Dinakar Iyer

Cysteine-Rich LIM-Only Proteins CRP1 and CRP2 Are Potent Smooth Muscle Differentiation Cofactors

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors

Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR+. However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca2+ transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.

Prevalence of diabetes, metabolic syndrome, and cardiovascular risk factors in US Asian Indians: results from a national study

BACKGROUND Although studies of immigrant Asian Indians in other countries show high rates of diabetes (DM), metabolic syndrome (MetS), and cardiovascular disease (CVD), no randomized, population-based studies of this rapidly growing ethnic group exist in the US. METHODS The sample comprised 1038 randomly selected Asian Indian immigrants, aged 18 years and older at seven US sites. Prevalence of diabetes and MetS (age-adjusted and sex-adjusted means) was estimated and ANOVA was used to calculate gender and group differences (normoglycemia/impaired fasting glucose/diabetes) for CVD risk factors. RESULTS The mean age was 48.2 years. The majority of respondents were male, married, educated, and with some form of health insurance. Prevalence of diabetes was 17.4%, and 33% of the respondents had prediabetes. Cardiovascular risk factors, especially high levels of triglycerides, total cholesterol, LDL cholesterol, homocysteine, and C-reactive protein, and low levels of HDL cholesterol, were also prevalent; elevated lipoprotein(a) was not observed. The age-adjusted prevalence of MetS was 26.9% by the original NCEP/ATP III criteria, 32.7% by the modified NCEP/ATP III criteria, and 38.2% by the IDF criteria. The MetS rates for women, but not for men, increased with age using all three criteria. There was a progressive worsening of all metabolic parameters as individuals progressed from normal to IFG to diabetes. CONCLUSION The prevalence rates of diabetes and MetS among US Asian Indians are higher than reported in earlier, nonrandomized, smaller surveys. These data provide a firm basis for future mechanistic and interventional studies.

Combination of niacin and fenofibrate with lifestyle changes improves dyslipidemia and hypoadiponectinemia in HIV patients on antiretroviral therapy: results of "heart positive," a randomized, controlled trial.

CONTEXT HIV patients on antiretroviral therapy (ART) have a unique dyslipidemia [elevated triglycerides and non-high-density lipoprotein-cholesterol (HDL-C), low HDL-C] with insulin resistance (characterized by hypoadiponectinemia). OBJECTIVE The aim was to test a targeted, comprehensive, additive approach to treating the dyslipidemia. DESIGN AND SETTING We conducted a randomized, double-blind, placebo-controlled, 24-wk trial of lifestyle modification, fenofibrate, and niacin in multiethnic HIV clinics at an academic center. PARTICIPANTS Hypertriglyceridemic adult patients were stratified on three combinations of ART classes. Subjects retained at the first measurement (2 wk) after entry were included in the analysis (n = 191). INTERVENTIONS Subjects were randomized into five treatment groups: usual care (group 1); low-saturated-fat diet and exercise (D/E; group 2); D/E + fenofibrate (group 3); D/E + niacin (group 4); or D/E + fenofibrate + niacin (group 5). MAIN OUTCOME MEASURES We measured changes in fasting triglycerides, HDL-C, and non-HDL-C (primary), and in insulin sensitivity, glycemia, adiponectin, C-reactive protein, energy expenditure, and body composition (secondary). Data were analyzed as a factorial set of treatment combinations using a mixed repeated measures model, last observation carried forward, and complete case approaches (groups 2-5), and as an unstructured set of treatments (groups 1-5). RESULTS Fenofibrate improved triglycerides (P = 0.002), total cholesterol (P = 0.02), and non-HDL-C (P = 0.003), whereas niacin improved HDL-C (P = 0.03), and both drugs decreased the total cholesterol-to-HDL-C ratio (P = 0.005-0.01). The combination of D/E, fenofibrate, and niacin provided maximal benefit, markedly reducing triglycerides (-52% compared to usual care; P = 0.003), increasing HDL-C (+12%; P < 0.001), and decreasing non-HDL-C (-18.5%; P = 0.003) and total cholesterol-to-HDL-C ratio (-24.5%; P < 0.001). Niacin doubled adiponectin levels. CONCLUSIONS A combination of fenofibrate and niacin with low-saturated-fat D/E is effective and safe in increasing HDL-C, decreasing non-HDL-C and hypertriglyceridemia, and ameliorating hypoadiponectinemia in patients with HIV/ART-associated dyslipidemia.

Rise of Plasma Ghrelin With Weight Loss is Not Sustained During Weight Maintenance

Objective: Ghrelin is postulated to be an orexigenic signal that promotes weight regain after weight loss (WL). However, it is not known whether this putative effect of ghrelin is sustained after weight stabilization. The objective of this study was to investigate the relationship of plasma ghrelin concentrations to active WL and weight maintenance in obese subjects.

Human adipose tissue as a reservoir for memory CD4+ T cells and HIV

Objective:The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. Design:We examined memory CD4+ T cells and HIV DNA in adipose tissue–stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4+ T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4+ T cells. Methods:AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4+ T-cell activation and HIV production were measured by flow cytometry and ELISA. Results:AT-SVF CD3+ T cells were activated (>60% CD69+) memory CD4+ and CD8+ T cells in uninfected and HIV-infected persons, but the AT-SVF CD4+/CD8+ ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4+ T-cell activation and HIV production approximately two to three-fold in synergy with gamma-chain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-&agr;1&bgr;1. Adipocytes also enhanced T-cell viability. Conclusion:Adipose tissues of ART-treated patients harbour activated memory CD4+ T cells and HIV DNA. Adipocytes promote CD4+ T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV.

Regulation of Cardiac Specific nkx2.5 Gene Activity by Small Ubiquitin-like Modifier

The cardiac specific homeobox gene nkx2.5, a member of the nk-2 class family, plays a central role in cardiogenesis and is a target of the small ubiquitin-like modifier (SUMO). Nkx2.5 was modified by SUMO on its 51st amino acid, a lysine residue conserved across species but absent in other nk-2 members. Conversion of this lysine to an arginine (K51R) substantially reduced Nkx2.5 DNA binding and also its transcriptional activity. Unexpectedly, mutant K51R was targeted by ubiquitin. E3 ligase PIAS proteins PIAS1, PIASx, and PIASy, but not PIAS3, enhanced SUMO-1 attachment to Nkx2.5 on the primary SUMO acceptor site. SUMO-2 linkage to Nkx2.5 was catalyzed only by PIASx and not by other PIAS proteins. SUMO conjugation stabilized the formation of Nkx2.5-containing complexes that led to robust transcriptional activation. Thus, SUMO modification serves as a positive regulator for Nkx2.5 transcriptional activity.

Pathogenesis of A−β+ Ketosis-Prone Diabetes

A−β+ ketosis-prone diabetes (KPD) is an emerging syndrome of obesity, unprovoked ketoacidosis, reversible β-cell dysfunction, and near-normoglycemic remission. We combined metabolomics with targeted kinetic measurements to investigate its pathophysiology. Fasting plasma fatty acids, acylcarnitines, and amino acids were quantified in 20 KPD patients compared with 19 nondiabetic control subjects. Unique signatures in KPD—higher glutamate but lower glutamine and citrulline concentrations, increased β-hydroxybutyryl-carnitine, decreased isovaleryl-carnitine (a leucine catabolite), and decreased tricarboxylic acid (TCA) cycle intermediates—generated hypotheses that were tested through stable isotope/mass spectrometry protocols in nine new-onset, stable KPD patients compared with seven nondiabetic control subjects. Free fatty acid flux and acetyl CoA flux and oxidation were similar, but KPD had slower acetyl CoA conversion to β-hydroxybutyrate; higher fasting β-hydroxybutyrate concentration; slower β-hydroxybutyrate oxidation; faster leucine oxidative decarboxylation; accelerated glutamine conversion to glutamate without increase in glutamate carbon oxidation; and slower citrulline flux, with diminished glutamine amide–nitrogen transfer to citrulline. The confluence of metabolomic and kinetic data indicate a distinctive pathogenic sequence: impaired ketone oxidation and fatty acid utilization for energy, leading to accelerated leucine catabolism and transamination of α-ketoglutarate to glutamate, with impaired TCA anaplerosis of glutamate carbon. They highlight a novel process of defective energy production and ketosis in A−β+ KPD.

Leptin replacement therapy does not improve the abnormal lipid kinetics of hypoleptinemic patients with HIV-associated lipodystrophy syndrome

Patients with HIV-associated dyslipidemic lipodystrophy (HADL) have characteristic lipid kinetic defects: accelerated lipolysis, blunted fat oxidation and increased hepatic fatty acid reesterification. HADL patients with lipoatrophy also have leptin deficiency. Small or non-randomized studies have suggested that leptin replacement improves glucose metabolism in HADL, with very limited data regarding its effects on the lipid kinetic abnormalities. We performed a randomized, double-blind, placebo-controlled, dose-escalating (0.02 mg/kg/d for two months; 0.04 mg/kg/d for a further two months) study of the effects of metreleptin on lipid kinetics in 17 adults with HADL, hypertriglyceridemia and hypoleptinemia. Rates of lipolysis, intra-adipocyte and intrahepatic reesterification and fatty acid oxidation were measured using infusions of (13)C(1)-palmitate and (2)H(5)-glycerol, and indirect calorimetry. Fasting lipid profiles and glucose and insulin responses to oral glucose challenge were also measured. Metreleptin treatment induced significant, dose-dependent increases in fasting plasma leptin levels. There was no significant change in total lipolysis, net lipolysis, adipocyte or hepatic re-esterification or fatty acid oxidation, or in fasting triglyceride or HDL-C concentrations, with metreleptin treatment. Metreleptin decreased fasting non-HDL-C levels (P<.01) and area-under-the-curve for glucose (P<.05). In hypoleptinemic HADL patients, treatment with metreleptin at 0.02 or 0.04 mg/kg/d does not improve abnormal fasting lipid kinetics, or triglyceride or HDL-C levels. Metreleptin does, however, improve glycemia and non-HDL-C in these patients. These results suggest a dissociation between leptins effects on glucose metabolism compared to those on lipid kinetics in HADL.

HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Reciprocal Effects on PPAR/GR Co-Regulation

HIV-1 Vpr induces the cardinal metabolic defects of HIV-associated lipodystrophy and hepatosteatosis in vivo. Chewing the Fat with HIV Protein HIV infection is infamously known for its devastating immunosuppressive effects; however, immunosuppression is not the whole story. Patients whose viral load is well controlled with antiretroviral therapy (ART) are still at risk for a variety of chronic metabolic complications, including adipose dysfunction. ART drugs have been implicated in some of these chronic complications, but others, such as decreased body fat and altered fat distribution, occur in even untreated patients. Now, Agarwal et al. demonstrate that, at least in mice, the HIV protein viral protein R (Vpr) may directly contribute to adipose dysfunction. The authors began with the observation that Vpr circulates in the blood of HIV-infected patients on ART, even those with no detectable viral load. Paired with the knowledge that Vpr can coactivate the glucocorticoid receptor (GR) and co-repress peroxisome proliferator–activated receptor γ (PPARγ), both of which are involved in metabolic regulation, they hypothesized the Vpr itself may play a pathogenic role in adipose dysfunction in these individuals. They took their studies into two mouse models of Vpr expression that lacked HIV infection: transgenic and pharmacologic. Vpr alone could disrupt PPAR/GR co-regulation and cell cycle control in adipose depots and liver to produce adipose dysfunction and hepatosteatosis. If these mechanisms hold true in humans, they could lead to targeted treatment of these metabolic complications with Vpr inhibitors, GR antagonists, or PPARγ/PPARα agonists. Viral infections, such as HIV, have been linked to obesity, but mechanistic evidence that they cause adipose dysfunction in vivo is lacking. We investigated a pathogenic role for the HIV-1 accessory protein viral protein R (Vpr), which can coactivate the glucocorticoid receptor (GR) and co-repress peroxisome proliferator–activated receptor γ (PPARγ) in vitro, in HIV-associated adipose dysfunction. Vpr circulated in the blood of most HIV-infected patients tested, including those on antiretroviral therapy (ART) with undetectable viral load. Vpr-mediated mechanisms were dissected in vivo using mouse models expressing the Vpr transgene in adipose tissues and liver (Vpr-Tg) or infused with synthetic Vpr. Both models demonstrated accelerated whole-body lipolysis, hyperglycemia and hypertriglyceridemia, and tissue-specific findings. Fat depots in these mice had diminished mass, macrophage infiltration, and blunted PPARγ target gene expression but increased GR target gene expression. In liver, we observed blunted PPARα target gene expression, steatosis with decreased adenosine monophosphate–activated protein kinase activity, and insulin resistance. Similar to human HIV-infected patients, Vpr circulated in the serum of Vpr-Tg mice. Vpr blocked differentiation in preadipocytes through cell cycle arrest, whereas in mature adipocytes, it increased lipolysis with reciprocally altered association of PPARγ and GR with their target promoters. These results delineate a distinct pathogenic sequence: Vpr, released from HIV-1 in tissue reservoirs after ART, can disrupt PPAR/GR co-regulation and cell cycle control to produce adipose dysfunction and hepatosteatosis. Confirmation of these mechanisms in HIV patients could lead to targeted treatment of the metabolic complications with Vpr inhibitors, GR antagonists, or PPARγ/PPARα agonists.