Dennis C. Mynarcik

Association of severe insulin resistance with both loss of limb fat and elevated serum tumor necrosis factor receptor levels in HIV lipodystrophy.

&NA;HIV‐lipodystrophy (HIV‐LD) is characterized by the loss of body fat from the limbs and face, an increase in truncal fat, insulin resistance, and hyperlipidemia, factors placing affected patients at increased risk for vascular disease. This study evaluated insulin sensitivity and inflammatory status associated with HIV‐LD and provides suggestions about its etiology. Insulin sensitivity and immune activation markers were assessed in 12 control subjects and 2 HIV‐positive groups, 14 without and 15 with LD syndrome. Peripheral insulin sensitivity (mostly skeletal muscle) was determined with the hyperinsulinemic‐euglycemic clamp. Circulating insulin‐like growth factor (IGF) binding protein‐1 (IGFBP‐1) and free fatty acid (FFA) levels, and their response to insulin infusion were indicative of insulin responsiveness of liver and adipose tissue, respectively. Serum levels of soluble type 2 tumor necrosis factor‐&agr; (TNF‐&agr;) receptor (sTNFR2) were used as an indicator of immune activation. HIV‐LD study subjects had significantly reduced (twofold) peripheral insulin sensitivity, but normal levels of FFA and reduced levels of IGFBP‐1, relative to the nonlipodystrophy groups, indicating that the loss of insulin sensitivity was more pronounced in skeletal muscle than in liver or fat. The significant loss of peripheral fat in the HIV‐LD group (34%; p < .05) closely correlated with the reduced peripheral insulin sensitivity (p = .0001). Levels of sTNFR2 were elevated in all HIV‐infected study subjects, but they were significantly higher in those with lipodystrophy than without, and sTNFR2 levels strongly correlated with the reduction in insulin sensitivity (p = .0001). Loss of peripheral fat, normal levels of FFA, and reduced levels of IGFBP‐1 indicate that insulin resistance in HIV‐LD is distinct from type 2 diabetes and obesity. The relationship between the degree of insulin resistance and sTNFR2 levels suggests an inflammatory stimulus is contributing to the development of HIV‐associated lipodystrophy.

Adiponectin and leptin levels in HIV-infected subjects with insulin resistance and body fat redistribution.

Summary: In this study, we sought to determine the relationship between serum levels of leptin and adiponectin (Acrp30) in patients with HIV‐associated lipodystrophy (HIV‐LD). Three groups of subjects were studied; HIV‐positive subjects with lipodystrophy (HIV‐LD; n = 22), HIV‐positive subjects without lipodystrophy (HIV; n = 17), and ethnicity‐ and body mass index‐matched healthy control subjects (n = 20). Although total body fat from dual energy x‐ray absorptiometry was similar in all three groups, the HIV‐LD group had a significantly lower mean proportion of body fat in the limbs ± SEM (37.2% ± 2.2%) than either controls (49.8% ± 1.5%) or HIV subjects (45.7% ± 2.0%). The HIV‐LD group also had the lowest mean insulin sensitivity ± SEM (5.11 ± 0.59 mg of glucose/[kg of lean body mass • min] vs. 10.2 ± 0.72 mg of glucose/[kg of lean body mass • min] in controls and 8.64 ± 0.69 mg of glucose/[kg of lean body mass • min] in the HIV group). Leptin levels were similar in all three groups and were significantly correlated to total body fat (r = 0.86; p < .001), but these levels did not correlate with either insulin sensitivity or limb fat. Mean Acrp30 levels ± SEM were lowest in the HIV‐LD group (5.43 ± 0.44 &mgr;g/mL vs. 11.2 ± 1.4 &mgr;g/mL in the HIV group and 14.9 ± 1.8 &mgr;g/mL in control subjects). Further, Acrp30 levels were positively correlated with insulin sensitivity (r = 0.610; p < .001) and limb fat (r = 0.483; p < .001). However, the correlation between limb fat and insulin sensitivity disappeared when Acrp30 level and other potential mediators were removed from the association, suggesting that a deficiency in Acrp30 in subjects with HIV‐LD may be part of the mechanism for the reduced insulin sensitivity.

Improved insulin sensitivity and body fat distribution in HIV-infected patients treated with rosiglitazone: A pilot study

Summary: The insulin‐sensitizing drugs thiazolidinediones (TZDs), such as rosiglitazone, improve insulin sensitivity and also promote adipocyte differentiation in vitro. The authors hypothesized that TZDs might be beneficial to patients with HIV disease to improve insulin sensitivity and the distribution of body fat by increasing peripheral fat. The ability of rosiglitazone (8 mg/d) to improve insulin sensitivity (from hyperinsulinemic‐euglycemic clamp) and to improve body fat distribution (determined from computed tomography measurements of visceral adipose tissue [VAT] and subcutaneous adipose tissue [SAT]) was determined in 8 HIV‐positive patients. Before treatment, the insulin sensitivity of the patients was reduced to approximately 34% of that in control subjects. The rate of glucose disposal during a hyperinsulinemic‐euglycemic clamp (Rd) was 3.8 ± .4 (SEM) mg glucose/kg lean body mass/min compared with 11.08 ± 1.1 (p < .001) in healthy age‐ and body mass index (BMI)‐matched control subjects. After rosiglitazone treatment of 6 to 12 weeks, Rd increased to 5.99 ± .9 (p = .02), an improvement of 59 ± 22%. SAT increased by 23 ± 10% (p = .05), and, surprisingly, VAT was decreased by 21 ± 8% (p = .04) with a trend for increased SAT/VAT that failed to reach statistical significance. There were no significant changes in blood counts, viral loads, or CD4 counts with rosiglitazone treatment. The study demonstrates that rosiglitazone therapy improves insulin resistance and body fat distribution in some patients with HIV disease.

Identification of Common Ligand Binding Determinants of the Insulin and Insulin-like Growth Factor 1 Receptors INSIGHTS INTO MECHANISMS OF LIGAND BINDING

Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2–3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. cDNAs encoding alanine-substituted secreted recombinant IGF-1 receptors were expressed in 293 EBNA cells, and the ligand binding properties of the expressed proteins were evaluated. Mutation of Phe701 resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe714, produces a 140-fold reduction in affinity for insulin. Mutation of Asp8, Asn11, Phe58, Phe692, Glu693, His697, and Asn698 produces a 3.5–6-fold reduction in affinity for IGF-1. In contrast, alanine mutation of the corresponding amino acids of the insulin receptor with the exception of Asp12 produces reductions in affinity that are 50-fold or greater. The affinity of insulin for these mutants relative to wild type receptor was similar to that of their relative affinity for IGF-1 with two exceptions; the IC50 values for insulin binding to the mutants of Arg10, which has normal affinity for IGF-1, and His697, which has a 6-fold reduction in affinity for IGF-1, were both at least 2 orders of magnitude greater than for wild type receptor. The K d values for insulin of the corresponding alanine mutants of the insulin receptor, Arg14 and His710, are 2–3 orders of magnitude greater than for wild type receptor. However, in contrast, the relative affinity of des(25–30)[PheB25α-carboxamide]insulin for these IGF-1 receptor mutants is reduced only 4- and 50-fold, respectively.

Alanine-scanning Mutagenesis of a C-terminal Ligand Binding Domain of the Insulin Receptor α Subunit

A recent affinity labeling study has suggested that amino acids 704-717 of the C terminus of the insulin receptor represent a contact site for insulin. To determine whether these amino acids are part of a ligand binding site, we have performed alanine-scanning mutagenesis of this region. Mutant cDNAs encoding recombinant secreted receptors were transiently expressed in 293 EBNA cells, and their insulin binding properties were evaluated. Of the 14 residues in this region only 4 amino acids, Asp-707, Val-712, Pro-716, and Arg-717, could be mutated to alanine without compromising insulin binding. The reduction in affinity resulting from the individual mutation of the remaining amino acids varied from an increase in Kd to 3.69 × 10-9M (Asn-711) to greater than 10-6M (Thr-704, Phe-705, Glu-706, and His-710); the Kd of native secreted recombinant receptor is 0.56 × 10-9M.

Influence of Age on the Association of Retinol-binding Protein 4 With Metabolic Syndrome

Objective: The relationships of retinol‐binding protein 4 (RBP4) with insulin sensitivity and body fat distribution have been investigated in a few recent studies with conflicting results. This may have been due to differences in ages of the subjects in the different studies. The aim of this study was to investigate whether the association of RBP4 and insulin sensitivity and percent trunk fat are influenced by age.

Endothelial Adhesion Molecules Are Associated with Inflammation in Subjects with HIV Disease

BACKGROUND The presence of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and soluble vascular cell adhesion molecule-1 (VCAM-1) is associated with elevated risk of cardiovascular disease. Subjects with human immunodeficiency virus (HIV) disease have multiple risk factors for cardiovascular disease, including elevated serum lipid levels, insulin resistance, and elevated levels of ICAM-1 and VCAM-1. This study assessed the variables associated with elevated adhesion molecule levels in this patient population. METHODS Serum levels of ICAM-1 and VCAM-1 were assessed in 31 subjects without HIV disease and 52 subjects with HIV disease. Pearson correlation indicated a significant relationship between ICAM concentration and other variables, including CD4+ cell count, HIV viral burden, insulin sensitivity, and serum lipid level. Multiple regression modeling was used to determine the strengths of association among the variables. RESULTS Subjects with HIV disease had elevated levels of ICAM-1 and VCAM-1. Pearson correlation analysis revealed significant associations between ICAM-1 and VCAM-1 level and insulin sensitivity, plasma lipid level, and presence of type 2 soluble receptor for tumor necrosis factor-alpha (sTNFR2). With multiple regression modeling to control for interdependence, only sTNFR2, a marker of inflammation, was an independent predictor of ICAM-1 and VCAM-1 levels. CONCLUSIONS The study suggests that many of the variables associated with ICAM-1 and VCAM-1 levels can be related to their impact on inflammation.

Analog Binding Properties of Insulin Receptor Mutants IDENTIFICATION OF AMINO ACIDS INTERACTING WITH THE COOH TERMINUS OF THE B-CHAIN OF THE INSULIN MOLECULE

Recent studies utilizing alanine scanning mutagenesis have identified a major ligand binding domain of the secreted recombinant insulin receptor composed of two subdomains, one between amino acids 1 and 120 and the other between amino acids 704 and 716. In order to obtain a more detailed characterization of these subdomains, we examined the binding of an insulin superanalog, des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 α-carboxamide]insulin, to alanine mutants of the ligand binding determinants of these subdomains. cDNAs encoding mutant secreted recombinant receptors were transiently expressed in 293 EBNA cells, and the binding properties for this analog of the expressed receptors were evaluated. In general des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 α-carboxamide]insulin binding correlated with insulin binding, suggesting that both peptides bound to the receptor in a similar manner. Alanine mutations of eight amino acids (Asn15, Phe64, Phe705, Glu706, Tyr708, Leu709, Asn711, and Phe714) of the receptor produced the most profound decreases in affinity for des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 α-carboxamide]insulin, suggesting that interactions with these amino acids contributed the major part of the free energy of the ligand-receptor interaction. Mutation of Arg14 and His710 to Ala produced receptors with undetectable insulin binding but an affinity for des-(B25-30)-[His-A8, Asp-B10, Tyr-B25 α-carboxamide]insulin only 8-23-fold less than for native receptor. Further analog studies were performed to elucidate this paradox. The receptor binding potencies of His-A8 and Asp-B10 insulins for these receptor mutants appeared to parallel their relative potencies for native receptor. In contrast the receptor binding potency of des-(B25-30)-[Tyr-B25 α-carboxamide]insulin was disproportionately increased for these mutants when compared with its potency for native receptor.

Insulin-like growth factor system in patients with HIV infection: effect of exogenous growth hormone administration.

The purpose of this study was to characterize changes in the levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (BP) 1, 2, and 3 in HIV-infected adults throughout the course of their disease, and to assess the responsiveness of the IGF system components to growth hormone (GH) administration (6 mg/day) for 2 weeks. Healthy control study subjects (n = 10) were compared with patients who were either HIV-positive (n = 9), had AIDS without weight loss (n = 13), or had AIDS with >10% weight loss (n = 6), all of whom had been free of acute illness for at least 3 months. Under basal conditions, fasting serum concentrations of epinephrine, norepinephrine, cortisol, glucagon, insulin, IGF-I, and IGFBP-3 were not significantly different among the four groups. The serum concentrations of IGFBP-1 and IGFBP-2 were significantly higher in AIDS patients with wasting than in the other three groups (p < .05). In addition, there was a statistically significant positive correlation between the levels of IGFBP- 1 (p = .004) and IGFBP-2 (p = .03) and the stage of disease. Following GH administration, the serum concentrations of insulin and IGF-I were increased in all groups (p < .05). In addition, the increases in insulin levels correlated with stage of disease (p = .004). The responses of the IGFBPs were more variable. GH administration significantly increased the levels of IGFBP-3 in all groups except the patients with AIDS wasting, whereas the levels of IGFBP-1 were significantly decreased in controls and AIDS patients. These results demonstrate that there is a continuum of both elevations in the IGFBPs and altered metabolic responsiveness in patients infected with HIV that increases with the severity of the disease. These data also demonstrate that AIDS patients, who are free from secondary infection, respond to administration of GH by significantly increasing hepatic IGF-I production.

Delivery of rosiglitazone from an injectable triple interpenetrating network hydrogel composed of naturally derived materials

An in situ gelable and biodegradable triple-interpenetrating network (3XN) hydrogel, completely devoid of potentially cytotoxic extraneous small molecule crosslinkers, is formulated from partially oxidized dextran (Odex), teleostean and N-carboxyethyl chitosan (CEC). Both the rheological profile and mechanical strength of the 3XN hydrogel approximate the combined characteristics of the three individual hydrogels composed of the binary partial formulations (i.e., Odex/CEC, Odex/teleostean, and CEC/teleostean). The 3XN hydrogel is considerably more resistant to fibroblast-mediated degradation compared to each partial formulation in cell culture models; this is attributable to the interpenetrating triple-network structure. The presence of teleostean in the 3XN hydrogel imparts cell affinity, constituting an environment amenable to fibroblast growth. in vivo subdermal injection into mouse model shows that the 3XN hydrogel does not induce extensive inflammatory response nor is there any evidence of tissue necrosis, further confirming the non-cytotoxicity of the hydrogel and its degradation byproducts. Importantly, the capability of the 3XN hydrogel to serve as a sustained drug delivery vehicle is confirmed using rosiglitazone as a model drug. The presence of rosiglitazone profoundly changes the cell/tissue interactions with the subdermally injected 3XN hydrogel. Rosiglitazone suppresses both the inflammatory response and tissue repair in a dose-dependent manner and considerably moderated the hydrogel degradation.