Mandarino, Lawrence J.

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 μg · kg−1 · h−1) increased plasma growth hormone nearly threefold (to ≃9 ng/ml) within the range observed during sleep and exercise. This increased plasma insulin (14 ± 1 versus 8 ± 1 μU/ml, P < 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 μu/ml, P < 0.01) and stimulation of glucose utilization (half-maximal response at 98 ± 8 versus 52 ± 8 μU/ml, P / 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.

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 μU/ml) and glucagon levels (214 ± 31 versus 158 ± 21 pg/ml) than normal subjects, P < 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 < 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.

To determine whether receptor and/or postreceptor abnormalities of insulin action were responsible for insulin resistance in nonobese patients with non-insulindependent 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 μU/ml vs. 58 ± 2 μU/ml in the nondiabetics P < 0.001) but a normal maximal response to insulin. Although monocyte insulin binding was decreased in the diabetics (P < 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 ± 90μu.U · ml−1 · 10 mirr1 in the nondiabetic subjects, P < 0.001) and was more strongly correlated with fasting plasma glucose (r = 0.92, P < 0.001) and intravenous glucose tolerance (Kivgtt) (r = 0.98, P < 0.001) than was insulin sensitivity (Km) (r = 0.23, NS, and r = 0.57, P < 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.

To assess the effects of endogenous somatostatin on pancreatic islet A- and B-cell function, isolated rat islets were incubated in antisomatostatin γ-globulin to bind endogenously released somatostatin, and the insulin and glucagon secretion of these islets was compared with that of islets incubated in γ-globulin isolated from nonimmune serum. Islets incubated in antisomatostatin γ-globulin released significantly more insulin at 4, 8,16, and 32 mM glucose and significantly more glucagon at 8,16, and 32 mM glucose, P < 0.05–0.005. For glucose-stimulated insulin release the threshold was decreased, the Vmax was increased, but the apparent Km was unaltered; for glucose-suppression of glucagon release the threshold was increased, maximal suppression was decreased, but the apparent Ki was unaltered. The augmentative effect of the antisomatostatin γ-globulin was most prominent at 4 mM glucose for insulin release and at 8 mM glucose for glucagon release, but was not limited to glucose since both insulin and glucagon release stimulated by arginine were also augmented by antisomatostatin γ-globulin. These results provide evidence that endogenous somatostatin may act as a physiologic local regulator of both insulin and glucagon secretion and that its effect on insulin and glucagon secretion is dependent on the prevailing glucose concentration.

PurposeThe goal of this work was to detect disease‐related microstructural changes to the liver using magnetic resonance imaging. Chronic liver disease can cause microstructural changes in the liver that reduce plasma access to the perisinusoidal space—the site of exchange between the blood plasma and the hepatic parenchyma. The reduced plasma access to the perisinusoidal space inhibits hepatic function and contributes to the ∼30,000 chronic liver disease‐related deaths per year.MethodsThe extracellular matrix‐specific cationized ferritin magnetic resonance imaging probe was injected intravenously into healthy rats and a rat model of the chronic liver disease non‐alcoholic steatohepatitis. Rats were subsequently imaged with ‐weighted magnetic resonance imaging.ResultsThis work demonstrates that the binding of cationized ferritin to the perisinusoidal extracellular matrix is reduced by 55% in a rat model of non‐alcoholic steatohepatitis compared to healthy controls. This reduced binding is detectable in vivo with magnetic resonance imaging. Immunofluorescence and electron microscopy indicated that the reduced binding is due to inhibited macromolecular access to the perisinusoidal space caused by non‐alcoholic steatohepatitis‐related microstructural changes.ConclusionsThe reduced accumulation of intravenously injected cationized ferritin may report on changes in macromolecular access to the liver parenchyma in chronic liver disease. Magn Reson Med 70:1728–1738, 2013. © 2013 Wiley Periodicals, Inc.

Insulin resistance in skeletal muscle is a prominent feature of obesity and type 2 diabetes. The association between mitochondrial changes and insulin resistance is well known. More recently, there is growing evidence of a relationship between inflammation, extracellular remodeling, and insulin resistance. The intent of this review is to propose a potentially novel mechanism for the development of insulin resistance, focusing on the underappreciated connections among inflammation, extracellular remodeling, cytoskeletal interactions, mitochondrial function, and insulin resistance in human skeletal muscle. Several sources of inflammation, including expansion of adipose tissue resulting in increased lipolysis and alterations in pro- and anti-inflammatory cytokines, contribute to the insulin resistance observed in obesity and type 2 diabetes. In the experimental model of lipid oversupply, an inflammatory response in skeletal muscle leads to altered expression extracellular matrix-related genes as well as nuclear encoded mitochondrial genes. A similar pattern also is observed in “naturally” occurring insulin resistance in muscle of obese nondiabetic individuals and patients with type 2 diabetes mellitus. More recently, alterations in proteins (including α-actinin-2, desmin, proteasomes, and chaperones) involved in muscle structure and function have been observed in insulin-resistant muscle. Some of these cytoskeletal proteins are mechanosignal transducers that allow muscle fibers to sense contractile activity and respond appropriately. The ensuing alterations in expression of genes coding for mitochondrial proteins and cytoskeletal proteins may contribute to the mitochondrial changes observed in insulin-resistant muscle. These changes in turn may lead to a reduction in fat oxidation and an increase in intramyocellular lipid, which contributes to the defects in insulin signaling in insulin resistance.

Abstract The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.

OBJECTIVE The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. RESEARCH DESIGN AND METHODS Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. RESULTS NADH- and FADH2-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-dl-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). CONCLUSIONS We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.