Insulin sensitivity is not associated with palmitoleate availability in obese humans

We evaluated whether insulin resistance in obese people is associated with decreased plasma palmitoleate availability. Palmitoleate content (percentage and absolute concentrations) in FFA and VLDL was measured in obese subjects who were either insulin resistant (IR) or insulin sensitive (IS), based on assessment of multiorgan (skeletal muscle, liver, and adipose tissue) insulin sensitivity by using the hyperinsulinemic-euglycemic clamp procedure in conjunction with infusion of stable isotopically labeled tracers. Plasma palmitoleate concentration and the relative contribution of palmitoleate to total plasma FFA concentration in the IS group (0.018 ± 0.002 mmol/l and 4.4% ± 0.2%, respectively) were not significantly different than values in the IR group (0.023 ± 0.003 mmol/l and 4.4% ± 0.4%, respectively). Plasma VLDL-triglyceride palmitoleate concentration and the proportion of VLDL fatty acids as palmitoleate in the IS group (0.09 ± 0.02 mmol/l and 5.7 ± 0.3%, respectively) were also not significantly different than those in the IR group (0.16 ± 0.04 mmol/l and 5.0% ± 0.4%, respectively). These data demonstrate that decreased palmitoleate in plasma and in VLDL is not associated with insulin resistance in skeletal muscle, liver, or adipose tissue in obese people. We evaluated whether insulin resistance in obese people is associated with decreased plasma palmitoleate availability. Palmitoleate content (percentage and absolute concentrations) in FFA and VLDL was measured in obese subjects who were either insulin resistant (IR) or insulin sensitive (IS), based on assessment of multiorgan (skeletal muscle, liver, and adipose tissue) insulin sensitivity by using the hyperinsulinemic-euglycemic clamp procedure in conjunction with infusion of stable isotopically labeled tracers. Plasma palmitoleate concentration and the relative contribution of palmitoleate to total plasma FFA concentration in the IS group (0.018 ± 0.002 mmol/l and 4.4% ± 0.2%, respectively) were not significantly different than values in the IR group (0.023 ± 0.003 mmol/l and 4.4% ± 0.4%, respectively). Plasma VLDL-triglyceride palmitoleate concentration and the proportion of VLDL fatty acids as palmitoleate in the IS group (0.09 ± 0.02 mmol/l and 5.7 ± 0.3%, respectively) were also not significantly different than those in the IR group (0.16 ± 0.04 mmol/l and 5.0% ± 0.4%, respectively). These data demonstrate that decreased palmitoleate in plasma and in VLDL is not associated with insulin resistance in skeletal muscle, liver, or adipose tissue in obese people. An increase in circulating free fatty acids (FFA) impairs insulin action in skeletal muscle and liver and likely contributes to the insulin resistance associated with obesity (1Boden G. Interaction between free fatty acids and glucose metabolism.Curr. Opin. Clin. Nutr. Metab. Care. 2002; 5: 545-549Crossref PubMed Scopus (177) Google Scholar). However, data from studies conducted in rodents have shown that infusing palmitoleic acid (16:1n–7), which is normally produced in adipose tissue and the liver through lipogenesis, followed by desaturation of palmitic acid (16:0) by the stearoyl-CoA desaturase (SCD1) gene, augments skeletal muscle insulin signaling and increases insulin-mediated muscle glucose uptake (2Cao H. Gerhold K. Mayers J.R. Wiest M.M. Watkins S.M. Hotamisligil G.S. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.Cell. 2008; 134: 933-944Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). The importance of circulating palmitoleic acid in regulating insulin sensitivity in humans is unclear because of conflicting data from different studies, which have found that the percentage of palmitoleate in plasma FFA or lipids correlated directly with insulin sensitivity (3Stefan N. Kantartzis K. Celebi N. Staiger H. Machann J. Schick F. Cegan A. Elcnerova M. Schleicher E. Fritsche A. Circulating palmitoleate strongly and independently predicts insulin sensitivity in humans.Diabetes Care. 2010; 33: 405-407Crossref PubMed Scopus (111) Google Scholar) or was greater in insulin-resistant (IR) than insulin-sensitive (IS) subjects (4Kusunoki M. Tsutsumi K. Nakayama M. Kurokawa T. Nakamura T. Ogawa H. Fukuzawa Y. Morishita M. Koide T. Miyata T. Relationship between serum concentrations of saturated fatty acids and unsaturated fatty acids and the homeostasis model insulin resistance index in Japanese patients with type 2 diabetes mellitus.J. Med. Invest. 2007; 54: 243-247Crossref PubMed Scopus (59) Google Scholar, 5Pelikanova T. Kazdova L. Chvojkova S. Base J. Serum phospholipid fatty acid composition and insulin action in type 2 diabetic patients.Metabolism. 2001; 50: 1472-1478Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 6Vessby B. Tengblad S. Lithell H. Insulin sensitivity is related to the fatty acid composition of serum lipids and skeletal muscle phospholipids in 70-year-old men.Diabetologia. 1994; 37: 1044-1050Crossref PubMed Scopus (294) Google Scholar, 7Ho R.C. Davy K.P. Hickey M.S. Summers S.A. Melby C.L. Behavioral, metabolic, and molecular correlates of lower insulin sensitivity in Mexican-Americans.Am. J. Physiol. Endocrinol. Metab. 2002; 283: E799-E808Crossref PubMed Scopus (21) Google Scholar, 8Okada T. Furuhashi N. Kuromori Y. Miyashita M. Iwata F. Harada K. Plasma palmitoleic acid content and obesity in children.Am. J. Clin. Nutr. 2005; 82: 747-750Crossref PubMed Scopus (123) Google Scholar). Moreover, these studies did not measure absolute palmitoleate concentration, which provides a better assessment of palmitoleate availability to peripheral tissues, or palmitoleate within very low-density lipoprotein (VLDL), which may be an important contributor to tissue fatty acid delivery. The purpose of the present study was to determine whether impaired insulin-mediated skeletal muscle glucose uptake was associated with a decrease in plasma palmitoleic acid availability in obese people. Accordingly, we determined plasma free palmitoleic acid concentration, which is derived primarily from palmitoleate syn­thesized in adipose tissue, and palmitoleic acid content in VLDL, which is derived primarily from palmitoleate synthesized in the liver. Study subjects were specifically selected to be either IS or IR based on the increase in glucose uptake during a hyperinsulinemic-euglycemic clamp procedure. A total of 39 overweight and obese (body mass index [BMI], 36.1 ± 0.7 kg/m2; age 42 ± 2 years) men and women who previously underwent a hyperinsulinemic-euglycemic clamp procedure and an evaluation of lipoprotein kinetics (9Fabbrini E. Magkos F. Mohammed B.S. Pietka T. Abumrad N.A. Patterson B.W. Okunade A. Klein S. Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity.Proc. Natl. Acad. Sci. USA. 2009; 106: 15430-15435Crossref PubMed Scopus (738) Google Scholar) participated in this study. Among these 39 subjects, 20 subjects, who comprised the lowest and highest quartiles in skeletal muscle insulin sensitivity, assessed as the increase in glucose disposal during insulin infusion, were used to establish an IR group (n = 10; age, 41 ± 4 years old; 3 men and 7 women; ≤135% increase in glucose disposal) and an IS group (n = 10; age, 44 ± 3 years old; 10 women; ≥261% increase in glucose disposal). All subjects completed a comprehensive medical evaluation, including an oral glucose tolerance test at screening. No subject had any history or evidence of diabetes or other serious chronic diseases or consumed >10 g/day of alcohol. Subjects gave their written informed consent before participating in this study, which was approved by the Human Research Protection Office of Washington University School of Medicine. Percent body fat and intra-abdominal adipose tissue volume were determined by using dual-energy X-ray absorptiometry and magnetic resonance imaging (10Korenblat K.M. Fabbrini E. Mohammed B.S. Klein S. Liver, muscle, and adipose tissue insulin action is directly related to intrahepatic triglyceride content in obese subjects.Gastroenterology. 2008; 134: 1369-1375Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Hepatic tissue, skeletal muscle, and adipose tissue insulin sensitivity levels were measured by using a two-stage hyperinsulinemic-euglycemic clamp procedure in conjunction with stable isotopically labeled glucose and palmitate tracer infusions, as described previously (9Fabbrini E. Magkos F. Mohammed B.S. Pietka T. Abumrad N.A. Patterson B.W. Okunade A. Klein S. Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity.Proc. Natl. Acad. Sci. USA. 2009; 106: 15430-15435Crossref PubMed Scopus (738) Google Scholar). Subjects were admitted to the Clinical Research Unit on the evening before the clamp procedure, consumed a standard meal at 1900 h, and then fasted until completion of the clamp procedure the next day. At 0600 h, the following morning, a primed, continuous infusion of [6,6-2H2]glucose and a continuous infusion of [2,2-2H2]palmitate were started and continued for 9.5 h. After 3.5 h of tracer infusion (basal period), a two-stage euglycemic-hyperinsulinemic clamp procedure was started and continued for 6 h. Insulin was infused at a rate of 20 mU ∙ m−2 body surface area (BSA) ∙ min−1 during stage 1 (3.5–5.5 h) and at a rate of 50 mU ∙ m−2 BSA ∙ min−1 during stage 2 (5.5–9.5 h) of the clamp procedure. Euglycemia (100 mg/dl) was maintained by infusing 20% dextrose enriched to 2.5% with [6,6-2H2]glucose at various rates. Glucose and palmitate tracer-to-tracee ratios were determined by using gas chromatography-mass spectrometry (10Korenblat K.M. Fabbrini E. Mohammed B.S. Klein S. Liver, muscle, and adipose tissue insulin action is directly related to intrahepatic triglyceride content in obese subjects.Gastroenterology. 2008; 134: 1369-1375Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 11Fabbrini E. Mohammed B.S. Magkos F. Korenblat K.M. Patterson B.W. Klein S. Alterations in adipose tissue and hepatic lipid kinetics in obese men and women with nonalcoholic fatty liver disease.Gastroenterology. 2008; 134: 424-431Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Adipose tissue and skeletal muscle insulin sensitivity levels were assessed as the relative decrease in palmitate rate of appearance (Ra) in plasma during stage 1 (low-dose insulin) and the relative increase in glucose rate of disappearance (Rd) from plasma during stage 2 (high-dose insulin) of the clamp procedure (9Fabbrini E. Magkos F. Mohammed B.S. Pietka T. Abumrad N.A. Patterson B.W. Okunade A. Klein S. Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity.Proc. Natl. Acad. Sci. USA. 2009; 106: 15430-15435Crossref PubMed Scopus (738) Google Scholar). Hepatic insulin sensitivity was calculated as the hepatic insulin sensitivity index (HISI) (9Fabbrini E. Magkos F. Mohammed B.S. Pietka T. Abumrad N.A. Patterson B.W. Okunade A. Klein S. Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity.Proc. Natl. Acad. Sci. USA. 2009; 106: 15430-15435Crossref PubMed Scopus (738) Google Scholar). To measure palmitoleate content in adipose tissue, subcutaneous abdominal adipose tissue biopsies were performed and tissue was obtained by aspiration through a 4-mm liposuction cannula at 1 h after the basal stage of the hyperinsulinemic-euglycemic clamp procedure was started. Immediately after being collected, adipose tissue samples were rinsed in ice-cold saline, frozen in liquid nitrogen, and stored at −80°C until final analyses were performed. Blood samples obtained during a study conducted to measure hepatic lipoprotein kinetics, performed ∼1 week apart from the clamp procedure, were used to assess circulating palmitoleate content in plasma FFA and VLDL. Subjects were admitted to the Clinical Research Unit in the evening before the study and consumed a standard meal. The following morning, after subjects fasted overnight, 17 blood samples were obtained over 12 h (0600–1800 h) of continued fasting to determine free palmitoleate concentrations, plasma FFA composition, and palmitoleate in VLDL lipid by using gas chromatography (11Fabbrini E. Mohammed B.S. Magkos F. Korenblat K.M. Patterson B.W. Klein S. Alterations in adipose tissue and hepatic lipid kinetics in obese men and women with nonalcoholic fatty liver disease.Gastroenterology. 2008; 134: 424-431Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). To isolate VLDL particles, approximately 1.5 ml of plasma was transferred into OptiSeal polyallomer tubes (Beckman Instruments, Palo Alto, CA) overlaid with an NaCl-EDTA solution (1.006 g/ml) and centrifuged at 100,000 g for 16 h at 10°C in an Optima LE-80K preparative ultracentrifuge equipped with a type 50.4 Ti rotor (Beckman Instruments, Palo Alto, CA). The top layer containing VLDL was removed by tube slicing (CentriTube slicer; Beckman Instruments, Palo Alto, CA). Values from all samples for each subject were averaged to provide a robust measure of fatty acid composition in plasma and VLDL. Plasma-free palmitoleate (in mmol/l) was calculated as percent palmitoleate in plasma × FFA concentration. Palmitoleate in VLDL (in mmol/l) was calculated as the percentage of VLDL fatty acids as palmitoleate × VLDL-triglyceride (TG) concentration (in mmol/l) × 3 (because each TG contains 3 fatty acids). An estimate of adipose tissue palmitoleate content (in moles) was calculated as the percentage of palmitoleate × moles of fatty acids in adipose tissue TG, the latter calculated on the basis of total body fat mass and the average molecular mass of TG (861 g/mol). The product-to-precursor ratio of palmitoleate to palmitate (C16:1:C16:0) in VLDL-TG was used as an index of hepatic SCD1 activity, because SCD1 is the enzyme that mediates the desaturation of palmitate (C16:0) into palmitoleate (C16:1) (12Peter A. Cegan A. Wagner S. Lehmann R. Stefan N. Konigsrainer A. Konigsrainer I. Haring H.U. Schleicher E. Hepatic lipid composition and stearoyl-coenzyme A desaturase 1 mRNA expression can be estimated from plasma VLDL fatty acid ratios.Clin. Chem. 2009; 55: 2113-2120Crossref PubMed Scopus (104) Google Scholar). The adipose tissue SCD1 gene expression was determined by using quantitative real-time PCR. Total RNA was isolated from adipose tissues by using TRIzol (Invitrogen). RNA was quantified by using spectrophotometry (NanoDrop 1000, Thermo Scientific, Wilmington, DE), and cDNA was synthesized by using a TaqMan reverse transcription kit (Applied BioSystems). cDNA samples were then amplified by using SYBR Green PCR Master Mix (Applied BioSystems) on an ABI 7500 real-time PCR system (Applied BioSystems). Results were analyzed by comparing the cycle threshold (Ct) value of each sample after normalization to the housekeeping 36B4 gene. The changes, Δ, in Ct (ΔCt) were used to calculate the relative levels of each mRNA compared with that of the control gene from the various samples, using the formula. The forward and reverse primers used for SCD1 transcription were 5′-ACACTTGGGAGCCCTGTATG-3′ and 3′-GCAG­CC­GA­GCTTTGTAAGA-5′, respectively; and the forward and reverse primers used for 36B4 transcription were 5′-GTGAT­GTG­­CA­GCTGATCAAGACT-3′ and 3′-GATGACCAGCCCAAAGG­AGA-5′, respectively. All data sets were tested for normality according to the Shapiro-Wilk test. For normally distributed variables, comparisons between groups were performed by using parametric procedures. Levene's test was used to assess the equality of group variances on each dependent variable, and Student's t-test for unpaired samples was used to determine the significance of differences between groups. When variables were not normally distributed and could not be normalized by using standard mathematical transformations, datasets were ranked for all statistical analyses. Pearson correlation analysis was used to assess the relationships between variables for all available subjects (n = 39). Results are presented as means ± standard errors of the mean (SEM). A P value of ≤0.05 was considered statistically were performed by using The metabolic of the study subjects are shown in was a in insulin sensitivity, measured as the percent increase in muscle glucose uptake during the hyperinsulinemic-euglycemic clamp procedure, from to Plasma palmitoleate and palmitoleate in VLDL from to and from to respectively. study skeletal muscle insulin-mediated glucose uptake was greater in the IS than in the IR values for the and insulin-mediated of palmitate measure of adipose tissue insulin were also significantly greater in the IS that in the IR However, free palmitoleate and palmitoleate in VLDL, measured as either a proportion of total fatty acids or as absolute concentrations, were not significantly different between the groups. was a lower plasma palmitoleate availability as either free palmitoleate or within VLDL in the IS We a correlation between palmitoleate availability as either free palmitoleate or palmitoleate within VLDL and skeletal muscle insulin was correlated with plasma = P = and adipose tissue = P = palmitoleate of the study resistant sensitive of subjects ± 2 ± ± mass index ± 0.7 ± ± mass ± 1 ± ± fat ± ± ± increase in glucose ± ± ± of FFA ± 1 ± ± Insulin × ± 0.02 ± ± FFA ± 0.04 ± ± of plasma FFA as palmitoleate ± ± ± palmitoleate ± ± ± VLDL-TG ± ± ± of as palmitoleate ± ± ± VLDL-TG palmitoleate ± ± ± tissue and skeletal muscle insulin sensitivity levels were calculated as the relative decrease in FFA rate of appearance during stage 1 and the relative increase in glucose rate of disappearance during stage 2 of the clamp procedure, respectively. The hepatic insulin sensitivity index (HISI) was calculated as the of the hepatic insulin resistance index × insulin are presented as mean ± P and P values significantly different than the insulin resistant group in a Adipose tissue and skeletal muscle insulin sensitivity levels were calculated as the relative decrease in FFA rate of appearance during stage 1 and the relative increase in glucose rate of disappearance during stage 2 of the clamp procedure, respectively. The hepatic insulin sensitivity index (HISI) was calculated as the of the hepatic insulin resistance index × insulin are presented as mean ± P and P values significantly different than the insulin resistant group Palmitoleate in adipose tissue TG from to of total fatty acids in Plasma-free palmitoleate correlated directly with adipose tissue palmitoleate content = P = and with adipose tissue SCD1 gene expression = P = Adipose tissue palmitoleate content correlated directly with SCD1 gene expression = P = and VLDL palmitoleate concentration correlated directly with hepatic SCD1 activity, estimated by using the ratio in VLDL = P = palmitoleate correlated directly with total plasma FFA concentration = P = The from the present study demonstrate is that decreased palmitoleate availability is an important in the of liver, skeletal muscle, and adipose tissue insulin resistance in obese We found that free palmitoleate and palmitoleate in VLDL, which are the of circulating palmitoleate available for are not different in obese IS and obese IR subjects, by age, and percent body was a between circulating palmitoleate and skeletal muscle insulin sensitivity, because plasma palmitoleate with total plasma FFA concentration, which is associated with insulin from studies conducted in isolated and in a model have found palmitoleate insulin signaling and increases insulin-mediated skeletal muscle glucose uptake (2Cao H. Gerhold K. Mayers J.R. Wiest M.M. Watkins S.M. Hotamisligil G.S. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.Cell. 2008; 134: 933-944Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, N. M. K. of palmitate and palmitoleate on insulin action and glucose in skeletal muscle J. PubMed Scopus Google Scholar). These data to the that palmitoleate provides a between adipose tissue and that is in regulating systemic metabolic We found that adipose tissue palmitoleate content correlated directly with SCD1 gene and that plasma free palmitoleate was directly correlated with adipose tissue palmitoleate These are with the that plasma free palmitoleate is derived from adipose tissue fatty acid Palmitoleate can also be to peripheral tissues by lipoprotein of circulating which is produced by the liver. of palmitoleate is greater than free palmitoleate in the relative of palmitoleate in VLDL to be in the IS subjects, the absolute concentration of VLDL palmitoleate to be in the IR subjects because total VLDL-TG concentration was adipose tissue and liver are important of systemic palmitoleate availability. studies have evaluated the between plasma palmitoleate and insulin sensitivity in the from of these studies that palmitoleate is not associated with insulin sensitivity, as the percent plasma FFA as palmitoleate was greater in obese than in and was correlated with of T. Furuhashi N. Kuromori Y. Miyashita M. Iwata F. Harada K. Plasma palmitoleic acid content and obesity in children.Am. J. Clin. Nutr. 2005; 82: 747-750Crossref PubMed Scopus (123) Google serum palmitoleate composition in phospholipids was directly correlated with IR by the homeostasis model assessment of insulin in subjects with type 2 diabetes T. Kazdova L. Chvojkova S. Base J. Serum phospholipid fatty acid composition and insulin action in type 2 diabetic patients.Metabolism. 2001; 50: 1472-1478Abstract Full Text PDF PubMed Scopus (67) Google and palmitoleate in plasma was associated with peripheral insulin sensitivity in B. Tengblad S. Lithell H. Insulin sensitivity is related to the fatty acid composition of serum lipids and skeletal muscle phospholipids in 70-year-old men.Diabetologia. 1994; 37: 1044-1050Crossref PubMed Scopus (294) Google Scholar). data from a study conducted in overweight subjects that the percentage of plasma FFA as palmitoleate was directly associated with insulin sensitivity, measured as the glucose infusion rate to during a clamp procedure (3Stefan N. Kantartzis K. Celebi N. Staiger H. Machann J. Schick F. Cegan A. Elcnerova M. Schleicher E. Fritsche A. Circulating palmitoleate strongly and independently predicts insulin sensitivity in humans.Diabetes Care. 2010; 33: 405-407Crossref PubMed Scopus (111) Google Scholar). However, all these studies measured the percent palmitoleate in FFA or other lipids did not absolute plasma palmitoleate palmitoleate as a circulating the absolute plasma concentration is important than relative composition in regulating metabolic study, we measured the percent contribution and absolute concentration of palmitoleate within plasma FFA and VLDL, which provides a robust assessment of total palmitoleate availability than We evaluated whether decreased availability of the circulating of present in plasma FFA and in VLDL, was associated with insulin Accordingly, we selected groups of obese subjects, from a of subjects who were either IR or IS based on the increase in insulin-mediated muscle glucose uptake determined by using the hyperinsulinemic-euglycemic clamp procedure in conjunction with tracer these subjects were also found to be either insulin resistant or sensitive in the liver of insulin to glucose and adipose tissue of insulin to Subjects with type 2 diabetes were to the of diabetes on insulin An important of study is that we measured average palmitoleate content obtained from blood samples over a than a basal sample as measured in study was to the to an of palmitoleate by availability from free palmitoleate in plasma and palmitoleate in VLDL, we did not a free palmitoleate in plasma or in VLDL in the IS group compared with the IR However, study not the that plasma palmitoleate availability may be related to insulin sensitivity in subjects or that palmitoleate availability within muscle and liver tissue be different in IS and IR An of study is the of men within study which from differences in the between palmitoleate and glucose data not the that palmitoleate is an important of insulin sensitivity in humans and demonstrate that insulin resistance in obese people is not associated with a decrease in circulating these not the that palmitoleate can insulin sensitivity in as in (2Cao H. Gerhold K. Mayers J.R. Wiest M.M. Watkins S.M. Hotamisligil G.S. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.Cell. 2008; 134: 933-944Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). The and Pietka for the of the Clinical Research Unit for in the studies, and the study subjects for their body mass index body surface area threshold hepatic insulin sensitivity index insulin resistant insulin sensitive rate of appearance rate of disappearance triglyceride