Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.

The adenine nucleotide translocase (ANT) of the mitochondrial inner membrane exchanges ADP for ATP. Mitochondria were isolated from human vastus lateralis muscle (n = 9). Carboxyatractyloside titration of O2 consumption rate (Jo) at clamped [ADP] of 21 µM gave ANT abundance of 0.97 ±â€¯0.14 nmol ANT/mg and a flux control coefficient of 82% ±â€¯6%. Flux control fell to 1% ±â€¯1% at saturating (2 mM) [ADP]. The KmADP for Jo was 32.4 ±â€¯1.8 µM. In terms of the free (-3) ADP anion this KmADP was 12.0 ±â€¯0.7 µM. A novel luciferase-based assay for ATP production gave KmADP of 13.1 ±â€¯1.9 µM in the absence of ATP competition. The free anion KmADP in this case was 2.0 ±â€¯0.3 µM. Targeted proteomic analyses showed significant acetylation of ANT Lysine23 and that ANT1 was the most abundant isoform. Acetylation of Lysine23 correlated positively with KmADP, r = 0.74, P = 0.022. The findings underscore the central role played by ANT in the control of oxidative phosphorylation, particularly at the energy phosphate levels associated with low ATP demand. As predicted by molecular dynamic modeling, ANT Lysine23 acetylation decreased the apparent affinity of ADP for ANT binding.

Parallel Neurodegenerative Phenotypes in Sporadic Parkinson's Disease Fibroblasts and Midbrain Dopamine Neurons.

Understanding the mechanisms causing Parkinson's disease (PD) is vital to the development of much needed early diagnostics and therapeutics for this debilitating condition. Here, we report cellular and molecular alterations in skin fibroblasts of late-onset sporadic PD subjects, that were recapitulated in matched induced pluripotent stem cell (iPSC)-derived midbrain dopamine (DA) neurons, reprogrammed from the same fibroblasts. Specific changes in growth, morphology, reactive oxygen species levels, mitochondrial function, and autophagy, were seen in both the PD fibroblasts and DA neurons, as compared to their respective controls. Additionally, significant alterations in alpha synuclein expression and electrical activity were also noted in the PD DA neurons. Interestingly, although the fibroblast and neuronal phenotypes were similar to each other, they also differed in their nature and scale. Furthermore, statistical analysis revealed novel associations between various clinical measures of the PD subjects and the different fibroblast and neuronal data. In essence, these findings encapsulate spontaneous, in-tandem, disease-related phenotypes in both sporadic PD fibroblasts and iPSC-based DA neurons, from the same patient, and generates an innovative model to investigate PD mechanisms with a view towards rational disease stratification and precision treatments.

Parallel neurodegenerative phenotypes in sporadic Parkinson's disease fibroblasts and midbrain dopamine neurons.

Understanding the mechanisms causing Parkinson's disease (PD) is vital to the development of much needed early diagnostics and therapeutics for this debilitating condition. Here, we report cellular and molecular alterations in skin fibroblasts of late-onset sporadic PD subjects, that were recapitulated in matched induced pluripotent stem cell (iPSC)-derived midbrain dopamine (DA) neurons, reprogrammed from the same fibroblasts. Specific changes in growth, morphology, reactive oxygen species levels, mitochondrial function, and autophagy, were seen in both the PD fibroblasts and DA neurons, as compared to their respective controls. Additionally, significant alterations in alpha synuclein expression and electrical activity were also noted in the PD DA neurons. Interestingly, although the fibroblast and neuronal phenotypes were similar to each other, they differed in their nature and scale. Furthermore, statistical analysis revealed potential novel associations between various clinical measures of the PD subjects and the different fibroblast and neuronal data. In essence, these findings encapsulate spontaneous, in-tandem, disease-related phenotypes in both sporadic PD fibroblasts and iPSC-based DA neurons, from the same patient, and generates an innovative model to investigate PD mechanisms with a view towards rational disease stratification and precision treatments.

Global IRS-1 phosphorylation analysis in insulin resistance.

AIMS/HYPOTHESIS: IRS-1 serine phosphorylation is often elevated in insulin resistance models, but confirmation in vivo in humans is lacking. We therefore analysed IRS-1 phosphorylation in human muscle in vivo. METHODS: We used HPLC-electrospray ionisation (ESI)-MS/MS to quantify IRS-1 phosphorylation basally and after insulin infusion in vastus lateralis muscle from lean healthy, obese non-diabetic and type 2 diabetic volunteers. RESULTS: Basal Ser323 phosphorylation was increased in type 2 diabetic patients (2.1 ± 0.43, p ≤ 0.05, fold change vs lean controls). Thr495 phosphorylation was decreased in type 2 diabetic patients (p ≤ 0.05). Insulin increased IRS-1 phosphorylation at Ser527 (1.4 ± 0.17, p ≤ 0.01, fold change, 60 min after insulin infusion vs basal) and Ser531 (1.3 ± 0.16, p ≤ 0.01, fold change, 60 min after insulin infusion vs basal) in the lean controls and suppressed phosphorylation at Ser348 (0.56 ± 0.11, p ≤ 0.01, fold change, 240 min after insulin infusion vs basal), Thr446 (0.64 ± 0.16, p ≤ 0.05, fold change, 60 min after insulin infusion vs basal), Ser1100 (0.77 ± 0.22, p ≤ 0.05, fold change, 240 min after insulin infusion vs basal) and Ser1142 (1.3 ± 0.2, p ≤ 0.05, fold change, 60 min after insulin infusion vs basal). CONCLUSIONS/INTERPRETATION: We conclude that, unlike some aspects of insulin signalling, the ability of insulin to increase or suppress certain IRS-1 phosphorylation sites is intact in insulin resistance. However, some IRS-1 phosphorylation sites do not respond to insulin, whereas other Ser/Thr phosphorylation sites are either increased or decreased in insulin resistance.

Increased abundance of the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1) in patients with obesity and type 2 diabetes: evidence for altered adiponectin signalling.

AIMS/HYPOTHESIS: The adiponectin signalling pathway is largely unknown, but recently the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1), has been shown to interact directly with adiponectin receptor (ADIPOR)1. APPL1 is present in C2C12 myoblasts and mouse skeletal muscle, but its presence in human skeletal muscle has not been investigated. METHODS: Samples from type 2 diabetic, and lean and non-diabetic obese participants were analysed by: immunoprecipitation and western blot; HPLC-electrospray ionisation (ESI)-mass spectrometry (MS) analysis; peak area analysis by MS; HPLC-ESI-MS/MS/MS analysis; and RT-PCR analysis of APPL1 mRNA. RESULTS: Immunoprecipitation and western blot indicated a band specific to APPL1. Tryptic digestion and HPLC-ESI-MS analysis of whole-muscle homogenate APPL1 unambiguously identified APPL1 with 56% sequence coverage. Peak area analysis by MS validated western blot results, showing APPL1 levels to be significantly increased in type 2 diabetic and obese as compared with lean participants. Targeted phosphopeptide analysis by HPLC-ESI-MS/MS/MS showed that APPL1 was phosphorylated specifically on Ser(401). APPL1 mRNA expression was significantly increased in obese and type 2 diabetic participants as compared with lean participants. After bariatric surgery in morbidly obese participants with subsequent weight loss, skeletal muscle APPL1 abundance was significantly reduced (p < 0.05) in association with an increase in plasma adiponectin (p < 0.01), increased levels of ADIPOR1 (p < 0.05) and increased muscle AMP-activated protein kinase (AMPK) phosphorylation (p < 0.05). CONCLUSIONS/INTERPRETATION: APPL1 abundance is significantly higher in type 2 diabetic muscle; APPL1 is phosphorylated in vivo on Ser(401). Improvements in hyperglycaemia and hypoadiponectinaemia following weight loss are associated with reduced skeletal muscle APPL1, and increased plasma adiponectin levels and muscle AMPK phosphorylation.

Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle.

AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is linked to mitochondrial dysfunction in obesity and type 2 diabetes. Emerging evidence indicates that reversible phosphorylation regulates oxidative phosphorylation (OxPhos) proteins. The aim of this study was to identify and quantify site-specific phosphorylation of the catalytic beta subunit of ATP synthase (ATPsyn-beta) and determine protein abundance of ATPsyn-beta and other OxPhos components in skeletal muscle from healthy and insulin-resistant individuals. METHODS: Skeletal muscle biopsies were obtained from lean, healthy, obese, non-diabetic and type 2 diabetic volunteers (each group n = 10) for immunoblotting of proteins, and hypothesis-driven identification and quantification of phosphorylation sites on ATPsyn-beta using targeted nanospray tandem mass spectrometry. Volunteers were metabolically characterised by euglycaemic-hyperinsulinaemic clamps. RESULTS: Seven phosphorylation sites were identified on ATPsyn-beta purified from human skeletal muscle. Obese individuals with and without type 2 diabetes were characterised by impaired insulin-stimulated glucose disposal rates, and showed a approximately 30% higher phosphorylation of ATPsyn-beta at Tyr361 and Thr213 (within the nucleotide-binding region of ATP synthase) as well as a coordinated downregulation of ATPsyn-beta protein and other OxPhos components. Insulin increased Tyr361 phosphorylation of ATPsyn-beta by approximately 50% in lean and healthy, but not insulin-resistant, individuals. CONCLUSIONS/INTERPRETATION: These data demonstrate that ATPsyn-beta is phosphorylated at multiple sites in human skeletal muscle, and suggest that abnormal site-specific phosphorylation of ATPsyn-beta together with reduced content of OxPhos proteins contributes to mitochondrial dysfunction in insulin resistance. Further characterisation of phosphorylation of ATPsyn-beta may offer novel targets of treatment in human diseases with mitochondrial dysfunction, such as diabetes.

Hyperglycemia normalizes insulin-stimulated skeletal muscle glucose oxidation and storage in noninsulin-dependent diabetes mellitus.

The diminished ability of insulin to promote glucose disposal and storage in muscle has been ascribed to impaired activation of glycogen synthase (GS). It is possible that decreased glucose storage could occur as a consequence of decreased glucose uptake, and that GS is impaired secondarily. Muscle glucose uptake in 15 diabetic subjects was matched to 15 nondiabetic subjects by maintaining fasting hyperglycemia during infusion of insulin. Leg muscle glucose uptake, glucose oxidation (local indirect calorimetry), release of glycolytic products, and muscle glucose storage, as well as muscle GS and pyruvate dehydrogenase (PDH) were determined before and during insulin infusion. Basal leg glucose oxidation and PDH were increased in the diabetics. Insulin-stimulated leg glucose uptake in the diabetics (8.05 +/- 1.41 mumol/[min.100 ml leg tissue]) did not differ from controls (5.64 +/- 0.37). Insulin-stimulated leg glucose oxidation, nonoxidized glycolysis, and glucose storage (2.48 +/- 0.27, 0.68 +/- 0.15, and 5.04 +/- 1.34 mumol/[min.100 ml], respectively) were not different from controls (2.18 +/- 0.12, 0.62 +/- 0.16, and 2.83 +/- 0.31). PDH and GS in noninsulin-dependent diabetes mellitus (NIDDM) were also normal during insulin infusion. When diabetics were restudied after being rendered euglycemic by overnight insulin infusion, GS and PDH were reduced compared with hyperglycemia. Thus, fasting hyperglycemia is sufficient to normalize insulin-stimulated muscle glucose uptake in NIDDM, and glucose is distributed normally to glycogenesis and glucose oxidation, possibly by normalization of GS and PDH.

Interaction between glucose and free fatty acid metabolism in human skeletal muscle.

The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.

Rates of noninsulin-mediated glucose uptake are elevated in type II diabetic subjects.

Although insulin is extremely potent in regulating glucose transport in insulin-sensitive tissues, all tissues are capable of taking up glucose by facilitated diffusion by means of a noninsulin-mediated glucose uptake (NIMGU) system. Several reports have estimated that in the postabsorptive state the majority of glucose disposal occurs via a NIMGU mechanism. However, these estimates have been either derived or extrapolated in normal humans. In the present study we have directly measured NIMGU rates in 11 normal (C) and 7 Type II noninsulin-dependent diabetic subjects (NIDDM; mean +/- SE fasting serum glucose, 249 +/- 24 mg/dl). To accomplish this, the serum glucose was clamped at a desired level during a period of insulin deficiency induced by a somatostatin infusion (SRIF, 550 micrograms/h). With a concomitant [3-3H]glucose infusion, we could isotopically quantitate glucose disposal rates (Rd) during basal (basal insulin present) and insulin-deficient (SRIF) conditions. With this approach we found that (a) basal Rd was greater in NIDDM than in C, 274 +/- 31 vs. 150 +/- 7 mg/min, due to elevated hepatic glucose output, (b) NIMGU composes 75 +/- 5% of basal Rd in C and 71 +/- 4% in NIDDM, (c) NIDDMS have absolute basal NIMGU rates that are twice that of C (195 +/- 23 vs. 113 +/- 8 mg/min, P less than 0.05), (d) when C were studied under conditions of insulin deficiency (SRIF infusion) and at a serum glucose level comparable to that of the NIDDM group (250 mg/dl), their rates of NIMGU were the same as that of the NIDDM group (186 +/- 19 vs. 195 +/- 23 mg/min; NS). We conclude that (a) in the postabsorptive state, NIMGU is the major pathway for glucose disposal for both C and NIDDM; (b) for a given glucose level the efficiency of NIMGU (NIMGU divided by serum glucose level) is equal in C and NIDDM, but since basal Rd is elevated in NIDDMs their absolute basal rates of NIMGU are higher; and (c) elevated basal rates of NIMGU in NIDDM may play a role in the pathogenesis of the late complications of diabetes.

Effects of insulin infusion on human skeletal muscle pyruvate dehydrogenase, phosphofructokinase, and glycogen synthase. Evidence for their role in oxidative and nonoxidative glucose metabolism.

To determine whether activation by insulin of glycogen synthase (GS), phosphofructokinase (PFK), or pyruvate dehydrogenase (PDH) in skeletal muscle regulates intracellular glucose metabolism, subjects were studied basally and during euglycemic insulin infusions of 12, 30, and 240 mU/m2 X min. Glucose disposal, oxidative and nonoxidative glucose metabolism were determined. GS, PFK, and PDH were assayed in skeletal muscle under each condition. Glucose disposal rates were 2.37 +/- 0.11, 3.15 +/- 0.19, 6.71 +/- 0.44, and 11.7 +/- 1.73 mg/kg X min; glucose oxidation rates were 1.96 +/- 0.18, 2.81 +/- 0.28, 4.43 +/- 0.32, and 5.22 +/- 0.52. Nonoxidative glucose metabolism was 0.39 +/- 0.13, 0.34 +/- 0.26, 2.28 +/- 0.40, and 6.52 +/- 1.21 mg/kg X min. Both the proportion of active GS and the proportion of active PDH were increased by hyperinsulinemia. PFK activity was unaffected. Activation of GS was correlated with nonoxidative glucose metabolism, while activation of PDH was correlated with glucose oxidation. Sensitivity to insulin of GS was similar to that of nonoxidative glucose metabolism, while the sensitivity to insulin of PDH was similar to that of glucose oxidation. Therefore, the activation of these enzymes in muscle may regulate nonoxidative and oxidative glucose metabolism.

Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle.

The broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1-association with PI 3-kinase defines a key step in insulin resistance.

Fuel selection in human skeletal muscle in insulin resistance: a reexamination.

For many years, the Randle glucose fatty acid cycle has been invoked to explain insulin resistance in skeletal muscle of patients with type 2 diabetes or obesity. Increased fat oxidation was hypothesized to reduce glucose metabolism. The results of a number of investigations have shown that artificially increasing fat oxidation by provision of excess lipid does decrease glucose oxidation in the whole body. However, results obtained with rodent or human systems that more directly examined muscle fuel selection have found that skeletal muscle in insulin resistance is accompanied by increased, rather than decreased, muscle glucose oxidation under basal conditions and decreased glucose oxidation under insulin-stimulated circumstances, producing a state of "metabolic inflexibility." Such a situation could contribute to the accumulation of triglyceride within the myocyte, as has been observed in insulin resistance. Recent knowledge of insulin receptor signaling indicates that the accumulation of lipid products in muscle can interfere with insulin signaling and produce insulin resistance. Therefore, although the Randle cycle is a valid physiological principle, it may not explain insulin resistance in skeletal muscle.

Intracellular defects in glucose metabolism in obese patients with NIDDM.

Skeletal muscle insulin resistance in obese patients with non-insulin-dependent diabetes mellitus (NIDDM) is characterized by decreased glucose uptake. Although reduced glycogen synthesis is thought to be the predominant cause for this deficit, studies supporting this notion often have been conducted at supraphysiological insulin concentrations in which glucose storage is the overwhelming pathway of glucose disposal. However, at lower, more physiological insulin concentrations, decreased muscle glucose oxidation could play a significant role. This study was undertaken to determine whether, under euglycemic conditions, insulin resistance for leg muscle glucose uptake in NIDDM patients is due primarily to decreased glucose storage or to oxidation. The leg balance technique and leg indirect calorimetry were used under steady-state euglycemic conditions to estimate muscle glucose uptake, storage, and oxidation in eight moderately obese NIDDM patients and eight matched-control subjects. Leg muscle biopsies also were performed to determine whether alterations in muscle pyruvate dehydrogenase or glycogen synthase activities could explain defects in glucose oxidation or storage. At insulin concentrations of approximately 500-600 pM, leg glucose uptake, oxidation, and storage in the NIDDM group (2.03 +/- 0.42, 1.00 +/- 0.13, 0.66 +/- 0.36 mumol.min-1.100 ml-1) were significantly lower (P less than 0.05) than rates in control subjects (5.14 +/- 0.64, 1.92 +/- 0.17, 2.80 +/- 0.54). Pyruvate dehydrogenase and glycogen synthase activities were also decreased, consistent with the in vivo metabolic defects. The average deficit in leg glucose uptake in NIDDM was 3.11 +/- 0.42 mumol.min-1.100 ml-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased activation of skeletal muscle glycogen synthase by mixed-meal ingestion in NIDDM.

Glycogen synthase (GS) catalyzes the formation of glycogen in human skeletal muscle, the tissue responsible for disposal of a significant portion of an oral carbohydrate load. Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by fasting and postprandial hyperglycemia in conjunction with reduced rates of insulin-stimulated glucose disposal and storage in peripheral tissues, including muscle. Our objectives in this study were to determine whether ingestion of a mixed meal activates GS in control nondiabetic subjects and whether meal-related GS activation is reduced in NIDDM. To accomplish this, mixed formula meals were administered to 11 NIDDM and 9 age- and weight-matched nondiabetic control subjects. Plasma glucose and insulin values were measured before and for 90 min after meal ingestion. Skeletal muscle biopsies were performed just before and 90 min after meal ingestion for measurement of GS activity. Compared with control subjects, NIDDM subjects had significantly higher postprandial hyperglycemia and reduced postprandial hyperinsulinemia. GS was activated by meal ingestion in control subjects to a significantly greater extent than in NIDDM subjects. In NIDDM subjects, activation of GS was inversely correlated with fasting plasma glucose (r = .69, P less than .05). Therefore, NIDDM is characterized by reduced activation of a key step in the process of muscle glycogen repletion after a meal. Reduced activation of GS by a mixed meal in NIDDM may contribute to the reduced glucose disposal after a meal, thus contributing to the hyperglycemia observed in these subjects.

Effects of growth hormone on insulin action in man. Mechanisms of insulin resistance, impaired suppression of glucose production, and impaired stimulation of glucose utilization.

The present studies were undertaken to assess the mechanisms responsible for growth hormone-induced insulin resistance in man. The insulin dose-response characteristics for suppression of glucose production and stimulation of glucose utilization and their relationship to monocyte insulin binding were determined in six normal volunteers after 12-h infusion of growth hormone and 12-h infusion of saline. The infusion of growth hormone (2 micrograms . kg-1 . h-1) increased plasma growth hormone nearly threefold (to congruent to 9 ng/ml) within the range observed during sleep and exercise. This increased plasma insulin (14 +/- 1 versus 8 +/- 1 microunits/ml, P less than 0.005) concentrations without significantly altering plasma glucose concentrations or basal rates of glucose production and utilization. Insulin dose-response curves for both suppression of glucose production (half-maximal response at 37 +/- 3 versus 20 +/- 3 microunits/ml, P less than 0.01) and stimulation of glucose utilization (half-maximal response at 98 +/- 8 versus 52 +/- 8 microunits/ml, P less than 0.01) were shifted to the right with preservation of normal maximal responses to insulin. Monocyte insulin binding was unaffected. Thus, except at near maximal insulin receptor occupancy, the action of insulin on glucose production and utilization per number of monocyte insulin receptors occupied was decreased. These results indicate that increases in plasma growth hormone within the physiologic range can cause insulin resistance in man, which is due to decreases in both hepatic and extrahepatic effects of insulin. Assuming that insulin binding to monocytes reflects insulin binding in insulin sensitive tissues, this decrease in insulin action can be explained on the basis of a postreceptor defect.

Mechanism and significance of insulin resistance in non-insulin-dependent diabetes mellitus.

To determine whether receptor and/or postreceptor abnormalities of insulin action were responsible for insulin resistance in nonobese patients with non-insulin-dependent diabetes mellitus (NIDDM) and to assess the role of insulin resistance in their impaired glucose tolerance, insulin dose-response characteristics, insulin binding to monocytes, and insulin secretion were compared in 10 nonobese patients with NIDDM and six age-weight-matched nondiabetic volunteers. The insulin resistance of the diabetics was characterized by a shift to the right of their insulin dose-response curve (Km 81 +/- 4 microunits/ml vs. 58 +/- 2 microunits/ml in the nondiabetics P less than 0.001) but a normal maximal response to insulin. Although monocyte insulin binding was decreased in the diabetics (P less than 0.01), their response to insulin was appropriate for the number of insulin receptors occupied indicating normal postreceptor function. Insulin secretion was markedly reduced in diabetic subjects (52 +/- 22 vs. 471 +/- 90 microunits . ml-1 . 10 min-1 in the nondiabetic subjects, P less than 0.001) and was more strongly correlated with fasting plasma glucose (r = 0.92, P less than 0.001) and intravenous glucose tolerance (Kivgtt) (r = 0.98, P less than 0.001) than was insulin sensitivity (Km) (r = 0.23, NS, and r = 0.57, P less than 0.05, respectively). We conclude that in nonobese patients with NIDDM, insulin resistance is characterized by a shift to the right of the insulin dose-response curve, which can be accounted for solely by an insulin receptor defect. However, in these patients, impaired insulin secretion rather than insulin resistance appears to be the predominant metabolic abnormality.

Seven days of euglycemic hyperinsulinemia induces insulin resistance for glucose metabolism but not hypertension, elevated catecholamine levels, or increased sodium retention in conscious normal rats.

Epidemiological studies have suggested an association among chronic hyperinsulinemia, insulin resistance, and hypertension. However, the causality of this relationship remains uncertain. In this study, chronically catheterized conscious rats were made hyperinsulinemic for 7 days (approximately 90 mU/l, i.e., threefold over basal), while strict euglycemia was maintained (approximately 130 mg/dl, coefficient of variation < 10%) by using a modification of the insulin/glucose clamp technique. Control rats received vehicle infusion. Baseline mean arterial pressure and heart rate were 125 +/- 5 mmHg and 427 +/- 12 beats/min and remained unchanged during the 7-day infusion of insulin (127 +/- 7 mmHg; 401 +/- 12 beats/min) or vehicle (133 +/- 4 mmHg; 411 +/- 10 beats/min). Baseline plasma epinephrine (88 +/- 15 pg/ml), norepinephrine (205 +/- 31 pg/ml), and sodium balance (0.34 +/- 0.09 mmol) remained constant during the 7-day insulin or vehicle infusion. After 7 days of insulin or vehicle infusion, in vivo insulin action was determined in all rats using a 2-h hyperinsulinemic (1 mU/min) euglycemic clamp with [3-3H]glucose infusion to quantitate whole-body glucose uptake, glycolysis, glucose storage (total glucose uptake minus glycolysis), and hepatic glucose production. Compared with vehicle-treated rats, 7 days of sustained hyperinsulinemia resulted in a reduction (P < 0.01) in insulin-mediated glucose uptake, glucose storage, and glycolysis by 39, 62, and 26%, respectively. Hepatic glucose production was normally suppressed after 7 days of hyperinsulinemia. Neither insulin-stimulated glucose uptake nor glucose storage correlated with blood pressure or heart rate. In conclusion, 7 days of euglycemic hyperinsulinemia induces severe insulin resistance with respect to whole-body glucose metabolism but does not increase blood pressure, catecholamine levels, or sodium retention. This indicates that hyperinsulinemia-induced insulin resistance is not associated with the development of hypertension in rats who do not have a genetic predisposition for hypertension. Because hyperinsulinemia was initiated in normal rats under euglycemic conditions, additional (inherited or acquired) factors may be necessary to observe an effect of hyperinsulinemia and/or insulin resistance to increase blood pressure.

Familial insulin resistance and acanthosis nigricans. Presence of a postbinding defect.

Type A insulin resistance, associated with acanthosis nigricans and menstrual irregularity, has been ascribed to a decreased concentration of insulin receptors. We now report four affected females from one family, a mother and three daughters (including identical twins) who appear to have the type A syndrome. Two of the kindred had no apparent ovarian dysfunction, while the other two had hyperprolactinemia without other findings of polycystic ovary disease, suggesting a genetic disease with variable penetrance. All had normal erythrocyte and monocyte insulin binding. Insulin dose-response studies to assess glucose metabolism and insulin sensitivity were performed in the affected twins. The dose response to insulin was shifted to the right with a decrease in maximal response. These results are consistent with a postbinding defect in insulin action in these patients.

Pathogenesis of hypoglycemia in insulinoma patients: suppression of hepatic glucose production by insulin.

To determine the mechanism by which hyperinsulinemia causes hypoglycemia in insulinoma patients, rates of glucose production and utilization, and circulating levels of insulin, glucagon, alanine, lactate, and glycerol were measured in 6 insulinoma patients during development of fasting hypoglycemia and in 8 normal volunteers studied over an identical interval. Initially, insulinoma patients had a greater plasma insulin (42 +/- 9 versus 15 +/- 1 microunits/ml) and glucagon levels (214 +/- 31 versus 158 +/- 21 pg/ml) than normal subjects, P less than 0.05, but their plasma glucose levels (81 +/- 4 mg/dl) and rates of glucose production and utilization (1.71 +/- 0.08 and 1.74 +/- 0.08 mg/kg . min, respectively) were not significantly different from those of normal subjects (93 +/- 2 mg/dl, 1.93 +/- 0.11, and 1.92 +/- 0.13 mg/kg . min, respectively). During a subsequent 8-h fast, glucose production and glucose utilization decreased in both groups, but more markedly in insulinoma patients. Since glucose utilization exceeded glucose production to a greater extent in insulinoma patients than in normal subjects, plasma glucose decreased to 44 +/- 3 mg/dl in insulinoma patients, but only to 84 +/- 1 mg/dl in normal subjects (P less than 0.001). Glucose utilization in insulinoma patients never exceeded that of normal subjects. These results demonstrate that fasting hypoglycemia in the insulinoma patients is usually due to suppression of glucose production rather than to acceleration of glucose utilization, as is widely thought. A direct effect of insulin on the liver is probably responsible, since circulating levels of gluconeogenic precursors are normal and since plasma glucagon increases during development of hypoglycemia in insulinoma patients.

Insulin-induced hexokinase II expression is reduced in obesity and NIDDM.

NIDDM and obesity are characterized by decreased insulin-stimulated glucose uptake in muscle. It has been suggested that impaired glucose phosphorylation to glucose-6-phosphate, catalyzed in muscle by hexokinase (HK)II, may contribute to this insulin resistance. Insulin is known to increase HKII mRNA, protein, and activity in lean nondiabetic individuals. The purpose of this study was to determine whether defects in insulin-stimulated HKII expression and activity could contribute to the insulin resistance of obesity and NIDDM. Fifteen lean nondiabetic control subjects, 17 obese nondiabetic subjects, and 14 obese NIDDM patients were studied. Percutaneous muscle biopsies of the vastus lateralis were performed in conjunction with leg balance and local indirect calorimetry measurements before and at the end of a 3-h euglycemic-hyperinsulinemic clamp (40 or 240 mU x min(-1) x m[-2]). Leg glucose uptake in response to the 40-mU insulin infusion was higher in the lean control subjects (2.53 +/- 0.35 micromol x min(-1) per x 100 ml leg vol) than in obese (1.46 +/- 0.50) or NIDDM (0.53 +/- 0.25, P < 0.05) patients. In response to 240 mU insulin, leg glucose uptake was similar in all of the groups. In response to 40 mU insulin, HKII mRNA in lean control subjects was increased 1.48 +/- 0.18-fold (P < 0.05) but failed to increase significantly in the obese (1.12 +/- 0.24) or NIDDM (1.14 +/- 0.18) groups. In response to 240 mU insulin, HKII mRNA was increased in all groups (control subjects 1.48 +/- 0.18, P < 0.05 vs. basal, obese 1.30 +/- 0.16, P < 0.05, and NIDDM 1.25 +/- 0.14, P < 0.05). Under basal conditions, HKI and HKII activities did not differ significantly between groups. Neither the 40 mU nor the 240 mU insulin infusion affected HK activity. Total HKII activity was reduced in the obese subjects (4.33 +/- 0.08 pmol x min(-1) x g(-1) muscle protein) relative to the lean control subjects (5.00 +/- 0.08, P < 0.05). There was a further reduction in the diabetic patients (3.10 +/- 0.10, P < 0.01 vs. the control subjects, P < 0.01 vs. the obese subjects). Resistance to insulin's metabolic effects extends to its ability to induce HKII expression in obesity and NIDDM.