These findings indicate a relationship between RBP4, insulin sensitivity, and percent trunk fat in individuals who may not have features of insulin resistance.

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-alpha (TNF-alpha) 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.

Affinity labeling studies and mutational analyses have implicated the involvement of a predicted domain of the insulin receptor (L1, amino acids 1-119) in ligand binding. In order to obtain a higher resolution localization of this ligand binding site, we have performed alanine scanning mutagenesis of this domain. Alanine mutant cDNAs encoding a secreted recombinant insulin receptor extracellular domain were expressed transiently in adenovirus transformed human embryonic kidney cells and the affinity of the expressed receptor for insulin was determined. Mutation of 14 amino acids located in four discontinuous peptide segments to alanine was disruptive of insulin binding: Segment 1, amino acids 12-15; Segment 2, amino acids 34-44; Segment 3, amino acids 64-67; and Segment 4, amino acids 89-91. The quantitative contribution of the four segments to the free energy of insulin binding was 1 > 3 > 2 > 4. Of the 14 amino acids whose mutation compromised insulin binding, 3 are charged, 3 hydrophobic, 5 aromatic, and 3 are amides.

The high resolution crystal structure of an N-terminal fragment of the IGF-I receptor, has been reported. While this fragment is itself devoid of ligand binding activity, mutational analysis has indicated that its N terminus (L1, amino acids 1-150) and the C terminus of its cysteine-rich domain (amino acids 190 -300) contain ligand binding determinants. Mutational analysis also suggests that amino acids 692-702 from the C terminus of the ␣ subunit are critical for ligand binding. A fusion protein, formed from these fragments, binds IGF-I with an affinity similar to that of the whole extracellular domain, suggesting that these are the minimal structural elements of the IGF-I binding site. To further characterize the binding site, we have performed structure directed and alanine-scanning mutagenesis of L1, the cysteinerich domain and amino acids 692-702. Alanine mutants of residues in these regions were transiently expressed as secreted recombinant receptors and their affinity was determined. In L1 alanine mutants of The insulin-like growth factors I and II are essential for normal fetal and post-natal growth (1). They were originally identified as circulating polypeptides with potent mitogenic activity, which mediated many of the actions of growth hormone, and were later shown to be structurally homologous to proinsulin. It is now apparent that these growth factors are produced by many cell types and have paracrine and autocrine as well as endocrine functions. Targeted disruption of the gene for IGF-I 1 in transgenic mice results in both embryonic and post-natal growth retardation (2). In contrast, the effects of disruption of the IGF-II gene are confined to growth retardation during the embryonic period (2). In addition to being mitogens, it is now evident that these peptides play a crucial role in cell survival (3) and contribute to transformation and the maintenance of the malignant phenotype in many tumor systems (4). However, despite extensive study, the signal transduction mechanisms underlying the biological effects of these peptides remain to be elucidated.The mitogenic effects of these growth factors appear to be mediated by receptors belonging to the insulin receptor subclass of receptor tyrosine kinases (for review see Ref. (5)). The type 1 IGF receptor binds both peptides with high affinity; the affinity for IGF-I being greater than that for IGF-II. Transgenic experiments indicate that the growth-promoting effects of both peptides can be mediated by this receptor (2, 6). Such studies also point to the role of a second receptor in mediating the mitogenic effects of IGF-II (2, 6), and recent in vitro studies indicate that this is the A isoform of the insulin receptor (7); this receptor binds IGF-II with high affinity and can mediate the growth-promoting effects of the peptide (8).The receptors in this family are dimeric protein-tyrosine kinases with significant homology (5). In higher vertebrates there are three known members, the insulin receptor (9, 10), the type 1 IGF receptor (11), and the orphan in...

Insulin initiates signal transduction in target cells by binding to a specific cell surface receptor, which is a member of the growth factor receptor tyrosine kinase superfamily of proteins (1). Ligand binding leads to the activation of the receptor's tyrosine kinase activity and the initiation of intracellular signaling. Mutational studies of receptor signaling and the elucidation of the structure of the receptor's tyrosine kinase catalytic domain suggest that kinase activation is effected by intramolecular transphosphorylation of the constituent tyrosine kinase catalytic domains of the receptor heterotetramer (2, 3). The molecular details of the mechanism by which insulin binding initiates the transphosphorylation event remain obscure and will require an understanding of the molecular organization of the extracellular domain of the receptor and the molecular basis of insulin binding.The insulin receptor is composed of two disulfide-linked heterodimers, each of which is composed in turn of a 135-kDa ␣ subunit (entirely extracellular) linked by a disulfide bond to a 95-kDa ␤ subunit, which has an extracellular domain, a single ␣ helical transmembrane domain, and an intracellular domain containing the tyrosine kinase catalytic activity (1). While the tertiary structure of the extracellular domain has not been elucidated, the presence of several characteristic structural motifs can be predicted from inspection of the deduced amino acid sequence (4, 5). The ␣ subunit contains a cysteine-rich domain homologous to that of the epidermal growth factor receptor (4, 5), and there are also two fibronectin type III repeats; the first is composed of the C terminus of the ␣ subunit and the N terminus of the ␤ subunit, and the second is composed of the C terminus of the extracellular region of the ␤ subunit (6).Bajaj et al. (7) have proposed a hypothetical model of the tertiary structure of the receptor extracellular domain based on homologies between the primary structures of the epidermal growth factor and insulin receptor families of tyrosine kinases. This model predicts that there are two homologous globular domains flanking the cysteine-rich domains: domain L1 containing amino acids 1-119 and domain L2 containing amino acids 311-428. Each contains repeating structural motifs (I-V) composed of ␣ helix, ␤-turn-␤, and hypervariable structures. Since all deletions and insertions occur in the hypervariable structures in the sequence alignments obtained for these proteins with this model, it was suggested that these may represent components of ligand binding domains.This proposal has received support from recent experimental observations (8 -12). We have recently performed alanine-scanning mutagenesis of the L1 domain of the insulin receptor and have shown it it to contain a ligand binding domain composed of 14 amino acids organized in four discontinuous peptide segments (13). However, it is unlikely that this is the complete insulin binding site, as secreted recombinant receptors with deletions N-terminal to the C terminus of...

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 microg/mL vs. 11.2 +/- 1.4 microg/mL in the HIV group and 14.9 +/- 1.8 microg/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.

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.

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. Insulin and insulin-like growth factor 1 are circulating serum peptides that share nearly 50% sequence homology (for a review, see Ref. 1). Conservation of the predicted major secondary structural elements of both peptides suggests that their tertiary structures are similar (2). Crystallographic and solution NMR structural studies have shown this to be the case (3, 4). Their cognate receptors also exhibit significant overall sequence homology (1). However, despite the overall homology of receptors and ligands, insulin and IGF-1 1 only bind weakly to each other's receptors; the affinity of each peptide for the noncognate receptor is at least 3 orders of magnitude lower than for the cognate receptor (5). The molecular basis for this discrimination is at present unclear, as are the molecular mechanisms underlying ligand binding.As a consequence of the availability of a large number of naturally occurring and chemically and biosynthetically modified analogs of insulin, an extensive body of information regarding its receptor binding determinants has accumulated. A current consensus is that A2 isoleucine, A3 valine, B12 valine, B24 and B25 phenylalanine, A19 tyrosine, A21 asparagine, and the partially buried residues A16 and B15 leucine are the major determinants of the receptor binding site, with A8 threonine, B9 serine, B10 histidine, B13 glutamate, and B16 tyrosine making minor contributions.2 This forms a patch on the surface of the molecule overlapping its dimerization surface. More recent studies also suggest that a small patch formed from the residues A13 and B17 on the hexamerization surface of insulin may represent a topologically distinct receptor binding site located on the opposite side of the molecule (6). Much less information is available with regard to the receptor binding determinants of IGF-1. Studies by Bayne and Cascieri (7,8) implicate a role of the C region of the molecule in receptor binding; specifically, tyrosine 31 appears to be essential for high affinity binding (8). In addition, tyrosines 24 and 60, corresponding to B25 phenylalanine and A19 tyrosine of insulin, respectively, both essential for high affinity binding of insulin, play important roles in the receptor binding of IGF-1...