Marc K. Hellerstein

Consuming fructose-sweetened, not glucose-sweetened, beverages increases visceral adiposity and lipids and decreases insulin sensitivity in overweight/obese humans

Studies in animals have documented that, compared with glucose, dietary fructose induces dyslipidemia and insulin resistance. To assess the relative effects of these dietary sugars during sustained consumption in humans, overweight and obese subjects consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Although both groups exhibited similar weight gain during the intervention, visceral adipose volume was significantly increased only in subjects consuming fructose. Fasting plasma triglyceride concentrations increased by approximately 10% during 10 weeks of glucose consumption but not after fructose consumption. In contrast, hepatic de novo lipogenesis (DNL) and the 23-hour postprandial triglyceride AUC were increased specifically during fructose consumption. Similarly, markers of altered lipid metabolism and lipoprotein remodeling, including fasting apoB, LDL, small dense LDL, oxidized LDL, and postprandial concentrations of remnant-like particle-triglyceride and -cholesterol significantly increased during fructose but not glucose consumption. In addition, fasting plasma glucose and insulin levels increased and insulin sensitivity decreased in subjects consuming fructose but not in those consuming glucose. These data suggest that dietary fructose specifically increases DNL, promotes dyslipidemia, decreases insulin sensitivity, and increases visceral adiposity in overweight/obese adults.

Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia

BACKGROUND In many patients with human immunodeficiency virus (HIV) infection, therapy with potent antiretroviral drugs does not result in complete suppression of HIV replication. The effect of cessation of therapy in these patients is unknown. METHODS Sixteen patients who had a plasma HIV RNA level of more than 2500 copies per milliliter during combination antiretroviral-drug therapy were randomly assigned, in a 2:1 ratio, to discontinue or continue therapy. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility were measured weekly. Viral replicative capacity was measured at base line and at week 12. RESULTS Discontinuation of therapy for 12 weeks was associated with a median decrease in the CD4 cell count of 128 cells per cubic millimeter and an increase in the plasma HIV RNA level of 0.84 log copies per milliliter. Virus from all patients with detectable resistance at entry became susceptible to HIV-protease inhibitors within 16 weeks after the discontinuation of therapy. Drug susceptibility began to increase a median of six weeks after the discontinuation of therapy and was temporally associated with increases in plasma HIV RNA levels and decreases in CD4 cell counts. Viral replicative capacity, measured by means of a recombinant-virus assay, was low at entry into the study and increased after therapy was discontinued. Despite the loss of detectable resistance in plasma, resistant virus was cultured from peripheral-blood mononuclear cells in five of nine patients who could be evaluated. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility remained stable in the patients who continued therapy. CONCLUSIONS Despite the presence of reduced drug susceptibility, antiretroviral-drug therapy can provide immunologic and virologic benefit. This benefit reflects continued antiviral-drug activity and the maintenance of a viral population with a reduced replicative capacity.

Directly measured kinetics of circulating T lymphocytes in normal and HIV-1-infected humans

The dynamic basis for T-cell depletion in late-stage HIV-1 disease remains controversial. Using a new, non-radioactive, endogenous labeling technique1, we report direct measurements of circulating T-cell kinetics in normal and in HIV-1-infected humans. In healthy, HIV-1-seronegative subjects, CD4+ and CD8+ T cells had half-lives of 87 days and 77 days, respectively, with absolute production rates of 10 CD4+ T cells/μl per day and 6 CD8+ T cells/μl per day. In untreated HIV-1-infected subjects (with a mean CD4 level of 342 cells/μl), the half-life of each subpopulation was less than 1/3 as long as those of healthy, HIV-1-seronegative subjects but was not compensated by an increased absolute production rate of CD4+ T cells. After viral replication was suppressed by highly active antiretroviral therapy for 12 weeks, the production rates of circulating CD4+ and CD8+ T cells were considerably elevated; the kinetic basis of increased CD4 levels was greater production, not a longer half-life, of circulating cells. These direct measurements indicate that CD4+ T-cell lymphopenia is due to both a shortened survival time and a failure to increase the production of circulating CD4+ T cells. Our results focus attention on T-cell production systems in the pathogenesis of HIV-1 disease and the response to antiretroviral therapy.

In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells

Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.

Nutritional recommendations for the management of sarcopenia.

The Society for Sarcopenia, Cachexia, and Wasting Disease convened an expert panel to develop nutritional recommendations for prevention and management of sarcopenia. Exercise (both resistance and aerobic) in combination with adequate protein and energy intake is the key component of the prevention and management of sarcopenia. Adequate protein supplementation alone only slows loss of muscle mass. Adequate protein intake (leucine-enriched balanced amino acids and possibly creatine) may enhance muscle strength. Low 25(OH) vitamin D levels require vitamin D replacement.

Measurement of de novo hepatic lipogenesis in humans using stable isotopes.

Direct measurement of de novo lipogenesis has not previously been possible in humans. We measured de novo hepatic lipogenesis in normal men by means of stable isotopes and by combining the acetylated-xenobiotic probe technique with mass isotopomer analysis of secreted very low density lipoprotein-fatty acids (VLDL-FA). Sulfamethoxazole (SMX) was administered with [13C]acetate during an overnight fast followed by refeeding with intravenous glucose (7-10 mg/kg of weight per min), oral Ensure (7-10 mg of carbohydrate/kg of weight per min), or a high-carbohydrate mixed-meal breakfast (3.5 g of carbohydrate/kg of weight). Respiratory quotients remained less than 1.0. High-performance liquid chromatography/mass spectrometry-determined enrichments in SMX-acetate attained stable plateau values, and hepatic acetyl-coenzyme A (CoA) dilution rate did not increase with refeeding (approximately 0.024 mmol/kg per min). The fraction of VLDL-palmitate derived from de novo lipogenesis was only 0.91 +/- 0.27% (fasted) and 1.64-1.97% (fed). For stearate, this was 0.37 +/- 0.08% and 0.47-0.64%. Precursor enrichments predicted from isotopomer ratios were close to measured SMX-acetate enrichments, indicating that SMX-acetate samples the true lipogenic acetyl-CoA pool. Stearate synthesis was less than palmitate and the two did not move in parallel. Estimated total VLDL-FA synthesis is less than 500 mg/day. Thus, de novo hepatic lipogenesis is a quantitatively minor pathway, consistent with gas exchange estimates; fatty acid futile cycling (oxidation/resynthesis) is not thermogenically significant; and synthesis rates of different nonessential fatty acids by human liver are not identical in nonoverfed normal men. The contribution and regulation of de novo lipogenesis in other settings can be studied using this technique.

Interleukin-1-induced anorexia in the rat. Influence of prostaglandins.

The anorexia associated with acute and chronic inflammatory or infectious conditions is poorly understood. Our objectives were to explore the anorexigenic effects of interleukin-1 (IL-1) in the rat. Recombinant human (rh) IL-1 beta, murine (rm) IL-1 alpha and to a lesser extent rhIL-1 alpha significantly reduced food intake at greater than or equal to 4.0 micrograms/kg i.p. but not at lower doses, in young (200-250 g) meal-fed rats on chow diets. The anorexic effect appears to be mediated by prostaglandins since pretreatment with ibuprofen completely blocked it, and a fish oil based diet abolished it, in comparison to corn oil or chow diets. Fish oil feeding also decreased basal and IL-1 stimulated prostaglandin E2 production by tissues in vitro (liver, brain, peritoneal macrophages) and in the whole body. Constant intravenous infusions of lower doses of IL-1 also diminished food intake, though intravenous boluses did not (reflecting rapid renal clearance). Chronic daily administration of IL-1 caused persistent inhibition of food intake for 7-17 d in chow and corn oil fed rats, but had no effect in fish oil fed rats. There was an attenuation of the effect (tachyphylaxis) after 7 d in corn oil and chow fed rats, but slowed weight gain and lower final weights were observed after 17-32 d of daily IL-1. Old (18-20 mo Fisher 344) rats showed less sensitivity to IL-1 induced anorexia. In conclusion, IL-1 is anorexigenic in the rat, but this is influenced by the structural form of IL-1, the route and chronicity of administration, the source of dietary fat, and the age of the animal. The ability of prior fat intake to influence the anorexic response to IL-1 represents a novel nutrient-nutrient interaction with potential therapeutic implications.

A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility

Insulin resistance is necessary but not sufficient for the development of type 2 diabetes. Diabetes results when pancreatic beta-cells fail to compensate for insulin resistance by increasing insulin production through an expansion of beta-cell mass or increased insulin secretion. Communication between insulin target tissues and beta-cells may initiate this compensatory response. Correlated changes in gene expression between tissues can provide evidence for such intercellular communication. We profiled gene expression in six tissues of mice from an obesity-induced diabetes-resistant and a diabetes-susceptible strain before and after the onset of diabetes. We studied the correlation structure of mRNA abundance and identified 105 co-expression gene modules. We provide an interactive gene network model showing the correlation structure between the expression modules within and among the six tissues. This resource also provides a searchable database of gene expression profiles for all genes in six tissues in lean and obese diabetes-resistant and diabetes-susceptible mice, at 4 and 10 wk of age. A cell cycle regulatory module in islets predicts diabetes susceptibility. The module predicts islet replication; we found a strong correlation between (2)H(2)O incorporation into islet DNA in vivo and the expression pattern of the cell cycle module. This pattern is highly correlated with that of several individual genes in insulin target tissues, including Igf2, which has been shown to promote beta-cell proliferation, suggesting that these genes may provide a link between insulin resistance and beta-cell proliferation.

Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance

Low-fat, high-carbohydrate (LF/HC) diets commonly elevate plasma triglyceride (TG) concentrations, but the kinetic mechanisms responsible for this effect remain uncertain. Subjects with low TG (normolipidemic [NL]) and those with moderately elevated TG (hypertriglyceridemic [HTG]) were studied on both a control and an LF/HC diet. We measured VLDL particle and TG transport rates, plasma nonesterified fatty acid (NEFA) flux, and sources of fatty acids used for the assembly of VLDL-TG. The LF/HC diet resulted in a 60% elevation in TG, a 37% reduction in VLDL-TG clearance, and an 18% reduction in whole-body fat oxidation, but no significant change in VLDL-apo B or VLDL-TG secretion rates. Significant elevations in fasting apo B-48 concentrations were observed on the LF/HC in HTG subjects. In both groups, fasting de novo lipogenesis was low regardless of diet. The NEFA pool contributed the great majority of fatty acids to VLDL-TG in NL subjects on both diets, whereas in HTG subjects, the contribution of NEFA was somewhat lower overall and was reduced further in individuals on the LF/HC diet. Between 13% and 29% of VLDL-TG fatty acids remained unaccounted for by the sum of de novo lipogenesis and plasma NEFA input in HTG subjects. We conclude that (a) whole-food LF/HC diets reduce VLDL-TG clearance and do not increase VLDL-TG secretion or de novo lipogenesis; (b) sources of fatty acids for assembly of VLDL-TG differ between HTG and NL subjects and are further affected by diet composition; (c) the presence of chylomicron remnants in the fasting state on LF/HC diets may contribute to elevated TG levels by competing for VLDL-TG lipolysis and by providing a source of fatty acids for hepatic VLDL-TG synthesis; and (d) the assembly, production, and clearance of elevated plasma VLDL-TG in response to LF/HC diets therefore differ from those for elevated TG on higher-fat diets.

Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of 2H2O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% 2H2O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0–200 μg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose–response relationship up to the 100 μg dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose–response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the 2H2O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270–400 days and die-away values after 2H2O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1–1.5% new cells per day, whereas obese ad libitum-fed ob/ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term 2H2O enrichments in body water were achieved by daily 2H2O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body 2H2O enrichment (≈3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with 2H2O was 0.056 (CD4+) and 0.043 (CD8+) (replacement rate <0.1% per day). In summary, 2H2O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells.