Errors in measuring plasma free fatty acid concentrations with a popular enzymatic colorimetric kit.

OBJECTIVES: Our goal was to test whether an enzymatic, colorimetric assay, the WAKO NEFA kit, provides information equivalent to liquid chromatography (LC) LC-based measures of free fatty acid (FFA). DESIGN & METHODS: We reanalyzed nadir FFA samples from 109 volunteers from a previous study where we demonstrated that maximal suppression of FFA concentrations predicts metabolic abnormalities in humans; the results from the WAKO NEFA kit, which has been widely used for over three decades, could not replicate our findings. We conducted additional studies to directly compare results from this kit to our LC-mass spectrometry (LC/MS) method that was validated by our LC-UV detection method. RESULTS: Plasma samples with FFA concentrations ranging from 0.015 to 1.813 mmol/L were measured both by LC-mass spectrometry (LC/MS) and by the WAKO NEFA kit. Despite good overall agreement (R2 = 0.86), the slope was significantly different from 1.0 and the intercept was significantly different from zero. The results from the kit were especially discrepant with FFA concentrations <0.200 and >1.000 mmol/L. Some of the discrepancy was related to the use of oleate as the standard solution for the kit and the substrate specificity of the kit enzymes for different fatty acids. Despite attempts to improve the kit by modifying the reaction time, sample volume and the types of standard solutions, we could not obtain a satisfactory agreement between the WAKO NEFA results and LC/MS. CONCLUSIONS: The WAKO NEFA kit should not be used when high precision and accuracy of FFA concentrations over a wide range is required.

Glucose tolerance and free fatty acid metabolism in adults with variations in TCF7L2 rs7903146.

OBJECTIVE: TCF7L2 variant rs7903146 is associated with increased risk for type 2 diabetes. We investigated the effect of TCF7L2 variant rs7903146 and glucose tolerance on free fatty acid (FFA) metabolism. RESEARCH DESIGN AND METHODS: We recruited 120 individuals, half homozygous for the major CC allele and half homozygous for the minor TT allele at rs7903146; each underwent a 2-h, 75g oral glucose tolerance test (OGTT). Plasma glucose, insulin and free fatty acid concentrations were measured on blood collected before and during the OGTT. RESULTS: Total FFA concentrations and percent FA species during OGTT were not different in CC and TT carriers when males and females were considered together. However, monounsaturated fatty acid (MUFA) concentrations and percentages were greater in TT than CC females during the OGTT. TT carriers with high HOMA-IR had significantly greater fasting FFA concentrations, lower disposition index (DI) and greater AUC of glucose than high HOMA-IR CC carriers, whereas no such differences were observed in the low HOMA-IR group. We found that fasting (826±25 vs. 634±22µmol/L, P<0.0001) and OGTT plasma FFA concentrations were greater in IGT than NGT subjects, and the difference remained after adjusting for sex, age, BMI, and genotype. Finally, IGT subjects had greater MUFA concentrations and percentages than NGT subjects during OGTT. CONCLUSIONS: Despite similar fasting insulin and glucose, fasting plasma FFA are greater in IGT than NGT adults. Insulin resistance and sex influence plasma FFA responses amongst carriers of the minor T allele of TCF7L2 rs7903146.

A novel ELISA for measuring CD36 protein in human adipose tissue.

CD36 is a transmembrane protein present in many tissues that is believed to facilitate inward fatty acid transport. Western blotting is the most widely used method to measure tissue CD36 protein content, but it is time consuming, technically demanding, and semiquantitative. To more precisely measure adipose tissue CD36 content we developed an enzyme linked immunosorbent assay (ELISA) after establishing that: 1) the anti-CD36 antibodies gave a single distinct band on traditional Western blots, and 2) the vast majority of adipocyte CD36 resides in the plasma membrane. By using serial dilutions of each sample and including a calibrator sample and quality control sample on each plate, we could achieve inter- and intra-assay variability of ∼ 10%. We found that CD36 content in omental and abdominal subcutaneous adipose tissue varied over a 2-5-fold range depending upon the means of data expression (per units of tissue protein, weight, or lipid). Omental CD36 content in women decreased markedly (P = 0.01) as a function of fat cell size. For the most part, tissue CD36 content was not correlated with CD36 mRNA. This ELISA method for tissue CD36 content should enhance research into the role of this protein on tissue fatty acid uptake.

Intrasample variability of intramyocellular triacylglycerol.

Intrasample variability of intramyocellular triacylglycerol (imcTG) in the skeletal muscle of rats has been examined. Aliquoting after homogenization of muscle samples reduced imcTG variability considerably compared with aliquoting before homogenization. The results suggested that skeletal muscle samples be homogenized before aliquoting in order to reduce imcTG variability.

Acute hyperinsulinemia inhibits intramyocellular triglyceride synthesis in high-fat-fed obese rats.

Hyperinsulinemia is common in obesity, but whether it plays a role in intramyocellular triglyceride (imcTG) buildup is unknown. In this study, hyperinsulinemic-euglycemic clamp experiments were performed in overnight-fasted lean and high-fat-fed obese rats, awake, to determine the effect of insulin on imcTG synthesis (incorporation of [(14)C]glycerol, [(14)C]glucose, and [(3)H]oleate). Insulin infusion at 25 (low insulin) and 100 (high insulin) pmol/kg/min increased plasma insulin by 5- and 16-fold, respectively, whereas plasma and intramyocellular glycerol, FFAs, triglycerides, and glucose levels were maintained at their basal levels by co-infusion of exogenous glycerol, FFAs, and triglycerides at fixed rates and glucose at varying rates. In obese rats, insulin suppressed incorporation of glycerol into the imcTG-glycerol moiety dose dependently (P < 0.01-P < 0.001) in gastrocnemius and tibialis anterior, but only the high insulin suppressed it in soleus (P < 0.05). The low insulin suppressed glucose incorporation into imcTG-glycerol in all three muscles (P = 0.01-P < 0.01). However, the low insulin did not affect (P > 0.05) and the high insulin suppressed (P < 0.05-P < 0.01) fatty acid incorporation into imcTG in all three muscles. Insulin also suppressed glycerol incorporation in lean rats (P < 0.01-P < 0.04). On the other hand, imcTG pool size was not affected by insulin (P > 0.05). These observations suggest that acute hyperinsulinemia inhibits imcTG synthesis and thus does not appear to promote imcTG accumulation via the synthetic pathway, at least in the short term.

Muscle type-dependent responses to insulin in intramyocellular triglyceride turnover in obese rats.

OBJECTIVE: To understand the role of hyperinsulinemia in intramyocellular (imc) triglyceride (TG) accumulation and in regulating imcTG turnover. RESEARCH METHODS AND PROCEDURES: imcTG was first prelabeled by continuous infusion of [U-(14)C]glycerol (pulse), and then the rate of label loss from the prelabeled imcTG pool (turnover) in gastrocnemius, tibialis anterior, and soleus muscle of awake, high-fat-fed obese rats during the subsequent hyperinsulinemic-euglycemic clamp experiments (chase) was determined. RESULTS: Post-absorptive basal fractional imcTG turnover rate in soleus was 0.010 +/- 0.001/min, significantly lower than that in gastrocnemius (0.026 +/- 0.002/min, p < 0.001) or tibialis anterior (0.030 +/- 0.002/min, p < 0.0001), a pattern reciprocal to their imcTG pool size. Insulin infusion at 25 pmol/kg per minute resulted in pathophysiological hyperinsulinemia (5-fold increase over the baseline value). This caused an increase in imcTG turnover by 3-fold in soleus (0.029 +/- 0.006/min, p = 0.002) but a decrease in gastrocnemius (0.012 +/- 0.003/min, p = 0.001) and in tibialis anterior (0.0064 +/- 0.001/min, p < 0.0001). Pathophysiological hyperinsulinemia suppressed hormone-sensitive lipase activity in heart (p = 0.01) and mesenteric fat (p = 0.05) but not in skeletal muscle (p > 0.05). The pool size of imcTG was not affected by hyperinsulinemia. DISCUSSION: The results demonstrated muscle-type dependence in the response of imcTG turnover to hyperinsulinemia in the obesity model. The reciprocal insulin effects on imcTG turnover in oxidative vs. oxidative-glycolytic muscle indicated a possibility that oxidative muscle contributes more to insulin resistance under hyperinsulinemia if imcTG-fatty acid oxidation is a function of turnover. imcTG turnover does not seem to regulate imcTG pool size acutely.

Evidence for increased and insulin-resistant lipolysis in skeletal muscle of high-fat-fed rats.

The metabolic and isotopic profiles of glycerol in skeletal muscle were examined using awake, fasted lean and high-fat-induced obese rats, and hyperinsulinemic-euglycemic clamp was performed to assess the effect of insulin. During the clamp, Intralipid (no heparin; Fresnius Kabi Clayton, Clayton, NC), free fatty acids, glycerol, and glucose were coinfused to maintain their respective basal plasma levels in both groups. At steady-state, [U-(14)C]glycerol was infused intravenously for 120 minutes followed by muscle biopsy. The classical phenotypic characteristics of obesity, namely, reduced insulin-stimulated glucose uptake, a failure to suppress systemic lipolysis by insulin, and elevated plasma fatty acid concentration, were observed in the obese rats. Novel observations showed that in the basal state, the isotopic specific activity (S.A.) of glycerol (dpm/nmol) in gastrocnemius (0.03 v 0.12), soleus (0.05 v 0.12), and tibialis anterior (0.03 v 0.12) was significantly lower (all P <.003) in obese than in lean rats despite similar concentrations, indicating an active basal intramyocellular lipolysis. In addition, the lipolysis appeared resistant to insulin because the suppression of muscle glycerol during the clamp was 8%, 12%, and 8% in obese compared to 67%, 71%, and 63% in the lean control for gastrocnemius (P =.001), soleus (P =.007), and tibialis anterior (P =.004), respectively. The active intracellular lipolysis likely disturbs metabolic functions that may contribute to insulin resistance.

Substrate concentration and metabolism in left and right muscles of rats.

Skeletal muscle fiber heterogeneity among muscle groups is well known; however, laterality of muscle metabolism has not been addressed. In the present studies, metabolite concentrations in left and right gastrocnemius, tibialis anterior, quadriceps, and soleus muscles and their response to exogenous insulin have been compared in fasted awake rats. The results indicated that the concentrations of muscle free glycerol (P >.4), glycerol 3-phosphate (P >.1) nonesterified fatty acids (NEFA) (P >.6) and intramyocellular triglycerides (imcTG) (P >.08) are comparable between left and right of the same muscle, and are similar among mixed glycolytic-oxidative muscles. The concentration of free glycerol in soleus responded to exogenous insulin in a pattern distinct to that seen for the mixed muscles. The results support interchangeable use of left and right side of same muscles, and probably among different muscles of similar fiber type, but not muscles of different fiber types.

Dual tail catheters for infusion and sampling in rats as an efficient platform for metabolic experiments.

The authors present a technique for establishing a virtually stress-free dual-catheter system in rats, allowing researchers to conduct extended, complex experimental procedures involving the simultaneous infusion of multiple compounds and frequent collection of blood samples.