Christina Koutsari

Thematic review series: Patient-Oriented Research. Free fatty acid metabolism in human obesity

Adipose tissue lipolysis provides circulating FFAs to meet the bodys lipid fuel demands. FFA release is well regulated in normal-weight individuals; however, in upper-body obesity, excess lipolysis is commonly seen. This abnormality is considered a cause for at least some of the metabolic defects (dyslipidemia, insulin resistance) associated with upper-body obesity. “Normal” lipolysis is sex-specific and largely determined by the individuals resting metabolic rate. Women have greater FFA release rates than men without higher FFA concentrations or greater fatty acid oxidation, indicating that they have greater nonoxidative FFA disposal, although the processes and tissues involved in this phenomenon are unknown. Therefore, women have the advantage of having greater FFA availability without exposing their tissues to higher and potentially harmful FFA concentrations. Upper-body fat is more lipolytically active than lower-body fat in both women and men. FFA released by the visceral fat depot contributes only a small percentage of systemic FFA delivery. Upper-body subcutaneous fat is the dominant contributor to circulating FFAs and the source of the excess FFA release in upper-body obesity. We believe that abnormalities in subcutaneous lipolysis could be more important than those in visceral lipolysis as a cause of peripheral insulin resistance. Understanding the regulation of FFA availability will help to discover new approaches to treat FFA-induced abnormalities in obesity.

Sex- and Depot-Dependent Differences in Adipogenesis in Normal-Weight Humans

To elucidate cellular mechanisms of sex‐related differences in fat distribution, we determined body fat distribution (dual‐energy X‐ray absorptiometry and single‐slice abdominal computed tomography (CT)), adipocyte size, adipocyte number, and proportion of early‐differentiated adipocytes (aP2+CD68−) in the stromovascular fraction (SVF) in the upper and lower body of normal‐weight healthy men (n = 12) and premenopausal women (n = 20) (age: 18–49 years, BMI: 18–26 kg/m2). Women had more subcutaneous and less visceral fat than men. The proportion of early differentiated adipocytes in the subcutaneous adipose tissue SVF of women was greater than in men (P = 0.01), especially in the femoral depot, although in vitro adipogenesis, as assessed by peroxisome proliferator activated receptor‐γ (PPARγ) expression, was not increased in femoral preadipocytes cultured from women compared with men. In women, differentiation of femoral preadipocytes was less than that of abdominal subcutaneous preadipocytes (P = 0.04), and femoral subcutaneous preadipocytes tended to be more resistant to tumor necrosis factor‐α (TNFα)–induced apoptosis (P = 0.06). Thus, turnover and utilization of the preadipocyte pool may be reduced in lower vs. the upper‐body fat in women. Collectively, these data indicate that the microenvironment, rather than differences in inherent properties of preadipocytes between genders, may explain the gynoid obesity phenotype and higher percent body fat in women compared to men.

Direct Free Fatty Acid Uptake Into Human Adipocytes In Vivo: Relation to Body Fat Distribution

We sought to assess whether direct uptake of circulating free fatty acids (FFAs) by adipocytes occurs in vivo in overnight postabsorptive humans and, if so, whether there are regional differences in uptake between lean and obese women and men. We used bolus injections of radiolabeled FFA tracers followed by carefully timed adipose tissue biopsies. First, we validated a method to measure direct adipocyte FFA uptake and then quantitated this process using the modified methods in normal-weight postabsorptive men and women. We then used a continuous infusion of radiolabeled FFA to measure direct and indirect (VLDL) regional adipose tissue uptake in obese men and women. Direct FFA uptake was greater in women than men: 8.2 ± 0.6 vs. 4.0 ± 0.5% (P < 0.0001) of FFAs were taken up by subcutaneous adipose tissue, respectively. Abdominal subcutaneous fat took up FFAs more avidly than femoral fat in men, but this did not occur in women. Similar sex and regional difference were found to occur in obese women and men. Gene expression of fatty acid transporters was greater in abdominal than femoral fat in men (P < 0.05) but not in women (P = 0.80). We observed sex- and site-specific recycling of circulating FFAs into subcutaneous fat. This is a novel FFA disposal pathway that may also play a role in the development or maintenance of body fat distribution. Regional variations in facilitated fatty acid transport may contribute to this process.

Committed subcutaneous preadipocytes are reduced in human obesity

Aims/hypothesisThe aim of this study was to test whether the availability of committed preadipocytes in abdominal and femoral subcutaneous adipose tissue varies with obesity and body fat distribution.MethodsBody composition, fat cell size, committed preadipocytes and macrophages were measured in subcutaneous abdominal and femoral adipose depots of 17 lean, 16 upper-body-obese (UBO) and 13 lower-body-obese (LBO) women. Preadipocytes and macrophages were identified by simultaneous staining with the respective markers aP2 and CD68. In a subset of samples we measured preadipocyte proliferation, differentiation and susceptibility to apoptosis.ResultsAbdominal adipocytes were smaller in lean than in obese women. Committed preadipocytes represented a greater fraction of stromovascular cells in lean than in obese women but were similar between UBO and LBO women (abdomen: ∼30 ± 3 vs ∼17 ± 2%; thigh: ∼30 ± 3 vs ∼17 ± 2%). Preliminary data suggested that preadipocyte kinetics were similar in LBO and lean women, whereas preadipocytes of UBO women differentiated less and were more susceptible to apoptotic stimuli. The fraction of stromovascular cells that were macrophages was greater in both depots in obese women (UBO and LBO) than in normal-weight women, but the difference was not statistically significant.Conclusions/interpretationThe proportion of subcutaneous adipose tissue stromovascular cells that are committed preadipocytes is reduced with obesity. This could be due to greater recruitment of preadipocytes to adipogenesis or greater preadipocyte apoptosis, depending upon the obesity phenotype. These data are consistent with the concept that body fat distribution may be regulated partly through differences in adipogenesis.

Storage of Circulating Free Fatty Acid in Adipose Tissue of Postabsorptive Humans Quantitative Measures and Implications for Body Fat Distribution

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 < 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.

A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans

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.

Meal Fatty Acid Uptake in Visceral Fat in Women

OBJECTIVE—Differential meal fat uptake into adipose tissue depots may be a determinant of body fat distribution. RESEARCH DESIGN AND METHODS—We used the meal fat tracer/adipose tissue biopsy approach to compare the effects of meal fat content on the fat uptake into visceral and upper and lower body subcutaneous fat depots in 21 premenopausal women. [3H]triolein was used to trace the fate of fatty acids from a normal-fat or high-fat meal. RESULTS—The proportion of dietary fat uptake into the three depots did not differ between meals; visceral fat accounted for only ∼5% of meal fat disposal irrespective of visceral fat mass. For the women consuming the normal-fat meal, the uptake of meal fatty acid into femoral fat (milligrams meal fat per gram lipid) increased as a function of leg fat mass (r = 0.68, P < 0.05), which we interpret as increased efficiency of uptake. The opposite pattern was seen in omental fat with the normal-fat meal and in all depots after the high-fat meal. For both meals, ∼40% of meal fat was oxidized (3H2O production) after 24 h. CONCLUSIONS—We conclude that greater thigh adipose tissue in women is associated with greater efficiency of meal fat storage under conditions of energy balance, whereas the opposite is seen with visceral fat. These findings imply that different mechanisms may regulate fatty acid uptake in different depots, which may in turn impact on body fat distribution.

Plasma Free Fatty Acid Storage in Subcutaneous and Visceral Adipose Tissue in Postabsorptive Women

OBJECTIVE— We assessed the direct (VLDL-triglycerides [VLDL-TG] independent) storage of circulating free fatty acids (FFAs) in visceral and subcutaneous fat in postabsorptive women. RESEARCH DESIGN AND METHODS— Twelve women (BMI 29.6 ± 6.6 kg/m2) received an identical, intravenous bolus dose of [1-14C]oleate followed by timed subcutaneous fat biopsies (abdominal and femoral) and then omental fat biopsy during tubal ligation surgery. Regional fat masses were assessed by combining dual-energy X-ray absorptiometry and computed tomography scanning. Separately, we assessed the fraction of FFA tracer entering VLDL-TG over the time representing the delay in collecting omental fat. RESULTS— Site-specific fat specific activity (SA) (dpm/g lipid) decreased as a function of fat mass in both upper-body subcutaneous (UBSQ) and visceral fat depots. These patterns are consistent with dilution of a relatively fixed amount of FFA tracer within progressively greater amounts of fat. Interestingly, femoral SA did not vary as a function of lower-body subcutaneous (LBSQ) fat mass. [1-14C]oleate storage per million LBSQ adipocytes was positively associated with LBSQ fat mass, but no significant relationships were observed in UBSQ or visceral fat depot. The fraction of [1-14C]oleate stored in UBSQ, LBSQ, and visceral fat was 6.7 ± 3.2, 4.9 ± 3.4, and 1.0 ± 0.3%, respectively. Only ∼4% of the tracer traversed VLDL-TG over 9.5 h. CONCLUSIONS— The increase in FFA tracer storage per adipocyte as a function of LBSQ fat mass implies that LBSQ adipocytes, in contrast to UBSQ and omental adipocytes, store more FFA in women with greater adiposity. The direct FFA storage pathway might play a role in favoring lower-body fat accumulation in women.

Free Fatty Acid Storage in Human Visceral and Subcutaneous Adipose Tissue: Role of Adipocyte Proteins

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 < 0.001), and both were greater in women than men (P < 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.

Nonoxidative free fatty acid disposal is greater in young women than men

CONTEXT Large increases in systemic free fatty acid (FFA) availability in the absence of a corresponding increase in fatty acid oxidation can create a host of metabolic abnormalities. These adverse responses are thought to be the result of fatty acids being shunted into hepatic very low-density lipoprotein-triglyceride production and/or intracellular lipid storage and signaling pathways because tissues are forced to increase nonoxidative FFA disposal. OBJECTIVE The objective of the study was to examine whether variations in postabsorptive nonoxidative FFA disposal within the usual range predict insulin resistance and hypertriglyceridemia. DESIGN We measured: systemic FFA turnover using a continuous iv infusion of [9-10, (3)H]palmitate; substrate oxidation with indirect calorimetry combined with urinary nitrogen excretion; whole-body and peripheral insulin sensitivity with the labeled iv glucose tolerance test minimal model. SETTING the study was conducted at the Mayo Clinic General Clinical Research Center. PARTICIPANTS Participants included healthy, postabsorptive, nonobese adults (21 women and 21 men). INTERVENTIONS There were no interventions. MAIN OUTCOME MEASURES Nonoxidative FFA disposal (micromoles per minute), defined as the FFA disappearance rate minus fatty acid oxidation. RESULTS Women had 64% greater nonoxidative FFA disposal rate than men but a better lipid profile and similar insulin sensitivity. There was no significant correlation between nonoxidative FFA disposal and whole-body sensitivity, peripheral insulin sensitivity, or fasting serum triglyceride concentrations in men or women. CONCLUSIONS Healthy nonobese women have greater rates of nonoxidative FFA disposal than men, but this does not appear to relate to adverse health consequences. Understanding the sex-specific interaction between adipose tissue lipolysis and peripheral FFA removal will help to discover new approaches to treat FFA-induced abnormalities.