lipodystrophy) or receptor IR, where all insulin signaling is blocked at the insulin receptor (i.e. insulin receptor pathogenic variants, INSR). These differences in insulin signaling associate with differences in ASCVD risk factors, i.e. dyslipidemia and uric acid (high in lipodystrophy, low in INSR), but the incidence of ASCVD in these population is unknown. We hypothesized that patients with post-receptor IR due to lipodystrophy would have more major adverse cardiovascular events (MACE) vs those with receptor IR due to INSR. This is a retrospective chart review of patients with lipodystrophy and INSR aged ≥18 years. Our primary outcome was incidence of MACE (composite of cardiovascular death, non-fatal myocardial infarction, ischemic stroke and hospitalization for unstable angina, heart failure or revascularization). Patients with INSR (n=24) were younger than those with lipodystrophy (n=241) (32 ± 12 vs 40 ± 15 years, p=0.02) at first MACE or last follow-up. 62% of patients with INSR were female vs 82% with lipodystrophy (p=0.02); 50% with INSR were white vs 75% with lipodystrophy (p=0.0098). Of patients with lipodystrophy, 153 had partial, 79 had generalized, and 9 had progeroid forms of lipodystrophy. Nine patients with INSR had homozygous or compound heterozygous variants and 15 had heterozygous variants. MACE occurred in 33 patients with lipodystrophy (5 generalized, 24 partial, 4 progeroid; age 42.6 ± 12.7 years) and in 0 patients with INSR with a Hazard Ratio of 2.9 (95% CI 0.6-14.5, P=0.2). In patients with lipodystrophy, 9 patients had cardiovascular deaths (mean age 32.4 ± 15.4 years, 3 with progeroid syndromes); 11 had non-fatal myocardial infarcts with 2 patients having recurrent events; 8 were hospitalized for strokes or transient ischemic attacks; 12 were hospitalized for coronary revascularization with 7 patients having recurrent events; 5 were hospitalized due to heart failure. In addition to MACE, 4 patients with lipodystrophy had peripheral revascularization or amputation vs 0 with INSR. MACE events did not occur in patients with INSR and were highly prevalent at a young age in patients with lipodystrophy, suggesting higher risk of MACE in lipodystrophy. There was no statistical difference for MACE events between the 2 groups; however, statistical power was limited by the small sample size and young age of patients INSR. These data suggest that contributions from both increased and decreased insulin signal transduction in patients with post-receptor IR may lead to increased ASCVD risk. Presentation: Friday, June 16, 2023

SAT090 Serum Ketone Levels Are Disproportionate To Beta-Oxidation In Insulin Receptor Mutation Compared Vs Lipodystrophy Patients

Abstract Disclosure: R. Klein: None. S.T. Chung: None. R.J. Brown: None. Introduction: Ketogenic substrates mainly derive from fatty acid (FA) metabolism via hepatic β-oxidation (βOx). βOx catabolizes FAs to acetyl-CoA, which can make ATP via the TCA cycle or ketones. Ketones are cleared peripherally by monocarboxylate transporters (MCT). Insulin is a major regulator of FA metabolism and ketogenesis. Insulin resistance (IR) is classified as receptor mediated, in which all downstream signaling is blocked (mutations of the insulin receptor, INSR), or post receptor IR where some downstream signaling pathways are decreased and others are increased (i.e. obesity and lipodystrophy, LD). Prior data showed high FA flux in INSR and LD; however, only patients with LD develop hepatosteatosis. We hypothesized that FAs in INSR are selectively directed toward βOx, and not re-esterified to triglyceride, to explain absence of hepatosteatosis. Here we assess pathways leading to βOx and ketogenesis between the two groups. Background: This was a cross-sectional study of subjects with INSR (N=30) and LD (N=30) matched for age, sex, HbA1c and pubertal stage. Untargeted lipidomic and proteomic analysis was completed on serum after an 8-12 hour fast. Analyses were conducted by t-test and Mann-Whitney. P<0.05 was considered significant without adjustment for multiplicity in this hypothesis generating study. Results: Insulin was 400% higher in INSR vs LD; glucose was comparable. 36 acyl-carnitines were measured. In general, acyl-carnitines were comparable with 3% higher in INSR and 28% higher in LD. Ketoacids leucine and lysine were 126% and 114% higher in LD vs INSR. Analysis of glycolytic intermediates showed no difference in pyruvate levels, but an 9% higher hexokinase-1 (a rate limiting glycolytic enzyme) in LD. Of 10 TCA cycle intermediates, 20% were elevated in LD vs INSR, the remaining 80% were similar. Despite evidence of comparable to higher βOx intermediates in LD, the ketones acetoacetate and 3-hydroxybutyrate were 600% and 740% higher in INSR vs LD. Both monocarboxylate transporters measured, sodium-coupled MCT-1 (SCMCT-1) and MCT-4, were higher in LD vs INSR (by 34% and 1%). Conclusions: Formation of acyl-carnitines from FA is the first step in hepatic βOx, and acylcarnitine levels are typically proportional to ketones. We observed higher ketones in INSR vs LD despite evidence of comparable βOx, including acyl-carnitines and glycolytic and TCA intermediates. This suggests that elevated ketones in INSR may be due to lower clearance via monocarboxylate transporters rather than increased formation via βOx. Concentration of MCT-1, the primary transporter of ketones into muscle, was not available. However, MCT-4 and SCMCT-1 which reside in the brain and kidneys, respectively, were lower in INSR, perhaps leading to decreased ketone utilization. We hypothesize that MCTs may be negatively regulated by insulin, via a mechanism not mediated by the insulin receptor. Presentation: Saturday, June 17, 2023

190-OR: Lrtm1, a Novel Regulator of Insulin Signaling and Metabolism

Patients with obesity and metabolic syndrome, as well as individuals with pre-type 2 diabetes, exhibit hyperinsulinemia prior to development of hyperglycemia due to underlying skeletal muscle insulin resistance. To mimic the effect of chronic hyperstimulation of the insulin receptor (IR), we overexpressed the IR specifically in skeletal muscle (IRMOE). This led to skeletal muscle post-receptor desensitization, with increased IR and IRS-1(Tyr) phosphorylation but decreased phosphorylation of Akt Ser473 and Thr308. To identify the regulators involved in this post-receptor insulin resistance, we performed RNA sequencing of IRMOE muscle. Among the most upregulated genes was Lrtm1, a poorly characterized leucine rich repeat transmembrane molecule while highly expressed in the heart and skeletal muscle. Overexpression of Lrtm1 in C2C12 myoblasts led to increased phosphorylation of early insulin signaling molecules (the insulin receptor, IRS-1, and Shc), but reduced phosphorylation of the downstream molecules Akt (at both Ser473 and Thr308) and Erk Thr202/204 following insulin stimulation. Mice with knockout of Lrtm1 (Lrtm1KO) showed significant increases in percent lean mass and decreases in percent fat mass, as well as a slight reduction in overall body weight. In addition, Lrtm1KO mice were more active than controls in both metabolic cages and an open field behavioral tests. Lrtm1KO mice also exhibited improved glucose tolerance and insulin sensitivity, especially in males, even on normal chow diet. Most importantly, Lrtm1KO mice were protected from high fat diet induced obesity and metabolic syndrome, with the knockouts showing 18% less body weight gain, 30% lower fat mass, significantly improved glucose tolerance and improved insulin sensitivity. Thus, Lrtm1 is a novel regulator of post-receptor insulin signaling and glucose metabolism that may provide a new target for therapy insulin resistant prediabetes and type 2 diabetes. Disclosure G. Wang: Employee; Agilent. Y. Yu: None. W. Chen: None. W. Cai: None. X. Liu: None. C. Kahn: None.

Enhanced Insulin Signaling Through Its Receptor Is Needed for the Development of Hyperuricemia in Humans

Background: There is an association between insulin resistance (IR) and hyperuricemia but the direction of causalityand mechanisms are unclear. In obesity and lipodystrophy, IR is “selective”, with lack of insulin signalingcausing hyperglycemia, but enhanced insulin signaling causing dyslipidemia and possibly hyperuricemia. Rare human conditions exist in which there is extreme, non-selective, IR impairing all insulin signalingpathways (e.g. mutations of the insulin receptor, INSR). We hypothesized that hyperinsulinemia withenhanced signaling through the insulin receptor causes hyperuricemia in selective IR, whereas lack ofinsulin signaling through its receptor despite hyperinsulinemia results in normal uricemia in non-selective IR. Method: Cross-sectional retrospective analysis comparing fasting insulin and serum uric acid in patients withsevere IR that was either selective (due to lipodystrophy) or non-selective (due to INSR mutation orautoantibody to the insulin receptor). Visits were chosen based on availability of serum uric acid andinsulin. If multiple visits were available, the visit with the highest insulin value was chosen. Results: We analyzed data of 68 patients with selective IR and 14 patients with non-selective IR. Fasting seruminsulin median [interquartile range] was 23.4 [14, 53] mcU/mL in selective IR vs 215 [73.7, 604] in non-selective IR, p <0.0001. Serum uric acid was 4.8 [2, 6] mg/dL in selective IR vs 3.5 [2, 4] in non-selectiveIR, p=0.0003. Conclusion: Patients with selective IR due to lipodystrophy had elevated serum uric acid, consistent with thehyperuricemia seen in selective IR caused by obesity. By contrast, patients with non-selective IR hadlower serum uric acid despite more severe IR as evidenced by almost 10-fold higher fasting insulin. These observations are consistent with a model in which enhanced insulin signaling through its receptorleads to hyperuricemia in post-receptor IR. Further studies are needed to understand the mechanism bywhich insulin leads to hyperuricemia.

Muscle-Specific Insulin Receptor Overexpression Protects Mice From Diet-Induced Glucose Intolerance but Leads to Postreceptor Insulin Resistance.

Skeletal muscle insulin resistance is a prominent early feature in the pathogenesis of type 2 diabetes. In attempt to overcome this defect, we generated mice overexpressing insulin receptors (IR) specifically in skeletal muscle (IRMOE). On normal chow, IRMOE mice have body weight similar to that of controls but an increase in lean mass and glycolytic muscle fibers and reduced fat mass. IRMOE mice also show higher basal phosphorylation of IR, IRS-1, and Akt in muscle and improved glucose tolerance compared with controls. When challenged with high-fat diet (HFD), IRMOE mice are protected from diet-induced obesity. This is associated with reduced inflammation in fat and liver, improved glucose tolerance, and improved systemic insulin sensitivity. Surprisingly, however, in both chow and HFD-fed mice, insulin-stimulated Akt phosphorylation is significantly reduced in muscle of IRMOE mice, indicating postreceptor insulin resistance. RNA sequencing reveals downregulation of several postreceptor signaling proteins that contribute to this resistance. Thus, enhancing early insulin signaling in muscle by overexpression of the IR protects mice from diet-induced obesity and its effects on glucose metabolism. However, chronic overstimulation of this pathway leads to postreceptor desensitization, indicating the critical balance between normal signaling and hyperstimulation of the insulin signaling pathway.

1785-P: Mice with Muscle-Specific Insulin Receptor Overexpression Are Protected from Diet-Induced Obesity and Glucose Intolerance but Develop Post-Receptor Insulin Resistance

Skeletal muscle insulin resistance is a prominent feature of type 2 diabetes (T2D) that precedes and predicts the development of disease in high risk populations. In individuals with T2D or pre-T2D, this insulin resistance is due, at least in part, to a decrease in the number of functional insulin receptors (IR) that localize on the cell membrane. To restore the number of IR, we generated mice that specifically overexpress IR in skeletal muscle (IRMOE). On chow diet, IRMOE mice had similar body weight compared to control, but displayed reduced body fat and higher lean mass percentage. Basal phosphorylation of IR, IRS and Akt were significantly higher in muscles from IRMOE mice compared to control, resulting in their improved glucose tolerance. Surprisingly, however, after i.v. injection of insulin, stimulated Akt phosphorylation was significantly lower in IRMOE muscle compared to control, despite persistent higher phosphorylation in IR and IRS, indicating activation of negative regulators in the IR signaling pathway when IR is chronically activated. Consistent with this, euglycemic-hyperinsulinemic clamp studies revealed significantly lower glucose infusion rate in insulin stimulated IRMOE mice compared to control. Increased basal and decreased insulin stimulated Akt phosphorylation was also observed in muscles of IRMOE mice after challenge with HFD. However, when fed HFD, IRMOE mice displayed both improved glucose tolerance and insulin tolerance which was in part due to being resistant to diet-induced obesity, despite consuming more calories. Taken together, these data indicate that muscle specific IR overexpression can protect mice from diet-induced obesity and its effects on glucose metabolism, but that chronic overexpression of IR can lead to post-receptor desensitization indicating additional post-receptor regulators of insulin signaling, which could provide new targets for therapy of T2D. Disclosure G. Wang: None. W. Cai: None. T.M. Batista: None. S. Softic: None. C. Kahn: Advisory Panel; Self; MedImmune. Board Member; Self; Kaleido Biosciences. Consultant; Self; AntriaBio, Inc., Cobalt Therapeutics, Flagship Pioneering. Research Support; Self; Alnylam. Funding American Diabetes Association (9-17-CMF-016 to G.W.)

MON-159 Increased Gluconeogenesis and Lower Glycogenolysis in Patients with Non-Selective Compared Selective Insulin Resistance

Type 2 diabetes is characterized by increased hepatic glucose production and selective insulin resistance that occurs downstream of the insulin receptor. However, the differential pathways and exact molecules implicated in selective insulin signaling are unclear. To elucidate the hepatic mechanistic pathways affected by selective versus non-selective insulin resistance, we compared basal rates of hepatic glucose production and its components, gluconeogenesis and glycogenolysis, in patients with severe non-selective insulin resistance (insulin receptor mutations (INSR)) to patients with selective insulin resistance (lipodystrophy and type 2 diabetes). In 7 patients with INSR, 14 with lipodystrophy and 9 with type 2 diabetes, we measured rates of hepatic glucose production (via [6,6-2H2] glucose) and fractional gluconeogenesis (via 2H2O) after an overnight fast. Absolute rates of gluconeogenesis were calculated as the product of fractional gluconeogenesis and hepatic glucose production, and glycogenolysis were calculated as hepatic glucose production minus gluconeogenesis. In patients with INSR, lipodystrophy and type 2 diabetes, HbA1c (8.2 ± 2.8, 8.4 ± 1.9, 7.4 ± 0.9%) and fasting glucose (120 ± 70, 159 ± 55, 127 ± 32 mg/dl) were comparable (P>0.05). Fasting serum insulin was higher in INSR vs. lipodystrophy and type 2 diabetes (332 ± 355, 48 ± 50, 50 ± 16 μU/ml; P<0.01). Patients with INSR and lipodystrophy had higher hepatic glucose production vs. type 2 diabetes (24.0 ± 4.0, 24.1 ± 6.2, 15.8 ± 4.3 µmol/kgLEAN/min; P<0.01). Fractional gluconeogenesis was higher in INSR vs. lipodystrophy and type 2 diabetes (77.1 ± 9.4, 55.4 ± 11.5, 59.1 ± 5.9%; P<0.01). Absolute gluconeogenesis was highest in INSR, intermediate in lipodystrophy, and lowest in type 2 diabetes (19.1 ± 4.4, 13.5 ± 4.2, 9.5 ± 1.9 µmol/kgLEAN/min; P<0.01). Glycogenolysis was lowest in INSR, highest in lipodystrophy, and intermediate in type 2 diabetes (4.9 ± 2.2 vs. 10.6 ± 4.8, 6.3 ± 2.6 µmol/kgLEAN/min; P<0.01). Higher hepatic glucose production in lipodystrophy vs. type 2 diabetes is consistent with higher hepatic insulin resistance in patients with lipodystrophy, but comparable fractional rates of gluconeogenesis and glycogenolysis confirm similar mechanisms of selective post-receptor insulin resistance. By contrast, the markedly higher contribution of gluconeogenesis to hepatic glucose production in INSR may reflect complete loss of insulin signaling for glycogen accumulation and/or suppression of gluconeogenesis. Further studies should evaluate whether greater substrate and fuel availability for gluconeogenesis, and/or lower hepatic glycogen stores are important determinants of the components of glucose production in patients with non-selective insulin resistance due to INSR mutation.

21 Sensitivity of Comparing Serum Triglycerides Levels at “Fasting” and “60 Minutes After Oral Glucose Loading” to Screen and Estimate Insulin Resistance

Insulin function on cellular uptake of plasma triglycerides is affected by insulin potency (proinsulin percentage) and insulin resistances (prereceptor, receptor, and postreceptor insulin resistance types), while its function on plasma free fatty acids re-esterification into triglycerides is not affected by receptor or postreceptor resistance. Glucose loading in oral glucose tolerance test should lead to (1) suppression of cellular “hepatic” utilization of fatty acids, (2) suppression of further stimulation of lipolytic and hyper-glycemic hormones (such as glucagon, adrenaline, ACTH and cortisol, GH, TSH and FT3), and (3) stimulation of insulin secretion, which enhances re-esterification of plasma free fatty acids into triglycerides and induces cellular uptake of plasma triglycerides. Therefore, during oral glucose tolerance test, measuring the “fasting” serum triglycerides and “after 60 minutes” serum triglycerides levels will provide two snapshots of the circulatory triglycerides levels in one hour, and because there is no exogenous intake of lipids and there are almost no endogenous catabolic effects, the comparison between the two triglycerides levels will uncover any lack of concomitance of the rate of triglycerides formation with the rate of its uptake. In metabolic syndrome or diabetes mellitus type 2, where the insulin resistance is significant, the “after 60 minutes” serum triglycerides levels are increased compared to the basal fasting levels, meaning that excess circulatory triglycerides formation exceeds the excess triglycerides uptake by cells (hepatic and adipose tissues). While in normal patients or in diabetes mellitus type 1, where the insulin resistance is not significant, there are no remarkable dissimilarities between the triglycerides levels. Comparison of serum triglycerides levels of “fasting” and “after 60 minutes” in oral glucose tolerance test, is sensitive for screening for insulin resistance (especially receptor or postreceptor resistances); however, disturbances in lipolytic hormones (deficiency or excess) or somatostatin could affect the specificity.

Severe Insulin Resistance and Hypertriglyceridemia After Childhood Total Body Irradiation

To characterize the metabolic phenotype of 2 cases of normal weight young women who developed type 2 diabetes (T2D), severe insulin resistance (insulin requirement >200 units/day), marked hypertriglyceridemia (>2000 mg/dL), and hepatic steatosis beginning 9 years after undergoing total body irradiation (TBI) and bone marrow transplantation for childhood cancer. Fasting plasma glucose, insulin, free fatty acids (FFAs), leptin, adiponectin, resistin, TNFα, and IL-6 were measured in each case and in 8 healthy women; Case 1 was also assessed after initiating pioglitazone. Coding regions and splice junctions of PPARG, LMNA, and AKT2 were sequenced in Case 1 and of PPARG in Case 2 to evaluate for familial partial lipodystrophies. Genotyping of APOE was performed in Case 1 to rule out type III hyperlipoproteinemia. Both cases had elevated plasma levels of insulin, leptin, resistin, and IL-6, high-normal to elevated TNFα, and low to low-normal adiponectin in keeping with post-receptor insulin resistance and adipose tissue inflammation. Case 1 experienced a biochemical response to pioglitazone. No causative mutations for partial lipodystrophies or type III hyperlipoproteinemia were identified. Though metabolic derangements have previously been reported in association with TBI, few cases have described insulin resistance and hypertriglyceridemia as severe as that seen in our patients. We speculate that early childhood TBI may impede adipose tissue development leading to metabolic complications from an attenuated ability of adipose tissue to accommodate caloric excess, and propose that this extreme metabolic syndrome be evaluated for as a late complication of TBI.

A woman with severe lupus nephritis and difficult to control diabetes mellitus

A woman with severe lupus nephritis and difficult to control diabetes mellitus

What have we learned form monogenic forms of severe insulin resistance associated with PCOS/HAIRAN?

Whatever the origin of severe insulin resistance (primary insulin receptors defects or lipodystrophies), in vivo hyperinsulinemia has been clearly shown to promote ovarian growth and androgen synthesis independently of gonadotropins. In lipodystrophic syndromes, the endocrine deficiency of adipose tissue has been shown to play important pathophysiological roles in metabolic alterations. In particular leptin is decreased, contributing to the ectopic lipid storage in non-adipose cells, which inhibits insulin signalling (lipotoxicity). Finally, polycystic ovary syndrome (PCOS) features are not always present in insulin resistance syndromes with lipodystrophy. This is in favour of an aggravating, but not a primary role of post-receptor insulin resistance on ovary dysfunctions.

Serum and intracellular magnesium deficiency in patients with metabolic syndrome—Evidences for its relation to insulin resistance

This cross sectional study evaluated serum (SMg) and intramononuclear (MMg) magnesium in patients with metabolic syndrome without diabetes and correlated them with cardiovascular risk factors. 72 patients and 57 controls (blood donors) were studied. Hypomagnesemia (SMg < 1.7 mg/dL) was seen in 23.2% and intracellular depletion in 36.1% of the patients. SMg and MMg means were significantly lower in patients than in controls: 1.80 ± 0.18 mg/dL vs. 2.43 ± 0.43 mg/dL and 0.98 ± 0.55 μg/mg vs. 1.67 ± 0.64 μg/mg of protein ( P < 0.001). Inverse correlation was observed between, SMg and MMg with BMI; SMg with systolic blood pressure and waist circumference in women. Patients with acanthosis nigricans had lower SMg (1.75 ± 0.18 mg/dL vs. 1.85± 0.18 mg/dL, P < 0.05). Non-white people had lower SMg (1.78 ± 0.16 mg/dL vs. 1.92 ± 0.24 mg/dL, P = 0.007) and MMg (0.95 ± 0.59 μg/mg vs. 1.13 ± 0.42 μg/mg, P = 0.03). Patients with IR showed lower MgM means (0.84 ± 0.33 μg/mg vs. 1.14 ± 0.69 μg/mg, P < 0.05). The same occurred in patients with low HDL-c levels (0.92 ± 0.46 μg/mg vs. 1.20 ± 0.70 μg/mg, P = 0.03), and those with moderate and severe hepatic steatosis (0.77 ± 0.29 μg/mg vs. 1.21 ± 0.80 μg/mg, P < 0.05). In conclusion, magnesium depletion in serum and mononuclear cells is common in obese people with metabolic syndrome, and it is more evident in non-white people with insulin resistance. This depletion may contribute to a post-receptor insulin resistance.

Knock-down of LAR protein tyrosine phosphatase induces insulin resistance

To test the role of the leukocyte common antigen-related protein tyrosine phosphatase (LAR) as a regulator of insulin receptor (IR) signalling, an siRNA probe against LAR was developed. Knock-down of LAR induced post-receptor insulin resistance with the insulin-induced activation of PKB/Akt and MAP kinases markedly inhibited. The phosphorylation and dephosphorylation of the IR and insulin receptor substrate (IRS) proteins were unaffected by LAR knock-down. These results identify LAR as a crucial regulator of the sensitivity of two key insulin signalling pathways to insulin. Moreover, the siRNA probe provides a molecular tool of general applicability for further dissecting the precise targets and roles of LAR.

Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance.

Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor.

Insulin Stimulation of Mitogen-activated Protein Kinase, p90rsk, and p70 S6 Kinase in Skeletal Muscle of Normal and Insulin-resistant Mice: IMPLICATIONS FOR THE REGULATION OF GLYCOGEN SYNTHASE

Skeletal muscles from mice stimulated with insulin in vivo were used to evaluate relationships between the insulin receptor tyrosine kinase, mitogen-activated protein (MAP) kinase, p90rsk, p70 S6 kinase (p70S6k), and glycogen synthase. Two models of insulin resistance were also evaluated: (a) transgenic mice with a severe insulin receptor defect and (b) gold thioglucose (GTG) mice (obesity with minimal insulin receptor dysfunction). In normal mice, insulin stimulated MAP kinase (6-fold), p90rsk (RSK2, 5-fold), p70S6k (10-fold), and glycogen synthase (30-50% increase in fractional velocity). In transgenic mice, stimulation of MAP kinase and RSK2 were not detectable, whereas activation of p70S6k and glycogen synthase were preserved. In GTG mice, activation of MAP kinase, RSK2, p70S6k, and glycogen synthase were impaired. Since p70S6k and glycogen synthase were correlated, rapamycin was used to block p70S6k, and glycogen synthase activation was unaffected in normal mice; however, it was partially impaired in transgenic mice. (a) stimulation of p70S6k and glycogen synthase are selectively preserved in muscles with a severe insulin receptor kinase defect, indicating signal amplification in pathways leading to these effects; (b) MAP kinase-RSK2 and p70S6k activation are impaired in obese mice, suggesting multiple loci for postreceptor insulin resistance; (c) glycogen synthase was dissociated from MAP kinase and RSK2, indicating that they are not required for this effect of insulin; and (d) p70S6k is not essential for glycogen synthase activation, but it may participate in redundant signaling pathways leading to this effect of insulin.

Effect of immobilization on glucose transporter expression in rat hindlimb muscles

Three days after denervation, the expression of GLUT4 mRNA and protein decreases by approximately 50% in rat hindlimb muscles, while GLUT1 mRNA increases transiently by approximately 500%. Although postreceptor insulin resistance of glucose transport develops before GLUT4 declines, the latter likely contributes to the time-dependent increased severity of the resistance. To determine whether muscle inactivity contributes to changes in glucose transporter expression, one rat hindlimb was immobilized in a plaster cast for 3 days; the unencumbered hindlimb served as control. Muscle GLUT4 mRNA decreased by 32% ( P < .02) and GLUT4 protein by 40% ( P < .05) after 3 days' immobilization. There was no significant change in GLUT1 mRNA or skeletal muscle α-actin mRNA expression or in the total RNA concentration. The data suggest that electromyogenic and/or contractile activity regulates GLUT4 expression in skeletal muscle at a pretranslational step.

Inhibitors of insulin receptor tyrosine kinase in fibroblasts from diverse patients with impaired insulin action: evidence for a novel mechanism of postreceptor insulin resistance.

Insulin resistance is a major feature of noninsulin-dependent diabetes mellitus. This resistance appears to involve molecules, apart from the insulin receptor, that are capable of altering its function. Previously, we reported that dermal fibroblasts from a female patient with insulin resistance and noninsulin-dependent diabetes produced an inhibitor of insulin receptor tyrosine kinase activity. We have now studied inhibitors in fibroblasts from four additional patients (one male and three females) with severe insulin resistance. Although clinical features were diverse, these patients had in common normal fasting glucose values, with fasting and postprandial hyperinsulinemia. The fibroblast insulin receptor content was within the normal range, but both basal and insulin-stimulated tyrosine kinase activity in fibroblast extracts were markedly decreased compared to those in extracts of fibroblasts from nondiabetic subjects. Studies revealed that these fibroblasts contained a glycoprotein inhibitor of insulin receptor tyrosine kinase activity. This inhibitor was not found in extracts of either similar insulin-resistant patients with normal insulin receptors or insulin-resistant patients with insulin receptor abnormalities. The inhibitor was not adsorbed with antiserum to either tyrosine phosphatases or fetuin. These studies thus suggest that one or more unique inhibitors of insulin receptor tyrosine kinase are present in fibroblasts of certain patients with severe insulin resistance. The presence of insulin receptor tyrosine kinase inhibitors in target cells, therefore, may constitute a novel mechanism of postreceptor insulin resistance.

Okadaic acid, insulin, and denervation effects on glucose and amino acid transport and glycogen synthesis in muscle.

Effects of okadaic acid (OKA) and calyculin A, cell-permeating specific inhibitors of phosphoprotein phosphatases-1 and -2A, were studied in isolated rat hemidiaphragms. OKA stimulated glucose transport (half-maximum = approximately 0.1 microM; maximum = approximately 1 microM) but was less effective than 6 nM insulin. Insulin and OKA effects were not additive. OKA diminished or abolished glucose transport-stimulation by insulin. System A amino acid transport was also stimulated by OKA, insulin was more effective, and preexposure to OKA inhibited insulin stimulation. Calyculin A affected both transport systems similarly to OKA. OKA did not affect basal glycogen synthesis but abolished its stimulation by insulin. Denervated muscles develop post-receptor insulin resistance. Glucose transport and glycogen synthesis were essentially unresponsive to insulin 3 days postdenervation; however, glucose transport was stimulated by OKA similarly to controls. OKA did not affect glycogen synthesis in denervated muscle except for abolishing a small insulin effect. The data suggest similar acute regulation of glucose and system A amino acid transport in muscle. Enhanced Ser/Thr phosphorylation of unidentified protein(s) stimulates both processes but inhibits their full stimulation by insulin. Postdenervation insulin resistance likely reflects impaired signal transduction.

Inhibitors of insulin receptor tyrosine kinase in fibroblasts from diverse patients with impaired insulin action: evidence for a novel mechanism of postreceptor insulin resistance

Inhibitors of insulin receptor tyrosine kinase in fibroblasts from diverse patients with impaired insulin action: evidence for a novel mechanism of postreceptor insulin resistance