Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.

A quantitative study of vapor diffusions for crystallizations: rates and solvent parameter changes.

Vapor diffusion crystallizations are among the most versatile methods for growing X-ray quality crystals. While many experimental sections describe the successful use of various solvent combinations, the literature has been entirely lacking in quantitative data (rates, measures of solvent strength changes) that might allow more informed planning rather than simple trial-and-error approaches. We here report the diffusion-induced volume changes for 44 solvent combinations over the first 60 h under standardized conditions, plus six more combinations that exhibit little or no volume changes. Additionally, the inner and outer vial compositions at 24 h were determined, and the resulting changes in solvation parameters were quantified using Hansen solubility parameters. Some general preliminary effects of changes in volume ratios and scale are described. These results identify two dozen solvent combinations with larger changes in solvent parameters than the very commonly used diethyl ether/dichloromethane example. These results should allow a more informed approach to the execution of vapor diffusion crystallizations than has previously been possible.

Effects of Dietary Fructose Restriction on Liver Fat, De Novo Lipogenesis, and Insulin Kinetics in Children With Obesity.

BACKGROUND & AIMS: Consumption of sugar is associated with obesity, type 2 diabetes mellitus, nonalcoholic fatty liver disease, and cardiovascular disease. The conversion of fructose to fat in liver (de novo lipogenesis [DNL]) may be a modifiable pathogenetic pathway. We determined the effect of 9 days of isocaloric fructose restriction on DNL, liver fat, visceral fat (VAT), subcutaneous fat, and insulin kinetics in obese Latino and African American children with habitual high sugar consumption (fructose intake >50 g/d). METHODS: Children (9-18 years old; n = 41) had all meals provided for 9 days with the same energy and macronutrient composition as their standard diet, but with starch substituted for sugar, yielding a final fructose content of 4% of total kilocalories. Metabolic assessments were performed before and after fructose restriction. Liver fat, VAT, and subcutaneous fat were determined by magnetic resonance spectroscopy and imaging. The fractional DNL area under the curve value was measured using stable isotope tracers and gas chromatography/mass spectrometry. Insulin kinetics were calculated from oral glucose tolerance tests. Paired analyses compared change from day 0 to day 10 within each child. RESULTS: Compared with baseline, on day 10, liver fat decreased from a median of 7.2% (interquartile range [IQR], 2.5%-14.8%) to 3.8% (IQR, 1.7%-15.5%) (P < .001) and VAT decreased from 123 cm3 (IQR, 85-145 cm3) to 110 cm3 (IQR, 84-134 cm3) (P < .001). The DNL area under the curve decreased from 68% (IQR, 46%-83%) to 26% (IQR, 16%-37%) (P < .001). Insulin kinetics improved (P < .001). These changes occurred irrespective of baseline liver fat. CONCLUSIONS: Short-term (9 days) isocaloric fructose restriction decreased liver fat, VAT, and DNL, and improved insulin kinetics in children with obesity. These findings support efforts to reduce sugar consumption. ClinicalTrials.gov Number: NCT01200043.

Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice.

Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE: Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5-transduced cells were selected in HIV-1-infected animals. Moreover, we show that GPI-scFv X5-transduced CD4 T cells exerted a negative effect on virus replication in vivo We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections.

Short-term isocaloric fructose restriction lowers apoC-III levels and yields less atherogenic lipoprotein profiles in children with obesity and metabolic syndrome.

BACKGROUND AND AIMS: Dietary fructose may play a role in the pathogenesis of metabolic syndrome (MetS). In a recently published study of obese children with MetS, we showed that isocaloric fructose restriction reduced fasting triglyceride (TG) and LDL-cholesterol (LDL-C). In these ancillary analyses, we tested the hypothesis that these effects were also accompanied by improved quantitative and qualitative changes in LDL and HDL subclasses and their apolipoproteins; as well as change in VLDL, particularly apoC-III. METHODS: Obese children with MetS (n = 37) consumed a diet that matched self-reported macronutrient composition for nine days, with the exception that dietary fructose was reduced from 11.7 ± 4.0% to 3.8 ± 0.5% of daily calories and substituted with glucose (in starch). Participants underwent fasting biochemical analyses on Days 0 and 10. HDL and LDL subclasses were analyzed using the Lipoprint HDL and LDL subfraction analysis systems from Quantimetrix. RESULTS: Significant reductions in apoB (78 ± 24 vs. 66 ± 24 mg/dl) apoC-III (8.7 ± 3.5 vs. 6.5 ± 2.6 mg/dl) and apoE (4.6 ± 2.3 vs. 3.6 ± 1.1 mg/dl), all p < 0.001) were observed. LDL size increased by 0.87 Å (p = 0.008). Small dense LDL was present in 25% of our cohort and decreased by 68% (p = 0.04). Small HDL decreased by 2.7% (p < 0.001) and large HDL increased by 2.4% (p = 0.04). The TG/HDL-C ratio decreased from 3.1 ± 2.5 to 2.4 ± 1.4 (p = 0.02). These changes in fasting lipid profiles correlated with changes in insulin sensitivity. CONCLUSIONS: Isocaloric fructose restriction for 9 days improved lipoprotein markers of CVD risk in children with obesity and MetS. The most dramatic reduction was seen for apoC-III, which has been associated with atherogenic hypertriglyceridemia.

Glycosyl Phosphatidylinositol-Anchored C34 Peptide Derived From Human Immunodeficiency Virus Type 1 Gp41 Is a Potent Entry Inhibitor.

Lipid rafts of the plasma membrane have been shown to be gateways for HIV-1 budding and entry. In nature, many glycosyl-phosphatidylinositol (GPI) anchored proteins are targeted to the lipid rafts. In the present study we constructed two fusion genes, in which C34 peptide or AVF peptide control was genetically linked with a GPI-attachment signal. Recombinant lentiviruses expressing the fusion genes were used to transduce TZM.bl and CEMss-CCR5 cells. Here, we show that with a GPI attachment signal both C34 and AVF are targeted to the lipid rafts through a GPI anchor. GPI-C34, but not GPI-AVF, in transduced TZM.bl cells efficiently blocks the infection of diverse HIV-1 strains of various subtypes. GPI-C34-transduced CEMss-CCR5 cells are totally resistant to HIV-1 infection. Importantly, maximum percentage of inhibition (MPI) by GPI-C34 is comparable to, if not higher than, a very high concentration of soluble C34. Potent blocking by GPI-C34 is likely due to its high local concentration, which allows GPI-C34 to efficiently bind to the prehairpin intermediate and prevent its transition to six helical bundle, thereby interfering with membrane fusion and virus entry. Our findings should have important implications in GPI-anchor-based therapy against HIV-1.

Isocaloric fructose restriction and metabolic improvement in children with obesity and metabolic syndrome.

OBJECTIVE: Dietary fructose is implicated in metabolic syndrome, but intervention studies are confounded by positive caloric balance, changes in adiposity, or artifactually high amounts. This study determined whether isocaloric substitution of starch for sugar would improve metabolic parameters in Latino (n = 27) and African-American (n = 16) children with obesity and metabolic syndrome. METHODS: Participants consumed a diet for 9 days to deliver comparable percentages of protein, fat, and carbohydrate as their self-reported diet; however, dietary sugar was reduced from 28% to 10% and substituted with starch. Participants recorded daily weights, with calories adjusted for weight maintenance. Participants underwent dual-energy X-ray absorptiometry and oral glucose tolerance testing on Days 0 and 10. Biochemical analyses were controlled for weight change by repeated measures ANCOVA. RESULTS: Reductions in diastolic blood pressure (-5 mmHg; P = 0.002), lactate (-0.3 mmol/L; P < 0.001), triglyceride, and LDL-cholesterol (-46% and -0.3 mmol/L; P < 0.001) were noted. Glucose tolerance and hyperinsulinemia improved (P < 0.001). Weight reduced by 0.9 ± 0.2 kg (P < 0.001) and fat-free mass by 0.6 kg (P = 0.04). Post hoc sensitivity analysis demonstrates that results in the subcohort that did not lose weight (n = 10) were directionally consistent. CONCLUSIONS: Isocaloric fructose restriction improved surrogate metabolic parameters in children with obesity and metabolic syndrome irrespective of weight change.

Effect of a High-Fructose Weight-Maintaining Diet on Lipogenesis and Liver Fat.

CONTEXT: Consumption of high-fructose diets promotes hepatic fatty acid synthesis (de novo lipogenesis [DNL]) and an atherogenic lipid profile. It is unclear whether these effects occur independent of positive energy balance and weight gain. OBJECTIVES: We compared the effects of a high-fructose, (25% of energy content) weight-maintaining diet to those of an isocaloric diet with the same macronutrient distribution but in which complex carbohydrate (CCHO) was substituted for fructose. DESIGN, SETTING, AND PARTICIPANTS: Eight healthy men were studied as inpatients for consecutive 9-day periods. Stable isotope tracers were used to measure fractional hepatic DNL and endogenous glucose production (EGP) and its suppression during a euglycemic-hyperinsulinemic clamp. Liver fat was measured by magnetic resonance spectroscopy. RESULTS: Weight remained stable. Regardless of the order in which the diets were fed, the high-fructose diet was associated with both higher DNL (average, 18.6 ± 1.4% vs 11.0 ± 1.4% for CCHO; P = .001) and higher liver fat (median, +137% of CCHO; P = .016) in all participants. Fasting EGP and insulin-mediated glucose disposal did not differ significantly, but EGP during hyperinsulinemia was greater (0.60 ± 0.07 vs 0.46 ± 0.06 mg/kg/min; P = .013) with the high-fructose diet, suggesting blunted suppression of EGP. CONCLUSION: Short-term high-fructose intake was associated with increased DNL and liver fat in healthy men fed weight-maintaining diets.

Effects of pravastatin on angiogenic and placental hypoxic imbalance in a mouse model of preeclampsia.

In order to determine the effects of pravastatin (Pra) on angiogenic and placental hypoxic imbalance in a model of preeclampsia induced by overexpression of soluble fms-like tyrosine kinase 1 (sFlt-1), we randomly allocated pregnant CD1 mice to injection with adenovirus-carrying sFlt-1 or mFc (control). The sFlt-1 group received either Pra (sFlt-1 + Pra) or water (sFlt-1). Mice were sacrificed at day 18, and serum levels of sFlt-1 and soluble endoglin (sEng) were measured. Placental expression of placental (PLGF) and vascular endothelial (VEGF) growth factors and other markers of angiogenesis and hypoxia were assayed. We observed that Pra treatment in sFlt-1 mice reduced sFlt-1 and sEng concentrations at day 18 to levels similar to control group. Placental PLGF and VEGF expression were upregulated, and markers of hypoxia downregulated to levels similar to control group. Hence, Pra prevents the rise in circulating antiangiogenic factors in a mouse model of preeclampsia. Statins may represent a novel approach to prevention of preeclampsia.

Effect of residents' previous laparoscopic surgery experience on initial robotic suturing experience.

Objective. To assess the impact of gynecology residents' previous laparoscopic experience on the learning curve of robotic suturing techniques and the value of initial structured teaching in dry lab prior to surgery. Methods. Thirteen gynecology residents with no previous robotic surgery experience were divided into Group 1, consisting of residents with 2 or fewer laparoscopic experiences, and Group 2, consisting of residents with 3 or more laparoscopic experiences. Group 1 had a dry-laboratory training in suturing prior to their initial experience in the operating room. Results. For all residents, it took on average 382 ± 159 seconds for laparoscopic suturing and 326 ± 196 seconds for robotic suturing (P = 0.12). Residents in Group 1 had a lower mean suture time than residents in Group 2 for laparoscopic suturing (P = 0.009). The residents in Group 2, however, had a lower mean suture time on the robot compared to Group 1 (P = 0.5). Conclusion. Residents with previous laparoscopic suturing experience may gain more from a robotic surgery experience than those with limited laparoscopic surgery experience. In addition, dry lab training is more efficient than hands-on training in the initial phase of teaching for both laparoscopic and robotic suturing skills.

Potent and broad anti-HIV-1 activity exhibited by a glycosyl-phosphatidylinositol-anchored peptide derived from the CDR H3 of broadly neutralizing antibody PG16.

PG9 and PG16 are two recently isolated quaternary-specific human monoclonal antibodies that neutralize 70 to 80% of circulating HIV-1 isolates. The crystal structure of PG16 shows that it contains an exceptionally long CDR H3 that forms a unique stable subdomain that towers above the antibody surface to confer fine specificity. To determine whether this unique architecture of CDR H3 itself is sufficient for epitope recognition and neutralization, we cloned CDR H3 subdomains derived from human monoclonal antibodies PG16, PG9, b12, E51, and AVF and genetically linked them to a glycosyl-phosphatidylinositol (GPI) attachment signal. Each fusion gene construct is expressed and targeted to lipid rafts of plasma membranes through a GPI anchor. Moreover, GPI-CDR H3(PG16, PG9, and E51), but not GPI-CDR H3(b12 and AVF), specifically neutralized multiple clades of HIV-1 isolates with a great degree of potency when expressed on the surface of transduced TZM-bl cells. Furthermore, GPI-anchored CDR H3(PG16), but not GPI-anchored CDR H3(AVF), specifically confers resistance to HIV-1 infection when expressed on the surface of transduced human CD4(+) T cells. Finally, the CDR H3 mutations (Y100HF, D100IA, and G7) that were previously shown to compromise the neutralization activity of antibody PG16 also abolished the neutralization activity of GPI-CDR H3(PG16). Thus, we conclude that the CDR H3 subdomain of PG16 neutralizes HIV-1 when targeted to the lipid raft of the plasma membrane of HIV-1-susceptible cells and that GPI-CDR H3 can be an alternative approach for determining whether the CDR H3 of certain antibodies alone can exert epitope recognition and neutralization.

Enhanced uridine bioavailability following administration of a triacetyluridine-rich nutritional supplement.

BACKGROUND: Uridine is a therapy for hereditary orotic aciduria and is being investigated in other disorders caused by mitochondrial dysfunction, including toxicities resulting from treatment with nucleoside reverse transcriptase inhibitors in HIV. Historically, the use of uridine as a therapeutic agent has been limited by poor bioavailability. A food supplement containing nucleosides, NucleomaxX®, has been reported to raise plasma uridine to supraphysiologic levels. METHODOLOGY/PRINCIPAL FINDINGS: Single- and multi-dose PK studies following NucleomaxX® were compared to single-dose PK studies of equimolar doses of pure uridine in healthy human volunteers. Product analysis documented that more than 90% of the nucleoside component of NucleomaxX® is in the form of triacetyluridine (TAU). Single and repeated dosing with NucleomaxX® resulted in peak plasma uridine concentrations 1-2 hours later of 150.9 ± 39.3 µM and 161.4 ± 31.5 µM, respectively, levels known to ameliorate mitochondrial toxicity in vitro. C(max) and AUC were four-fold higher after a single dose of NucleomaxX® than after uridine. No adverse effects of either treatment were observed. CONCLUSIONS/SIGNIFICANCE: NucleomaxX®, containing predominantly TAU, has significantly greater bioavailability than pure uridine in human subjects and may be useful in the management of mitochondrial toxicity.

Vif substitution enables persistent infection of pig-tailed macaques by human immunodeficiency virus type 1.

Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells in vitro. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4(+) T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication in vivo. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease.

GPI-anchored single chain Fv--an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike.

BACKGROUND: Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify. RESULTS: In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs. CONCLUSIONS: Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy.

Effects of insulin-like growth factor (IGF)-I/IGF-binding protein-3 treatment on glucose metabolism and fat distribution in human immunodeficiency virus-infected patients with abdominal obesity and insulin resistance.

CONTEXT: HIV-infected patients on antiretroviral therapy are at increased risk for excess visceral adiposity and insulin resistance. Treatment with GH decreases visceral adiposity but worsens glucose metabolism. IGF-I, which mediates many of the effects of GH, improves insulin sensitivity in HIV-negative individuals. OBJECTIVE: Our objective was to determine whether IGF-I, complexed to its major binding protein, IGF-binding protein-3 (IGFBP-3), improves glucose metabolism and alters body fat distribution in HIV-infected patients with abdominal obesity and insulin resistance. METHODS: We conducted a pilot, open-label study in 13 HIV-infected men with excess abdominal adiposity and insulin resistance to assess the effect of 3 months of treatment with IGF-I/IGFBP-3 on glucose metabolism and fat distribution. Glucose metabolism was assessed by oral glucose tolerance test and hyperinsulinemic-euglycemic clamp. Endogenous glucose production (EGP), gluconeogenesis, whole-body lipolysis, and de novo lipogenesis (DNL) were measured with stable isotope infusions. Body composition was assessed by dual-energy x-ray absorptiometry and abdominal computed tomography scan. RESULTS: Glucose tolerance improved and insulin-mediated glucose uptake increased significantly during treatment. EGP increased under fasting conditions, and suppression of EGP by insulin was blunted. Fasting triglycerides decreased significantly in association with a decrease in hepatic DNL. Lean body mass increased and total body fat decreased, whereas visceral adipose tissue did not change. CONCLUSIONS: Treatment with IGF-I/IGFBP-3 improved whole-body glucose uptake and glucose tolerance, while increasing hepatic glucose production. Fasting triglycerides improved, reflecting decreased DNL, and visceral adiposity was unchanged.

Comparison of surrogate and direct measurement of insulin resistance in chronic hepatitis C virus infection: impact of obesity and ethnicity.

UNLABELLED: Studies using surrogate estimates show high prevalence of insulin resistance in hepatitis C infection. This study prospectively evaluated the correlation between surrogate and directly measured estimates of insulin resistance and the impact of obesity and ethnicity on this relationship. Eighty-six nondiabetic, noncirrhotic patients with hepatitis C virus (age = 48 +/- 7 years, 74% male, 44% white, 22% African American, 26% Latino, 70% genotype 1) were categorized into normal-weight (body mass index [BMI] < 25, n = 30), overweight (BMI = 25-29.9, n = 38), and obese (BMI > or = 30, n = 18). Insulin-mediated glucose uptake was measured by steady-state plasma glucose (SSPG) concentration during a 240-minute insulin suppression test. Surrogate estimates included: fasting glucose and insulin, glucose/insulin, homeostasis model assessment (HOMA-IR), quantitative insulin sensitivity check index (QUICKI), insulin (I-AUC) and glucose (G-AUC) area under the curve during oral glucose tolerance test, and the Belfiore and Stumvoll indexes. All surrogate estimates correlated with SSPG, but the magnitude of correlation varied (r = 0.30-0.64). The correlation coefficients were highest in the obese. I-AUC had the highest correlation among all ethnic and weight groups (r = 0.57-0.77). HOMA-IR accounted for only 15% of variability in SSPG in the normal weight group. The common HOMA-IR cutoff of < or =3 to define insulin resistance had high misclassification rates especially in the overweight group independent of ethnicity. HOMA-IR > 4 had the lowest misclassification rate (75% sensitivity, 88% specificity). Repeat HOMA-IR measurements had higher within-person variation in the obese (standard deviation = 0.77 higher than normal-weight, 95% confidence interval = 0.25-1.30, P = 0.005). CONCLUSION: Because of limitations of surrogate estimates, caution should be used in interpreting data evaluating insulin resistance especially in nonobese, nondiabetic patients with HCV.

The acute effects of HIV protease inhibitors on insulin suppression of glucose production in healthy HIV-negative men.

BACKGROUND: The effects of different HIV protease inhibitors (PIs) on peripheral insulin resistance have been described, but less is known about their effects on insulin suppression of endogenous glucose production (EGP). METHODS: We tested the acute effects of 3 PIs, indinavir, ritonavir, and amprenavir, on EGP quantified by stable isotope techniques during the hyperinsulinemic, euglycemic clamp in 3 similar placebo-controlled protocols. RESULTS: EGP was higher with indinavir in the hyperinsulinemic state than with placebo (4.1 +/- 1.3 vs. 2.2 +/- 0.8 microg x kg(-1) x min(-1), P = 0.04). A trend toward higher EGP was seen with ritonavir (3.6 +/- 0.3 vs. 3.0 +/- 0.5 microg x kg(-1) x min(-1), P = 0.08). There was no evidence that amprenavir blunted insulin suppression of EGP compared with placebo (2.9 +/- 0.04 vs. 3.2 +/- 0.7 microg x kg(-1) x min(-1), P = 0.63). CONCLUSIONS: Some PIs can acutely blunt the ability of insulin to suppress EGP, but, as with insulin resistance, the effects of PIs on EGP are drug-specific, not class-specific.

The effects of recombinant human leptin on visceral fat, dyslipidemia, and insulin resistance in patients with human immunodeficiency virus-associated lipoatrophy and hypoleptinemia.

CONTEXT: Leptin deficiency is associated with dyslipidemia and insulin resistance in animals and humans with lipoatrophy; leptin replacement ameliorates these abnormalities. OBJECTIVE: The objective of the study was to evaluate the effects of leptin therapy in lipoatrophic HIV-infected patients with dyslipidemia and hypoleptinemia. DESIGN: This was a 6-month, open-label, proof-of-principle pilot study. SETTING: Metabolic ward studies were performed before and 3 and 6 months after leptin treatment. PARTICIPANTS: Participants included eight HIV-infected men with lipoatrophy, fasting triglycerides greater than 300 mg/dl, and serum leptin less than 3 ng/ml. INTERVENTION: Recombinant human leptin was given by sc injection (0.01 mg/kg and 0.03 mg/kg twice daily for successive 3 month periods). OUTCOME MEASURES: Measures included fat distribution by magnetic resonance imaging and dual-energy X-ray absorptiometry; fasting lipids; insulin sensitivity by euglycemic hyperinsulinemic clamp; endogenous glucose production, gluconeogenesis, glycogenolysis, and whole-body lipolysis by stable isotope tracer studies; oral glucose tolerance testing; liver fat by proton magnetic resonance spectroscopy; and safety. RESULTS: Visceral fat decreased by 32% (P = 0.001) with no changes in peripheral fat. There were significant decreases in fasting total (15%, P = 0.012), direct low-density lipoprotein (20%, P = 0.002), and non-high-density lipoprotein (19%, P = 0.005) cholesterol. High-density lipoprotein cholesterol increased. Triglycerides, whole-body lipolysis, and free fatty acids decreased during fasting and hyperinsulinemia. Fasting insulin decreased. Endogenous glucose production decreased during fasting and hyperinsulinemia, providing evidence of improved hepatic insulin sensitivity. Leptin was well tolerated but decreased lean mass. CONCLUSIONS: Leptin treatment was associated with marked improvement in dyslipidemia. Hepatic insulin sensitivity improved and lipolysis decreased. Visceral fat decreased with no exacerbation of peripheral lipoatrophy. Results from this pilot study suggest that leptin warrants further study in patients with HIV-associated lipoatrophy.

Indinavir increases glucose production in healthy HIV-negative men.

In the absence of HIV infection, changes in adipose tissue and lipid levels, HIV protease inhibitor therapy increases fasting glucose levels,suggestive of hepatic insulin resistance. After 4 weeks of indinavir treatment in nine HIV-negative healthy men, fasting glucose production and glycogenolysis were significantly increased. During the euglycemic hyperinsulinemic clamp, indinavir blunted the ability of insulin to suppress glucose production. Therefore, indinavir worsens hepatic insulin sensitivity, increasing endogenous glucose production.

Effects of recombinant human growth hormone on hepatic lipid and carbohydrate metabolism in HIV-infected patients with fat accumulation.

We recently reported that treatment with a pharmacologic dose of recombinant human growth hormone (GH) resulted in a significant loss of body fat and gain in lean tissue in HIV-infected patients with syndromes of fat accumulation. However, insulin-mediated glucose disposal decreased transiently after one month of GH therapy. The present paper focuses on the changes of hepatic carbohydrate and fat metabolism associated with GH treatment in the same subjects. We assessed hepatic insulin sensitivity under both fasting and hyperinsulinemic-euglycemic clamp conditions prior to and after one and six months of GH treatment (3 mg/day) in five patients using stable isotope tracer techniques. Indirect calorimetry, and measurements of lipid concentrations. Fasting endogenous glucose production (EGP) increased significantly at one month (12.0 +/- 0.7 to 14.9 +/- 0.9 micromol/kg/min, P < 0.03), and the increase was sustained at six months of GH treatment (14.0 +/- 1.1 micromol/kg/min, NS). This increase in EGP was driven in part by increased glucogenesis (GNG) (3.5 +/- 0.9 to 5.2 +/- 0.9 and 5.8 +/-1.2 micromol/kg/min, n = 4, P < 0.01 and P < 0.01 at one and six months, respectively); small changes in hepatic glycogenolysis also contributed. Sustained increases in lipolysis and progressive decreases in hepatic fractional de novo lipogenesis (DNL) and triglyceride concentrations occurred with GH treatment. These changes were accompanied by an improved lipid profile with a significant increase in HDL cholesterol and significant decreases in total and LDL cholesterol and triglyceride levels, the latter consistent with the decrease in hepatic DNL. During a hyperinsulinemic-euglycemic glucose clamp, EGP and GNG were markedly suppressed compared to the corresponding time points under fasting conditions, albeit less so when measured after one month of GH treatment. Thus, in HIV-infected patients with abnormal fat distribution, pharmacologic doses of GH improved the overall lipid profile, but worsened glucose homeostasis under both fasting and hyperinsulinemic conditions. The combined implications of these positive and negative metabolic effects for cardiovascular disease risk remain unknown.