Koutsari, Christina

Women differ from men by the larger lower‐body subcutaneous fat mass and the tendency to gain more fat in that depot if obesity develops. We hypothesized that these differences in fat distribution may depend on preadipocyte availability. We identified the proportion of preadipocytes from the stromovascular (SV) cells in adipose tissue by immunofluorescent staining for aP2 (the adipocyte fatty acid trafficking protein) and CD68 (a macrophage marker). Macrophages are the other cell type, aside from preadipocytes, in the SV fraction of adipose tissue that express aP2. We determined the percent preadipocytes in the SV fraction of subcutaneous abdominal and femoral adipose tissue of 17 premenopausal lean women and 10 age‐matched lean men. Body fat compartments were measured by DEXA and CT scans of the abdomen. Compared to men, women had less visceral fat and more subcutaneous fat mass, predominantly in the lower‐body compartment: 7.0 ± 0.3 kg vs. 5.2 ± 0.4 kg, respectively (p < 0.01). Femoral adipocytes were significantly larger than abdominal adipocytes in both groups (lipid per fat cell in women and men: abdomen, 0.46 ± 0.03 and 0.44 ± 0.06 μg/cell; thigh, 0.66 ± 0.04 and 0.55 ± 0.08 μg/cell, respectively). The percent preadipocytes in the SV fraction from femoral subcutaneous adipose tissue was greater in women than in men (29.7 ± 2.6% vs. 19.3 ± 2.0%, p < 0.02) but similar when isolated from the abdominal depot (29.9 ± 3.3% vs. 27.2 ± 1.9%). Our findings suggest that the fraction of femoral stromovascular cells as preadipocytes is a potential factor for sex‐dependent differences in regional fat distribution. The study was funded by RO1 DK 45343‐13S to M. Jensen.

We investigated the effects of meal ingestion on intramyofibrillar (IMF) and subsarcolemmal (SS) ceramide metabolism in volunteers ranging from lean to obese. Thirty-eight women and men underwent a steady-state meal ingestion protocol that included a 6.5-h infusion of [U-13C]palmitate and muscle biopsies 1.5 and 6.5 h after starting the tracer infusion. We measured IMF and SS sphingolipid concentrations and the contribution of plasma palmitate to intramyocellular C16:0 ceramide by use of LC-MS-MS. In response to meal ingestion SS C24 ceramide concentrations, but not C14-C20 concentrations, increased significantly. IMF ceramide concentrations did not change. The increases in SS C24 ceramides were negatively related to parameters of insulin resistance. The fractional contribution of plasma palmitate to intramyocellular C16:0 ceramides in both IMF and SS fractions was inversely related to overweight status (β = –0.432, P = 0.0095 and β = –0.443, P = 0.0058, respectively). These data indicate that meal ingestion has differing effects on SS ceramide subspecies and suggest that the fractional de novo synthesis of intramyocellular ceramide from plasma palmitate in the postprandial condition is reduced in those who are overweight.

We investigated the relationship between insulin resistance markers and subsarcolemmal (SS) and intramyofibrillar (IMF) ceramide concentrations, as well as the contribution of plasma palmitate (6.5-h infusion of [U-13C]palmitate) to intramyocellular ceramides. Seventy-six postabsorptive men and women had muscle biopsies 1.5, 6.5, and 24 h after starting the tracer infusion. Concentrations and enrichment of muscle ceramides were measured by liquid chromatography-tandem mass spectrometry. We found that HOMA of insulin resistance, plasma insulin, and triglyceride concentrations were positively correlated with SS C16:0 and C18:1 ceramide, but not SS C14:0-Cer, C20:0-Cer, C24:0-Cer, and C24:1-Cer concentrations; IMF ceramide concentrations were not correlated with any metabolic parameters. The fractional contribution of plasma palmitate to 16:0 ceramide was greater in SS than IMF (SS, 18.2% vs. IMF, 8.7%; P = 0.0006). Plasma insulin concentrations correlated positively with the fractional contribution of plasma palmitate to SS 16:0 ceramide. The fractional contribution of plasma palmitate to intramyocellular SS 16:0 ceramide was positively correlated with SS C16:0 ceramide concentrations (γ = 0.435; P = 0.002). We conclude that skeletal muscle SS ceramides, especially C16 to C18 chain lengths and the de novo synthesis of intramyocellular ceramide from plasma palmitate are associated with markers of insulin resistance.

Insulin stimulates the translocation fatty acid transport protein 1 (FATP1) to plasma membrane, and thus greater free fatty acid (FFA) uptake, in adipocyte cell models. Whether insulin stimulates greater FFA clearance into adipose tissue in vivo is unknown. We tested this hypothesis by comparing direct FFA storage in subcutaneous adipose tissue during insulin versus niacin-medicated suppression of lipolysis. We measured direct FFA storage in abdominal and femoral subcutaneous fat in 10 and 11 adults, respectively, during euglycemic hyperinsulinemia or after oral niacin to suppress FFA compared with 11 saline control experiments. Direct palmitate storage was assessed using a [U-13C]palmitate infusion to measure palmitate kinetics and an intravenous palmitate radiotracer bolus/timed biopsy. Plasma palmitate concentrations and flux were suppressed to 23 ± 3 and 26 ± 5 µmol ⋅ L−1 (P = 0.91) and 44 ± 4 and 39 ± 5 µmol ⋅ min−1 (P = 0.41) in the insulin and niacin groups, respectively, much less (P &lt; 0.001) than the saline control group (102 ± 8 and 104 ± 12 µmol ⋅ min−1, respectively). In the insulin, niacin, and saline groups, abdominal palmitate storage rates were 0.25 ± 0.05 vs. 0.25 ± 0.07 vs. 0.32 ± 0.05 µmol ⋅ kg adipose lipid−1 ⋅ min−1, respectively (P = NS), and femoral adipose storage rates were 0.19 ± 0.06 vs. 0.20 ± 0.05 vs. 0.31 ± 0.05 µmol ⋅ kg adipose lipid−1 ⋅ min−1, respectively (P = NS). In conclusion, insulin does not increase FFA storage in adipose tissue compared with niacin, which suppresses lipolysis via a different pathway.

We measured the incorporation of systemic free fatty acids (FFA) into circulating very low-density lipoprotein triglycerides (VLDL-TGs) under postabsorptive, postprandial, and walking conditions in humans. Fifty-five men and 85 premenopausal women with BMI 18–24 (lean) and 27–36 kg/m2 (overweight/obese) received an intravenous bolus injection of [1,1,2,3,3-2H5]glycerol (to measure VLDL-TG kinetics) and either [1-14C]palmitate or [9,10-3H]palmitate to determine the proportion of systemic FFA that is converted to VLDL-TG. Experiments started at 0630 h after a 12-h overnight fast. In the postabsorptive protocol, participants rested and remained fasted until 1330 h. In the postprandial protocol, volunteers ingested frequent portions of a fat-free smoothie. In the walking protocol, participants walked on a treadmill for 5.5 h at ∼3× resting energy expenditure. Approximately 7% of circulating FFA was converted into VLDL-TG. VLDL-TG secretion rates (SRs) were not statistically different among protocols. Visceral fat mass was the only independent predictor of VLDL-TG secretion, explaining 33–57% of the variance. The small proportion of systemic FFA that is converted to VLDL-TG can confound the expected relationship between plasma FFA concentration and VLDL-TG SRs. Regulation of VLDL-TG secretion is complex in that, despite a broad spectrum of physiological FFA concentrations, VLDL-TG SRs did not vary based on different acute substrate availability.

RATIONALE Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre‐existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U‐ 13 C]palmitate into intramyocellular ceramides. METHODS We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and 13 C‐isotopic enrichment of 16:0‐ceramide. Chromatographic separation was performed using ultra‐performance liquid chromatography. The analysis was performed on a triple quadrupole mass spectrometer using a positive ion electrospray ionization source with selected reaction monitoring (SRM). RESULTS The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H] + . The [ 13 C 16 ]16:0‐ceramide was monitored as [M+16+H] + . By monitoring two different transitions of the [ 13 C 16 ]16:0‐ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U‐ 13 C]palmitate infusion. CONCLUSIONS LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo . Copyright © 2012 John Wiley & Sons, Ltd.

We measured subcutaneous adipose tissue free fatty acid (FFA) storage rates in postprandial and walking conditions to better understand the contributions of this pathway to body fat distribution. Palmitate tracers were infused intravenously and fat biopsies collected to measure palmitate storage in upper- (UBSQ) and lower-body subcutaneous (LBSQ) fat in 41 (17 men) and 40 (16 men) volunteers under postprandial and under postabsorptive walking conditions, respectively. Postprandial palmitate storage was greater in women than men in UBSQ (0.50 ± 0.25 vs. 0.33 ± 0.37 μmol ⋅ kg fat−1 ⋅ min−1; P = 0.007) and LBSQ fat (0.37 ± 0.25 vs. 0.22 ± 0.20 μmol ⋅ kg fat−1 ⋅ min−1; P = 0.005); storage rates were significantly greater in UBSQ than LBSQ fat in both sexes. During walking, UBSQ palmitate storage did not differ between sexes, whereas LBSQ storage was greater in women than men (0.40 ± 0.22 vs. 0.25 ± 0.15 μmol ⋅ kg fat−1 ⋅ min−1; P = 0.01). In women only, walking palmitate storage was significantly greater in LBSQ than UBSQ fat. Adipocyte CD36 and diacylglycerol acyltransferase (DGAT) correlated with LBSQ palmitate storage in the postprandial and walking condition, respectively. We conclude that UBSQ fat is the preferred postprandial FFA storage depot for both sexes, whereas walking favors storage in LBSQ fat in women. Transmembrane transport (CD36) and esterification into triglycerides (DGAT) may be rate-limiting steps for LBSQ FFA storage during feeding and exercise.

OBJECTIVE Because direct adipose tissue free fatty acid (FFA) storage may contribute to body fat distribution, we measured FFA (palmitate) storage rates and fatty acid (FA) storage enzymes/proteins in omental and abdominal subcutaneous fat. RESEARCH DESIGN AND METHODS Elective surgery patients received a bolus of [1-14C]palmitate followed by omental and abdominal subcutaneous fat biopsies to measure direct FFA storage. Long chain acyl-CoA synthetase (ACS) and diacylglycerol acyltransferase activities, CD36, fatty acid-binding protein, and fatty acid transport protein 1 were measured. RESULTS Palmitate tracer storage (dpm/g adipose lipid) and calculated palmitate storage rates were greater in omental than abdominal subcutaneous fat in women (1.2 ± 0.8 vs. 0.7 ± 0.4 μmol ⋅ kg adipose lipid−1 ⋅ min−1, P = 0.005) and men (0.7 ± 0.2 vs. 0.2 ± 0.1, P &lt; 0.001), and both were greater in women than men (P &lt; 0.0001). Abdominal subcutaneous adipose tissue palmitate storage rates correlated with ACS activity (women: r = 0.66, P = 0.001; men: r = 0.70, P = 0.007); in men, CD36 was also independently related to palmitate storage rates. The content/activity of FA storage enzymes/proteins in omental fat was dramatically lower in those with more visceral fat. In women, only omental palmitate storage rates were correlated (r = 0.54, P = 0.03) with ACS activity. CONCLUSIONS Some adipocyte FA storage factors correlate with direct FFA storage, but sex differences in this process in visceral fat do not account for sex differences in visceral fatness. The reduced storage proteins in those with greater visceral fat suggest that the storage factors we measured are not a predominant cause of visceral adipose tissue accumulation.

Long‐chain acyl‐coenzymes A (acyl‐CoAs) (LCACoA) are the activated forms of long‐chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl‐CoA ([U‐ 13 C]16‐CoA) and oleoyl‐CoA ([U‐ 13 C]18:1‐CoA) using ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14‐CoA, C16‐CoA, C16:1‐CoA, C18‐CoA, C18:1‐CoA, C18:2‐CoA, C20‐CoA). The molecules are separated on a reversed‐phase UPLC column using a binary gradient with ammonium hydroxide (NH 4 OH) in water and NH 4 OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl‐CoA ([U −13 C]16‐CoA) and oleoyl‐CoA ([U −13 C]18:1‐CoA) using ultra‐performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14‐CoA, C16‐CoA, C16:1‐CoA, C18‐CoA, C18:1‐CoA, C18:2‐CoA, C20‐CoA). The molecules are separated on a reversed‐phase UPLC column using a binary gradient with ammonium hydroxide (NH 4 OH) in water and NH 4 OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M + 2 + H] + and [U 13 C]16‐CoA and [U 13 C]18:1‐CoA were monitored as [M + 16 + H] + and [M + 18 + H] + , respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U 13 C]16‐CoA and [U 13 C]18:1‐CoA during a low dose intravenous infusion of [U 13 C]palmitate and [U 13 C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool. Copyright © 2011 John Wiley & Sons, Ltd.

OBJECTIVE Preferential upper-body fat gain, a typical male pattern, is associated with a greater cardiometabolic risk. Regional differences in lipolysis and meal fat storage cannot explain sex differences in body fat distribution. We examined the potential role of the novel free fatty acid (FFA) storage pathway in determining body fat distribution in postabsorptive humans and whether adipocyte lipogenic proteins (CD36, acyl-CoA synthetases, and diacylglycerol acyltransferase) predict differences in FFA storage. RESEARCH DESIGN AND METHODS Rates of postabsorptive FFA (palmitate) storage into upper-body subcutaneous (UBSQ) and lower-body subcutaneous (LBSQ) fat were measured in 28 men and 53 premenopausal women. Stable and radiolabeled palmitate tracers were intravenously infused followed by subcutaneous fat biopsies. Body composition was assessed with a combination of dual-energy X-ray absorptiometry and computed tomography. RESULTS Women had greater FFA (palmitate) storage than men in both UBSQ (0.37 ± 0.15 vs. 0.27 ± 0.18 μmol · kg−1 · min−1, P = 0.0001) and LBSQ (0.42 ± 0.19 vs. 0.22 ± 0.11 μmol · kg−1 · min−1, P &lt; 0.0001) fat. Palmitate storage rates were significantly greater in LBSQ than UBSQ fat in women, whereas the opposite was true in men. Plasma palmitate concentration positively predicted palmitate storage in both depots and sexes. Adipocyte CD36 content predicted UBSQ palmitate storage and sex-predicted storage in LBSQ fat. Palmitate storage rates per kilogram fat did not decrease as a function of fat mass, whereas lipolysis did. CONCLUSIONS The FFA storage pathway, which had remained undetected in postabsorptive humans until recently, can have considerable, long-term, and sex-specific effects on body fat distribution. It can also offer a way of protecting the body from excessive circulating FFA in obesity.