Biomedical subjects

R A Neese

Publications and source records attributed to R A Neese.

At least 37 records · Page 2Linked to original sources

PubMed1999

De novo lipogenesis, lipid kinetics, and whole-body lipid balances in humans after acute alcohol consumption.

BACKGROUND: Acute alcohol intake is associated with changes in plasma lipid concentrations and whole-body lipid balances in humans. The quantitative roles of hepatic de novo lipogenesis (DNL) and plasma acetate production in these changes have not been established, however. OBJECTIVE: We used stable-isotope mass spectrometric methods with indirect calorimetry to establish the metabolic basis of changes in whole-body lipid balances in healthy men after consumption of 24 g alcohol. DESIGN: Eight healthy subjects were studied and DNL (by mass-isotopomer distribution analysis), lipolysis (by dilution of [1,2,3,4-(13)C(4)]palmitate and [(2)H(5)]glycerol), conversion of alcohol to plasma acetate (by incorporation from [1-(13)C(1)]ethanol), and plasma acetate flux (by dilution of [1-(13)C(1)]acetate) were measured. RESULTS: The fractional contribution from DNL to VLDL-triacylglycerol palmitate rose after alcohol consumption from 2 +/- 1% to 30 +/- 8%; nevertheless, the absolute rate of DNL (0.8 g/6 h) represented <5% of the ingested alcohol dose; 77 +/- 13% of the alcohol cleared from plasma was converted directly to acetate entering plasma. Acetate flux increased 2.5-fold after alcohol consumption. Adipose release of nonesterified fatty acids into plasma decreased by 53% and whole-body lipid oxidation decreased by 73%. CONCLUSIONS: We conclude that the consumption of 24 g alcohol activates the hepatic DNL pathway modestly, but acetate produced in the liver and released into plasma inhibits lipolysis, alters tissue fuel selection, and represents the major quantitative fate of ingested ethanol.

Acetates↗

PubMed1999

Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.

Mass isotopomer distribution analysis (MIDA) is a technique for measuring the synthesis of biological polymers. First developed approximately eight years ago, MIDA has been used for measuring the synthesis of lipids, carbohydrates, and proteins. The technique involves quantifying by mass spectrometry the relative abundances of molecular species of a polymer differing only in mass (mass isotopomers), after introduction of a stable isotope-labeled precursor. The mass isotopomer pattern, or distribution, is analyzed according to a combinatorial probability model by comparing measured abundances to theoretical distributions predicted from the binomial or multinomial expansion. For combinatorial probabilities to be applicable, a labeled precursor must therefore combine with itself in the form of two or more repeating subunits. MIDA allows dilution in the monomeric (precursor) and polymeric (product) pools to be determined. Kinetic parameters can then be calculated (e.g., replacement rate of the polymer, fractional contribution from the endogenous biosynthetic pathway, absolute rate of biosynthesis). Several issues remain unresolved, however. We consider here the impact of various deviations from the simple combinatorial probability model of biosynthesis and describe the analytic requirements for successful use of MIDA. A formal mathematical algorithm is presented for generating tables and equations (APPENDIX), on the basis of which effects of various confounding factors are simulated. These include variations in natural isotope abundances, isotopic disequilibrium in the precursor pool, more than one biosynthetic precursor pool, incorrect values for number of subunits present, and concurrent measurement of turnover from exogenously labeled polymers. We describe a strategy for testing whether isotopic inhomogeneity (e.g., an isotopic gradient or separate biosynthetic sites) is present in the precursor pool by comparing higher-mass (multiply labeled) to lower-mass (single- and double-labeled) isotopomer patterns. Also, an algebraic correction is presented for calculating fractional synthesis when an incomplete ion spectrum is monitored, and an approach for assessing the sensitivity of biosynthetic parameters to measurement error is described. The different calculation algorithms published for MIDA are compared; all share a common model, use overlapping solutions to computational problems, and generate identical results. Finally, we discuss the major practical issue for using MIDA at present: quantitative inaccuracy of instruments. The nature and causes of analytic inaccuracy, strategies for evaluating instrument performance, and guidelines for optimizing accuracy and reducing impact on biosynthetic parameters are suggested. Adherence to certain analytic guidelines, particularly attention to concentration effects on mass isotopomer ratios and maximizing enrichments in the isotopomers of interest, reduces error. Improving instrument accuracy for quantification of isotopomer ratios is perhaps the highest priority for this field. In conclusion, MIDA remains the "equation for biosynthesis," but attention to potentially confounding factors and analytic performance is required for optimal application.

Isotopes↗

PubMed1999

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.

Adult↗

PubMed1998

Contribution of newly synthesized cholesterol to rat plasma and bile determined by mass isotopomer distribution analysis: bile-salt flux promotes secretion of newly synthesized cholesterol into bile.

To quantify the contribution of newly synthesized cholesterol to total plasma and biliary cholesterol under physiological conditions, unrestrained rats were infused intravenously with [1-13C]acetate (0. 6mmol/h per kg) from 12:00 to 24:00 h, and fractional and absolute cholesterol-synthesis rates were determined by mass isotopomer distribution analysis (MIDA). As bile diversion leads to changes in cholesterol metabolism, rats were equipped with permanent catheters in the bile duct and duodenum, allowing sampling of small amounts of bile from an intact enterohepatic circulation. For comparison, rats with chronic bile diversion were also studied. Fractional synthesis of plasma cholesterol was 10.8+/-1.7% (mean+/-S.D.) after 12 h in rats with intact circulation. Fractional synthesis of biliary cholesterol was significantly higher than that of plasma cholesterol, i.e. 16.5+/-2.0% (P<0.05) after 12 h. In contrast, no differences between fractional synthesis of cholesterol in plasma and bile were found in bile-diverted animals (31.8+/-2.1 and 33.1+/-3.3% respectively after 12 h). The calculated absolute rate of cholesterol biosynthesis increased from 53+/-10 to 221+/-19 micromol/day per kg after bile diversion. A comparison of MIDA results with those obtained from balance studies indicated that MIDA does not assess total body synthesis in rats, presumably because of incomplete equilibration of newly synthesized molecules with cholesterol in the plasma compartment. These studies demonstrate that the contribution of newly synthesized cholesterol to biliary cholesterol is higher than to plasma cholesterol under physiological conditions, probably reflecting bile-salt-induced secretion of newly formed cholesterol by the periportal hepatocytes.

Animals↗

PubMed1998

Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans.

A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H]thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2 and H9 cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.

Animals↗

PubMed1998

De novo lipogenesis predicts short-term body-composition response by bioelectrical impedance analysis to oral nutritional supplements in HIV-associated wasting.

We studied the effects of enteral supplements on protein and energy intakes, body composition, energy expenditure, and gastrointestinal histology in 49 subjects with human immunodeficiency virus-associated weight loss (12.7 +/- 0.9% of body wt). We also determined whether a stable-isotope mass spectrometric measurement at baseline might predict the short-term response of fat-free mass (FFM) measured by bioelectrical impedance analysis. Thirty-nine subjects completed the study after being randomly assigned to receive either a whole-protein-based (n = 22) or a peptide-based (n = 17) formula. A nonsupplemented, nonrandomly assigned group (n = 13) was followed concurrently. Both formulas were well tolerated. Voluntary intakes of energy and protein from nonsupplement sources decreased significantly during supplementation [by 819-1638 kJ (196-382 kcal)/d and 5.6-14.4 g protein/d, respectively; P < 0.01] but to a lesser extent than the intake from the supplement [2300-2510 kJ(550-600 kcal)/d and 19-28 g protein/d, respectively], so that net increases in intakes of protein and energy (P < 0.03), as well as of several vitamins and trace elements were increased. Nevertheless, the mean FFM did not increase for the group as a whole, although there was considerable interindividual heterogeneity. Changes in FFM at 6 wk were significantly inversely correlated (r = 0.65, P < 0.01) with baseline synthesis of fat (de novo hepatic lipogenesis), but not with other potential measures of energy intake (insulin-like growth factor 1 or its binding protein) or inflammation (soluble tumor necrosis factor receptors I or II). The prospective identification of FFM response by measurement of de novo hepatic lipogenesis supported the hypothesis that the subset of wasting patients whose FFM is unresponsive to nutrient supplementation have altered nutrient metabolism.

Adult↗

PubMed1998

The inhibition of gluconeogenesis following alcohol in humans.

Accurate quantification of gluconeogenic flux following alcohol ingestion in overnight-fasted humans has yet to be reported. [2-13C1]glycerol, [U-13C6]glucose, [1-2H1]galactose, and acetaminophen were infused in normal men before and after the consumption of 48 g alcohol or a placebo to quantify gluconeogenesis, glycogenolysis, hepatic glucose production, and intrahepatic gluconeogenic precursor availability. Gluconeogenesis decreased 45% vs. the placebo (0.56 +/- 0.05 to 0.44 +/- 0.04 mg. kg-1. min-1 vs. 0.44 +/- 0.05 to 0.63 +/- 0.09 mg. kg-1. min-1, respectively, P < 0. 05) in the 5 h after alcohol ingestion, and total gluconeogenic flux was lower after alcohol compared with placebo. Glycogenolysis fell over time after both the alcohol and placebo cocktails, from 1.46-1. 47 mg. kg-1. min-1 to 1.35 +/- 0.17 mg. kg-1. min-1 (alcohol) and 1. 26 +/- 0.20 mg. kg-1. min-1, respectively (placebo, P < 0.05 vs. baseline). Hepatic glucose output decreased 12% after alcohol consumption, from 2.03 +/- 0.21 to 1.79 +/- 0.21 mg. kg-1. min-1 (P < 0.05 vs. baseline), but did not change following the placebo. Estimated intrahepatic gluconeogenic precursor availability decreased 61% following alcohol consumption (P < 0.05 vs. baseline) but was unchanged after the placebo (P < 0.05 between treatments). We conclude from these results that gluconeogenesis is inhibited after alcohol consumption in overnight-fasted men, with a somewhat larger decrease in availability of gluconeogenic precursors but a smaller effect on glucose production and no effect on plasma glucose concentrations. Thus inhibition of flux into the gluconeogenic precursor pool is compensated by changes in glycogenolysis, the fate of triose-phosphates, and peripheral tissue utilization of plasma glucose.

Acetaminophen↗

PubMed1998

VLDL-triglyceride production after alcohol ingestion, studied using [2-13C1] glycerol.

We used [2-13C1]glycerol to characterize very low density lipoprotein (VLDL)-triglyceride kinetics and intrahepatic glycerol metabolism in normal men (n = 4) after alcohol (EtOH) ingestion. [2-13C1]glycerol was infused before and after the consumption of 48 g EtOH or a placebo. Three additional subjects also received [1-13C1]acetate in addition to the [2-13C1]glycerol with EtOH treatment. Incorporation of tracer into the glycerol or fatty acid moiety of VLDL-triglyceride was measured by gas chromatography-mass spectrometry and used to calculate VLDL-triglyceride production rates. Intrahepatic triose-phosphate enrichments were also calculated based on mass isotopomer distribution analysis of plasma glucose. There was no difference in VLDL-triglyceride production rates after 48 g EtOH (11.9 +/- 3.7 mg/kg/h) or placebo (14.7 +/- 3. 3 mg/kg/h). The VLDL-triglyceride rate constants calculated by kinetic modeling using the glycerol and acetate tracers in the combined isotope infusion subjects were very closely correlated (r 2 = 0.94). The peak VLDL-glycerol enrichments after EtOH were 22.5 +/- 3.3% versus 7.6 +/- 0.8% after placebo (P < 0.001), while intrahepatic triose-phosphate enrichments were 19.8 +/- 1.3% and 13. 1 +/- 1.2% (P < 0.001), respectively. Moreover, the calculated asymptotic VLDL-glycerol enrichments (representing the hepatic alpha-glycerol phosphate enrichment) were significantly higher after EtOH than placebo. The higher ratio of VLDL-glycerol to triose-phosphate labeling after EtOH suggests a metabolic block at glycerol 3-phosphate dehydrogenase. We conclude that consumption of 48 g EtOH does not increase VLDL-triglyceride production in normal men but does cause accumulation of tracer in hepatic alpha-glycerol phosphate.

Adult↗

PubMed1997

Hepatic gluconeogenic fluxes and glycogen turnover during fasting in humans. A stable isotope study.

Fluxes through intrahepatic glucose-producing metabolic pathways were measured in normal humans during overnight or prolonged (60 h) fasting. The glucuronate probe was used to measure the turnover and sources of hepatic UDP-glucose; mass isotopomer distribution analysis from [2-13C1]glycerol for gluconeogenesis and UDP-gluconeogenesis; [U-13C6]glucose for glucose production (GP) and the direct UDP-glucose pathway; and [1-2H1]galactose for UDP-glucose flux and retention in hepatic glycogen. After overnight fasting, GP (fluxes in milligram per kilogram per minute) was 2.19+/-0.09, of which 0.79 (36%) was from gluconeogenesis, 1.40 was from glycogenolysis, 0.30 was retained in glycogen via UDP-gluconeogenesis, and 0.17 entered hepatic UDP-glucose by the direct pathway. Thus, total flux through the gluconeogenic pathway (1.09) represented 54% of extrahepatic glucose disposal (2.02) and the net hepatic glycogen depletion rate was 0.93 (46%). Prolonging [2-13C1]glycerol infusion slowly increased measured fractional gluconeogenesis. In response to prolonged fasting, GP was lower (1. 43+/-0.06) and fractional and absolute gluconeogenesis were higher (78+/-2% and 1.11+/-0.07, respectively). The small but nonzero glycogen input to plasma glucose (0.32+/-0.03) was completely balanced by retained UDP-gluconeogenesis (0.31+/-0.02). Total gluconeogenic pathway flux therefore accounted for 99+/-2% of GP, but with a glycogen cycle interposed. Prolonging isotope infusion to 10 h increased measured fractional gluconeogenesis and UDP-gluconeogenesis to 84-96%, implying replacement of glycogen by gluconeogenic-labeled glucose. Moreover, after glucagon administration, GP (1.65), recovery of [1-2H1]galactose label in plasma glucose (25%) and fractional gluconeogenesis (91%) increased, such that 78% (0.45/0.59) of glycogen released was labeled (i.e., of recent gluconeogenic origin). In conclusion, hepatic gluconeogenic flux into glycogen and glycogen turnover persist during fasting in humans, reconciling inconsistencies in the literature and interposing another locus of control in the normal pathway of GP.

Carbon Isotopes↗

PubMed1997

Hepatic glucose-6-phosphatase flux and glucose phosphorylation, cycling, irreversible disposal, and net balance in vivo in rats. Measurement using the secreted glucuronate technique.

Measurement of hepatic glucose production (HGP) by standard isotope dilution reveals only the net release of glucose from the liver, not the flux across glucose-6-phosphatase ([G6Pase] or total hepatic glucose output), hepatic glucose cycling (HGC), irreversible glucose disposal into glycogen in the liver (hepatic Rd), or net hepatic glucose balance. We describe two independent isotopic techniques for measuring these parameters in vivo, both of which use secreted glucuronate (GlcUA). HGC can be quantified by measuring a correction factor for glucose label retained in hepatic glucose-6-phosphate (G6P), sampled as GlcUA. A complementary technique for measuring total hepatic glucose output is also described (reverse dilution), requiring administration of no labeled glucose but instead a labeled gluconeogenic precursor and unlabeled glucose. Hepatic Rd is calculated by multiplying the rate of appearance (Ra) of hepatic UDP-glucose ([UDP-glc] based on dilution of labeled galactose in GlcUA) times the direct entry of glucose into hepatic UDP-glc and the fraction of labeled UDP-glc retained in the liver. The sum of hepatic Rd plus HGC represents the total hepatic glucose phosphorylation rate. Rats received intravenous (i.v.) glucose infusions at a rate of 15 to 30 mg/kg/min after a 24-hour fast. Despite a suppression of net HGP more than 50%, total hepatic glucose output was not significantly decreased, because of increased HGC. Total hepatic glucose output calculated by reverse dilution yielded similar results during i.v. glucose infusions at 15 mg/kg/min, although values were higher than obtained by the correction-factor method at 30 mg/kg/min. The fraction of labeled UDP-glc released into blood glucose, representing a hepatic glycogen cycle, decreased from 35% (fasted) to nearly 0% (i.v. glucose 30 mg/kg/min). Hepatic Rd was 1.4, 4.6, and 7.5 mg/kg/min (fasted and i.v. glucose 15 and 30 mg/kg/min, respectively); total hepatic glucose phosphorylation increased substantially (from 4.2 to 8.5 to 12.7 mg/kg/min) and net hepatic glucose balance changed from negative to positive during i.v. glucose. In conclusion, hepatic G6Pase flux, glucose phosphorylation, HGC, disposal of glucose into glycogen, and net glucose balance can be measured noninvasively in vivo under various metabolic conditions by techniques involving the GlcUA probe.

Animals↗

PubMed1997

Altered fluxes responsible for reduced hepatic glucose production and gluconeogenesis by exogenous glucose in rats.

The net release of glucose from the liver, or hepatic glucose production (HGP), and apparent gluconeogenesis (GNG) are reduced by exogenous glucose. We investigated the changes in metabolic fluxes responsible. Flux through the hepatic GNG pathway was quantified by mass isotopomer distribution analysis (MIDA) from [2-13C]glycerol. Unidirectional flux across hepatic glucose-6-phosphatase (G-6-Pase), or total hepatic glucose output (THGO), and hepatic glucose cycling (HGC) were also measured by using glucuronate (GlcUA) to correct for glucose 6-phosphate (G-6-P) labeling. Infusion of glucose (15-30 mg.kg-1.min-1 iv) to 24 h-fasted rats caused two important metabolic alterations. First was a significant increase in hepatic glucose uptake and HGC: > 60% of THGO was from HGC. Second, although flux through hepatic G-6-P increased (from 15.7 to 17.7-22.7 mg.kg-1.min-1), the partitioning of G-6-P flux changed markedly [from 30-35% to 55-60% entering UDP-glucose (UDP-Glc), P < 0.01]. Total flux through the GNG pathway remained active during intravenous glucose, but increased partitioning into UDP-Glc lowered GNG flux plasma glucose by 50%. In summary, the suppression of HGP and GNG flux into glucose is not primarily due to reduced carbon flow through hepatic G-6-Pase or the hepatic GNG pathway. THGO persists, but hepatic G-6-P is derived increasingly from plasma glucose, and flow through GNG persists, but the partitioning coefficient of G-6-P into UDP-Glc doubles. These adjustments permit net HGP to fall despite increased total production of hepatic G-6-P during administration of glucose.

Animals↗

PubMed1997

Glucose homeostasis in children with falciparum malaria: precursor supply limits gluconeogenesis and glucose production.

To evaluate glucose kinetics in children with falciparum malaria, basal glucose production and gluconeogenesis and an estimate of the flux of the gluconeogenic precursors were measured in Kenyan children with uncomplicated falciparum malaria before (n = 11) and during infusion of alanine (1.5 mg/kg.min; n = 6). Glucose production was measured by [6,6-2H2]glucose, gluconeogenesis by mass isotopomer distribution analysis of glucose labeled by [2-13C]glycerol. Basal plasma glucose concentration ranged from 2.1-5.5 mmol/L, and basal glucose production ranged from 3.3-7.3 mg/kg.min. Glucose production was largely derived from gluconeogenesis (73 +/- 4%; range, 52-93%). During alanine infusion, plasma glucose increased by 0.4 mmol/L (P = 0.03), glucose production increased by 0.8 mg/kg.min (P = 0.02), and gluconeogenesis increased by 0.8 mg/kg.min (P = 0.04). We conclude that glucose production in children with uncomplicated falciparum malaria is largely dependent on gluconeogenesis. However, gluconeogenesis is potentially limited by insufficient precursor supply. These data indicate that in children with falciparum malaria, gluconeogenesis fails to compensate in the presence of decreased glycogen flux to glucose, increasing the risk of hypoglycemia.

Alanine↗

PubMed1996

Effects of dietary n-3 fatty acid supplementation in men with weight loss associated with the acquired immune deficiency syndrome: Relation to indices of cytokine production.

Cytokines may be involved in weight loss and disturbances of metabolism associated with human immunodeficiency virus (HIV) infection. Dietary n-3 fatty acids reduce the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) by peripheral blood mononuclear cells (PBMC) in normal humans and prevent IL-1 and TNF anorexia in animals. Accordingly, we studied the nutritional and metabolic effects of a 10-week trial of dietary fish oil (MaxEPA 18 g/day) in men with weight loss due to acquired immune deficiency syndrome (AIDS). Twenty men were enrolled, and 16 completed the 10-week supplementation period. Prior weight loss was 13.7 +/- 1.8 kg(17.4 +/- 1.6% body weight, means +/- SE). Food intake, body composition, blood chemistries, serum cytokine concentrations, in vitro production of IL-1 and TNF by PBMC, and clinical course were followed. A subset of subjects (n=12) underwent stable isotope infusions to measure de novo hepatic lipogenesis (DNL), an in vivo metabolic index that is influenced by cytokine presence and has previously been found to be elevated in AIDS. An unsupplemented group of men with AIDS wasting (10.4 +/- 2.4 kg weight loss, 13.1 +/- 2.2% body weight) was monitored for 10 weeks as controls. Baseline food intake (2,395 +/- 177 kcal/day and 95.1 +/- 7.2 g protein/day), body weight, percent fat, and fat-free mass were unchanged over the 10-week supplementation period. Serum triglycerides were reduced in hypertriglyceridemic subjects, confirming compliance with fish oil supplementation and suggesting that their hypertriglyceridemia was at least in part due to overproduction. Serum TNF and IL-1 were undetectable before or after fish oil supplementation. Serum interferon alpha (IFN) was measurable but did not change. In vitro production of IL-1 and TNF by PBMC was markedly reduced both at baseline and after fish oil supplementation in this population, even in the presence of new AIDS complications, compared with normal controls. The metabolic measurement DNL fell and weight was gained (2.1 +/- 1.3 kg) in subjects who did not develop new AIDS-related complications, but further increases in DNL and further weight loss were observed in subjects who developed a new AIDS complication (p<0.05 for interaction between new complication and change in DNL). No changes in body weight, food intake, serum triglycerides, serum cytokines, or DNL were observed in the unsupplemented group. We conclude that fish oil is a weak anticytokine agent that is unable to overcome the metabolic and nutritional consequences of acute AIDS-related complications but may exert a clinical anticytokine effect in stable AIDS patients. Cytokine production by PBMC is not a useful or reliable marker of in vivo cytokine activity in AIDS patients with weight loss. In contrast, an integrative functional index that is sensitive to cytokine presence in tissues (hepatic DNL) correlated with clinical response. These findings are relevant to the design of future studies of more potent anticytokine agents, such as thalidomide.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Regulation of hepatic de novo lipogenesis in humans.

The enzymatic pathway for synthesis of fatty acids from acetyl-coenzyme A, or de novo lipogenesis (DNL), is present in human liver and, to a lesser extent, in adipose tissue. Although the molecular and enzymatic regulation of the components for DNL are well characterized, the quantitative importance of the assembled pathway and its physiologic functions have remained uncertain. We review methods that have been used for measuring DNL in vivo, their limitations and the conclusions based on them. Two new methods for direct measurement of DNL in humans are discussed-mass isotopomer distribution analysis (MIDA), a mass spectrometric technique based on combinatorial probabilities, and 2H2O incorporation. Recent findings with these methods in a variety of dietary and hormonal settings are reviewed. In particular, we focus on the question of whether or not surplus carbohydrate energy is converted to fat by the liver in humans. A somewhat surprising model of the response to carbohydrate over-feeding emerges from these studies, with a number of implications for metabolic regulation in health and disease. We close by speculating on potential functions of DNL in physiology and pathophysiology if storage of surplus carbohydrate energy is not an important function of DNL. The availability of techniques for quantifying DNL in vivo should make it possible to resolve these uncertainties regarding its functions and regulation in humans.

Diet↗

PubMed1995

Gluconeogenesis and intrahepatic triose phosphate flux in response to fasting or substrate loads. Application of the mass isotopomer distribution analysis technique with testing of assumptions and potential problems.

We measured gluconeogenesis (GNG) in rats by mass isotopomer distribution analysis, which allows enrichment of the true biosynthetic precursor pool (hepatic cytosolic triose phosphates) to be determined. Fractional GNG from infused [3-13C]lactate, [1-13C]lactate, and [2-13C]glycerol was 88 +/- 2, 89 +/- 3, and 87 +/- 2%, respectively, after 48 h of fasting. [2-13C]Glycerol was the most efficient label and allowed measurement of rate of appearance of intrahepatic triose phosphate (Ra triose-P), by dilution. IV fructose (10-15 mg/kg/min) increased absolute GNG by 81-147%. Ra triose-P increased proportionately, but endogenous Ra triose-P was almost completely suppressed, suggesting feedback control. Interestingly, 15-17% of fructose was directly converted to glucose without entering hepatic triose-P. IV glucose reduced GNG and Ra triose-P. 24-h fasting reduced hepatic glucose production by half, but absolute GNG was unchanged due to increased fractional GNG (51-87%). Reduced hepatic glucose production was entirely due to decreased glycogen input, from 7.3 +/- 1.8 to 1.1 +/- 0.2 mg/kg/min. Ra triose-P fell during fasting, but efficiency of triose-P disposal into GNG increased, maintaining GNG constant. Secreted glucuronyl conjugates and plasma glucose results correlated closely. In summary, GNG and intrahepatic triose-P flux can be measured by mass isotopomer distribution analysis with [2-13C]glycerol.

Animals↗

PubMed1995

Short-term alterations in carbohydrate energy intake in humans. Striking effects on hepatic glucose production, de novo lipogenesis, lipolysis, and whole-body fuel selection.

Short-term alterations in dietary carbohydrate (CHO) energy are known to alter whole-body fuel selection in humans, but the metabolic mechanisms remain unknown. We used stable isotope-mass spectrometric methods with indirect calorimetry in normal subjects to quantify the metabolic response to six dietary phases (5 d each), ranging from 50% surplus CHO (+50% CHO) to 50% deficient CHO (-50% CHO), and 50% surplus fat (+50% fat). Fasting hepatic glucose production (HGP) varied by > 40% from deficient to surplus CHO diets (1.78 +/- 0.08 vs 2.43 +/- 0.09 mg/kg per min, P < 0.01). Increased HGP on surplus CHO occurred despite significantly higher serum insulin concentrations. Lipolysis correlated inversely with CHO intake as did the proportion of whole-body lipolytic flux oxidized. Fractional de novo hepatic lipogenesis (DNL) increased more than 10-fold on surplus CHO and was unmeasurable on deficient CHO diets; thus, the preceding 5-d CHO intake could be inferred from DNL. Nevertheless, absolute hepatic DNL accounted for < 5g fatty acids synthesized per day even on +50% CHO. Whole-body CHO oxidation increased sixfold and fat oxidation decreased > 90% on surplus CHO diets. CHO oxidation was highly correlated with HGP (r2= 0.60). HGP could account for 85% of fasting CHO oxidation on +25% CHO and 67% on +50% CHO diets. Some oxidation of intracellular CHO stores was therefore also occurring. +50% fat diet had no effects on HGP, DNL, or fuel selection. We conclude that altered CHO intake alters HGP specifically and in a dose-dependent manner, that HGP may mediate the effects of CHO on whole-body fuel selection both by providing substrate and by altering serum insulin concentrations, that altered lipolysis and tissue oxidation efficiency contribute to changes in fat oxidation, and that surplus CHO is not substantially converted by the liver to fat as it spares fat oxidation, but that fractional DNL may nevertheless be a qualitative marker of recent CHO intake.

Body Weight↗

PubMed1994

Metabolic interactions between surplus dietary energy intake and cigarette smoking or its cessation.

Cigarette smoking (CS) alters lipid metabolism and is associated clinically with an atherogenic lipid profile. We recently showed that, under controlled eucaloric dietary conditions, CS stimulates lipolysis without increasing oxidation of fat and that cessation of CS does not result in a rebound tendency to synthesize or store fat. We asked here whether the ad libitum intake of surplus dietary energy interacts with the metabolic effects of CS or its cessation. Eight male heavy smokers were allowed ad libitum food intake in a metabolic ward, 1 wk in CS phase and 1 wk in non-CS phase, followed by 4 wk of outpatient non-CS and a repeat 7-day study. De novo hepatic lipogenesis (DNL), lipolysis, substrate cycling of free fatty acids (FFA), hepatic glucose production, and energy expenditure were measured by using a multiple stable-isotope infusion protocol and indirect calorimetry. Surplus dietary energy intake (> 150% of predicted energy needs) occurred in five of eight subjects (2 subjs > 5,500 kcal/day, 3 subjs > 4,000 kcal/day) with weight gain of 1-4 kg/wk, but with no difference between CS and non-CS phases. Acute CS significantly increased (P < 0.05) serum FFA concentrations (58%), FFA flux (63%), and glycerol flux (36%); nonsignificantly increased extra-adipocyte (hepatic) esterification of FFA (125%, P = 0.10) and resting energy expenditure (4.1%, P = 0.22); and did not change adipocyte reesterification of FFA or whole body oxidation of fat. Basal metabolic parameters (after overnight abstention from CS) did not differ between phases. Fractional DNL correlated significantly with excess energy intake (r2 = 0.39) and with percentage of total energy needs provided by carbohydrate (r2 = 0.47). The absence or presence of CS did not affect the increase in fractional DNL in subjects with excess energy intake, however. We conclude that cessation of CS does not result in a rebound tendency to synthesis or storage of fat, even in the presence of positive short-term energy balance, contrary to previous suggestions. Moreover, stimulation of lipolysis by CS does not increase oxidation of fat and thereby protect against fat deposition under conditions of surplus energy intake. The prevention of weight gain after cessation of CS, whether or not nicotine is provided, should focus on energy balance (calorigenesis as well as intake) rather than specific alterations in lipid metabolism.

Adult↗

PubMed1994

Effects of cigarette smoking and its cessation on lipid metabolism and energy expenditure in heavy smokers.

The relationship between thermogenic and potentially atherogenic effects of cigarette smoking (CS) and its cessation was investigated. Heavy smokers (n = 7, serum cotinine > 200 ng/ml, > 20 cigarettes/d) were maintained on isoenergetic, constant diets for 2 wk, 1 wk with and 1 wk without CS. Stable isotope infusions with indirect calorimetry were performed on day 7 of each phase, after an overnight fast. CS after overnight abstention increased resting energy expenditure by 5% (not significant vs. non-CS phase; P = 0.18). CS increased the flux of FFA by 77%, flux of glycerol by 82%, and serum FFA concentrations by 73% (P < 0.02 for each), but did not significantly affect fat oxidation. Hepatic reesterification of FFA increased more than threefold (P < 0.03) and adipocyte recycling increased nonsignificantly (P = 0.10). CS-induced lipid substrate cycles represented only 15% (estimated 11 kcal/d) of observed changes in energy expenditure. De novo hepatic lipogenesis was low (< 1-2 g/d) and unaffected by either acute CS or its chronic cessation. Hepatic glucose production was not affected by CS, despite increased serum glycerol and FFA fluxes. Cessation of CS caused no rebound effects on basal metabolic fluxes. In conclusion, a metabolic mechanism for the atherogenic effects of CS on serum lipids (increased hepatic reesterification of FFA) has been documented. Increased entry of FFA accounts for CS-induced increases in serum FFA concentrations. The thermogenic effect of CS is small or absent in heavy smokers while the potentially atherogenic effect is maintained, and cessation of CS does not induce a rebound lipogenic milieu that specifically favors accrual of body fat in the absence of increased food intake.

Adipocytes↗