Dissociation between adipose tissue fluxes and lipogenic gene expression in ob/ob mice.

Recent evidence has been presented that expression of lipogenic genes is downregulated in adipose tissue of ob/ob mice as well as in human obesity, suggesting a functionally lipoatrophic state. Using (2)H(2)O labeling, we measured three adipose tissue biosynthetic processes concurrently: triglyceride (TG) synthesis, palmitate de novo lipogenesis (DNL), and cell proliferation (adipogenesis). To determine the effect of the ob/ob mutation (leptin deficiency) on these parameters, adipose dynamics were compared in ob/ob, leptin-treated ob/ob, food-restricted ob/ob, and lean control mice. Adipose tissue fluxes for TG synthesis, de novo lipogenesis (DNL), and adipogenesis were dramatically increased in ob/ob mice compared with lean controls. Low-dose leptin treatment (2 microg/day) via miniosmotic pump suppressed all fluxes to control levels or below. Food restriction in ob/ob mice only modestly reduced DNL, with no change in TG synthesis or adipogenesis. Measurement of mRNA levels in age-matched ob/ob mice showed generally normal expression levels for most of the selected lipid anabolic genes, and leptin treatment had, with few exceptions, only modest effects on their expression. We conclude that leptin deficiency per se results in marked elevations in flux through diverse lipid anabolic pathways in adipose tissue (DNL, TG synthesis, and cell proliferation), independent of food intake, but that gene expression fails to reflect these changes in flux.

Sources of plasma glucose and liver glycogen in fasted ob/ob mice.

Alterations in intrahepatic carbohydrate fluxes in ob/ob mice and the effects of acute leptin administration were studied in vivo by use of a dual-isotope tracer infusion. Metabolic sources of plasma glucose (gluconeogenesis (GNG) and glycogenolysis) and hepatic glycogen (GNG, direct synthesis and pre-existing) were determined in 20-h-fasted mice infused with [2-13C1]glycerol and [U13C6]glucose for 3 h. Total glucose output (TGO) and the rate of appearance (Ra) of plasma glycerol were measured by isotope dilution. GNG, the direct pathway of hepatic glycogen synthesis and hepatic triose-phosphate flux were determined by mass isotopomer distribution analysis (MIDA). Serum glucose, insulin, leptin and liver glycogen concentrations were also measured. After a 24-h fast, ob/ob mice had 2-fold higher TGO, 2.5-fold elevated liver glycogen content and markedly higher glycogenolytic flux to glucose, absolute GNG and direct glycogen synthesis rates (10-fold increased) compared to the control group. Ob/ob mice also had elevated triose-phosphate flux compared to controls (40 vs. 22 mg/kg lean body mass/min). A model of intrahepatic flux distributions in control and ob/ob mice is presented. In summary, elevated fasting plasma glucose concentrations are due to increased TGO in ob/ob mice, which is maintained by both increased GNG and increased glycogenolysis. Furthermore, the ob/ob mice have major alterations in fasting hepatic carbohydrate fluxes into triose-phosphate pools and glycogen. We support the model that actions of leptin on hepatic glucose metabolism require insulin or other factors.

Measurement of TG synthesis and turnover in vivo by 2H2O incorporation into the glycerol moiety and application of MIDA.

A method is presented for measurement of triglyceride (TG) synthesis that can be applied to slow-turnover lipids. The glycerol moiety of TG is labeled from 2H2O, and mass isotopomer distribution analysis (MIDA) is applied. Mice and rats were given 4-8% 2H2O in drinking water; TG-glycerol was isolated from adipose and liver during < or =12-wk of 2H2O labeling. Mass isotopomer abundances in the glycerol moiety of TG were measured by GC-MS. The combinatorial pattern of isotopomers revealed the number of H atoms in glycerol incorporating label from 2H2O (n) to be 3.8-4.0 of a possible 5 for adipose tissue and 4.6-4.8 for liver TG. Hepatic TG-glycerol in fact reached 97% predicted maximal value of label incorporation (4.4-4.6 x body 2H2O enrichment), indicating near-complete replacement of the liver TG pool. Label incorporation into adipose tissue revealed turnover of mesenteric TG to be faster (k = 0.21 day-1) than other depots (k = 0.04-0.06 day-1) in mice. TG isolated from subcutaneous depots of growing adult rats plateaued at 85-90% of calculated maximal values at 12 wk (k = 0.05 day-1), excluding significant dilution by unlabeled alpha-glycerol phosphate. Turnover of plasma TG, modeled from 2H incorporation over 60 min, was 0.06 min-1 (half-life 11.5 min). In summary, use of 2H2O labeling with MIDA of TG-glycerol allows measurement of new alpha-glycerol phosphate-derived TG synthesis and turnover. The hypothesis that mesenteric TG is more lipolytically active than other depots, previously difficult to prove by isotope dilution techniques, was confirmed by this label incorporation approach.

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 (2)H(2)O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% (2)H(2)O 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 microg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose-response relationship up to the 100 microg 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 (2)H(2)O 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 (2)H(2)O 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 obob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term (2)H(2)O enrichments in body water were achieved by daily (2)H(2)O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body (2)H(2)O enrichment ( approximately 3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with (2)H(2)O was 0.056 (CD4(+)) and 0.043 (CD8(+)) (replacement rate <0.1% per day). In summary, (2)H(2)O 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.

Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry.

We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [(2)H(3)]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M(+1) and M(+2) mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3-10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.

Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation.

Methods for measuring rates of DNA synthesis, and thus cell proliferation, in humans had not been available until recently. We (D. C. Macallan, C. A. Fullerton, R. A. Neese, K. Haddock, S. S. Park, and M. K. Hellerstein, 1998, Proc. Natl. Acad. Sci. USA 95, 708-713) recently developed a stable isotope-mass spectrometric technique for measuring DNA synthesis by labeling the deoxyribose (dR) moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway. The original analytic approach had limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentose-tetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after several weeks), greater GC/MS signal, and much less abundance sensitivity of isotope ratios (i.e., less dependence of mass isotopomer abundances on the amount of material injected into the mass spectrometer source), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with 2H2O enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1.0 microg DNA (ca. 2 x 10(5) cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned.

Lipoprotein secretion and triglyceride stores in the heart.

The genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in mouse and human heart tissue. Why the heart would express these "lipoprotein assembly" genes has been unclear. Here we demonstrate that the beating mouse heart actually secretes spherical lipoproteins. Moreover, increased cardiac production of lipoproteins (e.g., in mice that express a human apolipoprotein B transgene) was associated with increased triglyceride secretion from the heart and decreased stores of triglycerides within the heart. Increased cardiac production of lipoproteins also reduced the pathological accumulation of triglycerides that occurs in the hearts of mice lacking long-chain acyl coenzyme A dehydrogenase. In contrast, blocking heart lipoprotein secretion (e.g., in heart-specific microsomal triglyceride transfer protein knockout mice) increased cardiac triglyceride stores. Thus, heart lipoprotein secretion helps regulate cardiac triglyceride stores and may protect the heart from the detrimental effects of surplus lipids.

Effect of nicotinic acid administration on hepatic very low density lipoprotein-triglyceride production.

Our objective was to examine very low density lipoprotein-triglyceride (VLDL-TG) kinetics after chronic and acute administration of nicotinic acid (NA). Incorporation of [1,2,3,4-(13)C(4)]palmitate and [2-(13)C(1)]glycerol into VLDL-TG was measured in five healthy, normolipidemic women. Each subject was studied twice; the 4-day hospital stays were separated by 1 mo, during which time doses of NA were increased to 2 g/day (500 mg, 4 times/day). During posttreatment study, 500 mg of NA were administered acutely at 0800. Under baseline postabsorptive conditions, incorporation curves from (13)C-labeled free fatty acid (FFA) and (13)C-labeled glycerol were superimposable, and VLDL-TG kinetics were in agreement (t(1/2) = 1.4 +/- 0.3 and 1.3 +/- 0.3 h, and production rates = 27.2 +/- 6.1 and 28.5 +/- 5.3 g/day, respectively). In the postabsorptive state after chronic NA therapy, VLDL-TG concentrations and production rates were lower despite a trend toward elevated plasma FFA concentrations and fluxes. After the acute dose of NA, plasma FFA concentrations and flux fell dramatically, and there was a virtual halt to VLDL-TG production, which continued throughout the 6-h period after NA, despite a marked rebound overshoot in serum FFA concentrations and flux after hour 2. Plasma homocysteine concentrations increased 68% (P < 0.001) in the NA phase, consistent with chronic increased transmethylation demand. We conclude that 1) NA acutely and chronically decreases VLDL-TG production rate in normal women; 2) the acute effect on VLDL-TG production is associated with an initial suppression of lipolysis but persists for several hours after the antilipolytic action of NA has abated and is observed in the basal postabsorptive state, when lipolytic rates are not reduced; and 3) the effect of NA on VLDL-TG production, therefore, cannot be completely explained by its antilipolytic actions.

Dependence of plasma alpha-tocopherol flux on very low-density triglyceride clearance in humans.

To evaluate the effect of dietary fat-induced alterations in triglyceride (TG) metabolism on plasma and very low-density lipoprotein (VLDL)-alpha-tocopherol, nine healthy males (mean +/- SEM, age: 36 +/- 3 years, BMI: 24.7 +/- 1.1) consumed a 35%-fat diet (control) for one week followed by a 15% low-fat, high-carbohydrate diet for 5 weeks. After each dietary phase, the subjects ingested an evening meal along with a 50 mg capsule of (2)H(6)-RRR-alpha-tocopheryl acetate; blood samples were drawn over a 24 h period while the subjects remained fasted. Low-fat feeding increased fasting plasma TG concentrations by 53% (116 +/- 27 to 178 +/- 32, mg/dl, p < 0.0001) primarily by reducing VLDL-TG clearance. Total plasma alpha-tocopherol concentrations (labeled + unlabeled) were unchanged (25.8 +/- 2.3 vs. 26.4 +/- 3.0 nmol/ml plasma) and no differences between the diets were observed for plasma (2)H(6)-alpha-tocopherol concentration (4.8 +/- 0.6 nmol/ml, for both diets) or enrichments (18.1 +/- 1.8% average for both diets). However, low-fat feeding significantly increased the amount of alpha-tocopherol in the VLDL fraction (43%, p = 0.04) in concert with elevations in VLDL-apoB and TG. The alpha-tocopherol and TG content of VLDL varied in parallel in individual subjects and fractional replacement rates and clearance of alpha-tocopherol and TG in VLDL were closely correlated. Kinetic parameters were decreased by 32-39% from high-fat to low-fat. These data suggest that vitamin E bioavailability is similar between a 15 and 35% fat diet, with a redistribution of alpha-tocopherol in lipoproteins occurring during low-fat feeding (increased in the VLDL fraction, reduced in the other lipoproteins), and transfer of alpha-tocopherol from VLDL depends upon TG removal from the particle, consistent with previous observations in vitro and in animal studies.

Effect of dietary energy restriction on glucose production and substrate utilization in type 2 diabetes.

A total of 8 obese subjects with type 2 diabetes were studied while on a eucaloric diet and after reduced energy intake (25 and then 75% of requirements for 10 days each). Weight loss was 2, 3, and 3 kg after 5, 10, and 20 days, respectively; all of the weight lost was body fat. Fasting blood glucose (FBG) levels fell from 11.9 +/- 1.4 at baseline to 8.9 +/- 1.6, 7.9 +/- 1.4, and 8.8 +/- 1.3 mmol/l at days 5, 10, and 20, respectively (P < 0.05, baseline vs. 5, 10, and 20 days). Endogenous glucose production (EGP) was 22 +/- 2, 18 +/- 2, 17 +/- 2, and 22 +/- 2 pmol x kg(-1) lean body mass (LBM) x min(-1) (P < 0.05, days 5 and 10 vs. baseline). Gluconeogenesis measured by mass isotopomer distribution analysis provided 31 +/- 4, 41 +/- 5, 40 +/- 4, and 33 +/- 4%, respectively, of the EGP (NS); absolute glycogenolytic contribution to the EGP was 15 +/- 2, 11 +/- 2, 11 +/- 2, and 15 +/- 2 pmol x kg(-1) LBM x min(-1), respectively (P < 0.001, baseline vs. days 5 and 10 and day 10 vs. day 20). The blood glucose clearance rate increased significantly at day 20 (P < 0.05). Neither lipolysis nor flux of plasma nonesterified fatty acids were altered compared with baseline. In conclusion, severe energy restriction per se independent of major changes in body composition reduces both FBG concentration and EGP in type 2 diabetes, the reduction in EGP results entirely from a reduction of glycogenolytic input into blood glucose, and the duration of reduced glycogenolysis is short-lived after relaxation of energy restriction even without weight gain, but effects on plasma glucose clearance persist and partially maintain the improvement in fasting glycemia.

Effects of isoenergetic overfeeding of either carbohydrate or fat in young men.

Ten pairs of normal men were overfed by 5 MJ/d for 21 d with either a carbohydrate-rich or a fat-rich diet (C- and F-group). The two subjects in each pair were requested to follow each other throughout the day to ensure similar physical activity and were otherwise allowed to maintain normal daily life. The increase in body weight, fat free mass and fat mass showed great variation, the mean increases being 1.5 kg, 0.6 kg and 0.9 kg respectively. No significant differences between the C- and F-group were observed. Heat production during sleep did not change during overfeeding. The RQ during sleep was 0.86 and 0.78 in the C- and F-group respectively. The accumulated faecal loss of energy, DM, carbohydrate and protein was significantly higher in the C- compared with the F-group (30, 44, 69 and 51% higher respectively), whereas the fat loss was the same in the two groups. N balance was not different between the C- and F-group and was positive. Fractional contribution from hepatic de novo lipogenesis, as measured by mass isotopomer distribution analysis after administration of [1-(13)C]acetate, was 0.20 and 0.03 in the C-group and the F-group respectively. Absolute hepatic de novo lipogenesis in the C-group was on average 211 g per 21 d. Whole-body de novo lipogenesis, as obtained by the difference between fat mass increase and dietary fat available for storage, was positive in six of the ten subjects in the C-group (mean 332 (SEM 191)g per 21 d). The change in plasma leptin concentration was positively correlated with the change in fat mass. Thus, fat storage during overfeeding of isoenergetic amounts of diets rich in carbohydrate or in fat was not significantly different, and carbohydrates seemed to be converted to fat by both hepatic and extrahepatic lipogenesis.

[Effects of excessive isocaloric intake of either carbohydrate or fat on body composition, fat mass, de novo lipogenesis and energy expenditure in normal young men]. / Virkninger af isokalorisk overindtagelse af kulhydrat eller fedt på legemsvaegt, fedtmasse, de novo lipogenese og energiomsaetning hos normale unge maend.

Ten pairs of normal young men were overfed by 5 MJ per day for 21 days with either a carbohydrate-rich or a fat-rich diet (C- and F-group). The two subjects of a pair were requested to follow each other throughout the day to ensure similar physical activity. The increase in body weight and fat mass were not significantly different between the C- and the F-group. Heat production during sleep did not change during overfeeding. The accumulated faecal loss of energy, dry matter, carbohydrate and protein was significantly higher in the C- than in the F-group. Hepatic de novo lipogenesis was 212 g per 21 days in the C-group and was too low to be determined in the F-group. Whole body de novo lipogenesis was positive in six of the ten subjects in the C-group (mean: 332 g per 21 days). It is concluded that the increase in body weight and fat mass during overfeeding of isocaloric amounts of diets rich in carbohydrate or in fat was not significantly different, and that surplus of carbohydrate seemed to be converted to fat both by hepatic and extrahepatic de novo lipogenesis.

Effects of nicotinic acid on fatty acid kinetics, fuel selection, and pathways of glucose production in women.

Chronic nicotinic acid (NA) ingestion effectively lowers lipid levels, but adverse effects on glucose metabolism have been reported. Our goal was to investigate acute and chronic effects of NA on lipolysis and glucose metabolism in women. Healthy normolipidemic volunteers (n = 5) were studied twice; four-day hospital stays were separated by 1 mo, during which time subjects took increasing doses of NA to 2 g/day (500 mg, 4 times). In the second study, 500 mg of NA was given at 0800. Rates of appearance (R(a)) of free fatty acid (FFA), glycerol, and glucose were determined by isotope dilution (of [1,2,3,4-(13)C(4)]palmitate, [2-(13)C(1)]glycerol, and [U-(13)C(6)]glucose). Mass isotopomer distribution analysis was used to measure gluconeogenesis and glycogenolysis. Fasting FFA concentrations ([FFA]), R(a) FFA, and R(a) glycerol were nonsignificantly elevated after 1 mo. Acute NA induced a significant reduction followed by a rebound overshoot of [FFA], R(a) FFA, and R(a) glycerol. Whole body fat oxidation fell initially and then increased back to basal levels; endogenous glucose production (EGP) increased in parallel with carbohydrate oxidation and then returned to basal values. The increased EGP was due entirely to increased glycogenolysis, not gluconeogenesis. We conclude that chronic effects of NA on FFA metabolism are complex (acute suppression followed by overshoot of R(a) FFA and [FFA] on top of a trend toward basal elevations), that responses after NA are consistent with operation of a glucose-fatty acid cycle in peripheral tissues, and that secondary effects on EGP were through changes in glycogenolysis, not gluconeogenesis.

Relationship between carbohydrate-induced hypertriglyceridemia and fatty acid synthesis in lean and obese subjects.

We previously reported that a eucaloric, low fat, liquid formula diet enriched in simple carbohydrate markedly increased the synthesis of fatty acids in lean volunteers. To examine the diet sensitivity of obese subjects, 7 obese and 12 lean volunteers were given two eucaloric low fat solid food diets enriched in simple sugars for 2 weeks each in a random-order, cross-over design (10% fat, 75% carbohydrate vs. 30% fat, 55% carbohydrate, ratio of sugar to starch 60:40). The fatty acid compositions of both diets were matched to the composition of each subject's adipose tissue and fatty acid synthesis measured by the method of linoleate dilution in plasma VLDL triglyceride. In all subjects, the maximum % de novo synthesized fatty acids in VLDL triglyceride 3;-9 h after the last meal was higher on the 10% versus the 30% fat diet. There was no significant difference between the dietary effects on lean (43+/-13 vs. 12+/-13%) and obese (37+/-15 vs. 6+/-6%) subjects, despite 2-fold elevated levels of insulin and reduced glucagon levels in the obese. Similar results were obtained for de novo palmitate synthesis in VLDL triglyceride measured by mass isotopomer distribution analysis after infusion of [(13)C]acetate. On the 10% fat diet, plasma triglycerides (fasting and 24 h) were increased and correlated with fatty acid synthesis. Triglycerides were higher when fatty acid synthesis was constantly elevated rather than having diurnal variation.Thus, eucaloric, solid food diets which are very low in fat and high in simple sugars markedly stimulate fatty acid synthesis from carbohydrate, and plasma triglycerides increase in proportion to the amount of fatty acid synthesis. However, this dietary effect is not related to body mass index, insulin, or glucagon levels.

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion.

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.

The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis.

A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes.

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.

Metabolic adaptations to dietary fat malabsorption in chylomicron-deficient mice.

A mouse model of chylomicron deficiency was recently developed; these mice express a human apolipoprotein (apo) B transgene in the liver but do not synthesize any apoB in the intestine. Despite severe intestinal fat malabsorption, the mice maintain normal concentrations of plasma lipids and liver-derived apoB 100-containing lipoproteins. We investigated the metabolic mechanisms by which plasma lipid levels are kept normal. De novo lipogenesis (DNL) and cholesterogenesis were measured by mass isotopomer distribution analysis (MIDA). Plasma non-esterified fatty acid (NEFA) fluxes and hepatic re-esterification of labelled plasma NEFA were also measured. Hepatic and plasma triacylglycerol (TG) concentrations and plasma NEFA fluxes were not different between chylomicron-deficient mice and controls. The contribution from DNL to the hepatic TG pool was only modestly higher in chylomicron-deficient mice [12+/-2.1% (n=7) compared with 3.7+/-1.0% (n=9); means+/-S.E.M.], whereas cholesterogenesis was markedly elevated. The fractional contribution from plasma NEFA to hepatic TG was greatly elevated in the chylomicron-deficient animals (62% compared with 23%). Accordingly, 73% of hepatic TG was neither from DNL nor from plasma NEFA in controls, presumably reflecting prior contribution from chylomicron remnants, compared with only 26% in the chylomicron-deficient group. The long-term contribution from DNL to adipose fat stores reached approximately the same steady-state values (approximately 30%) in the two groups. Body fat accumulation was much lower in chylomicron-deficient animals; thus, whole-body absolute DNL was significantly lower. We conclude that plasma and hepatic TG pools and hepatic secretion of apoB-containing particles are maintained at normal levels in chylomicron-deficient mice, not by de novo fatty acid synthesis, but by more avid re-esterification of plasma NEFA, replacing the normally predominant contribution from chylomicrons, and that some dietary fat can be absorbed by apoB-independent mechanisms.

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.

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.