Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism

Abstract

Dysregulation of lipid metabolism in individual tissues leads to systemic disruption of insulin action and glucose metabolism. Utilizing quantitative lipidomic analyses and mice deficient in adipose tissue lipid chaperones aP2 and mal1, we explored how metabolic alterations in adipose tissue are linked to whole-body metabolism through lipid signals. A robust increase in de novo lipogenesis rendered the adipose tissue of these mice resistant to the deleterious effects of dietary lipid exposure. Systemic lipid profiling also led to identification of C16:1n7-palmitoleate as an adipose tissue-derived lipid hormone that strongly stimulates muscle insulin action and suppresses hepatosteatosis. Our data reveal a lipid-mediated endocrine network and demonstrate that adipose tissue uses lipokines such as C16:1n7-palmitoleate to communicate with distant organs and regulate systemic metabolic homeostasis.

Figure 1. Whole body lipid profiling

A. Dendrograms of hierarchical clustering of subjects using significant metabolites by one-way ANOVA. R: regular diet, HF: high fat diet. B. Scatter plots of principal components from principal component analysis on subjects. Only fatty acids significant (p-value<0.05) from a one-way ANOVA were included. C. Strength of differences between diets in the WT and FABP^−/− (KO) mice over all fatty acids in all lipid classes. Each point on a line indicates the number of p values of one group that are smaller than the p value of equal ranking of the other group.

Figure 2. Adipose de novo lipogenesis and palmitoleate production

A. Difference of major fatty acids in diacylglycerol, free fatty acid, phospholipid and triglyceride fractions in adipose tissue between the mean of WT and FABP^−/− mice. B. Lipid class composition analysis for FAs in adipose tissue. The top 36 metabolites are shown. The F-statistics from a one-way ANOVA are displayed as red diamonds over the distribution of F-statistics from permuted data. The black line is the maximum F statistic observed in 100 permutations. The heat map displays the observed data, centered to the mean of the control group and scaled by the standard deviation of all observations. C. Plasma FAs in WT or FABP^−/− (KO) mice. D. Percentile suppression of palmitoleate by HFD in adipose tissue of WT or KO mice. Error bars represent the SEM.

Figure 3. Metabolic regulation by plasma lipids

A. SCD-1 promoter activities in control hepatocytes (No lipid) or hepatocytes treated with plasma lipids extracted from WT or FABP^−/− (KO) mice. B. SCD-1 promoter activities in hepatocytes treated with fatty acid mixtures resembling the ratio of plasma fatty acids from WT mice (WT Mix), KO mice (KO Mix), KO mice with the concentration of palmitoleate of WT mice (KO Mix, 16:1n7), or KO mice with the concentration of palmitoleate, palmitate, and stearate of WT mice (KO Mix, 16:1n7, + 16:0 18:0). Asterisk, significantly different from WT mix; a and b, significantly different from KO Mix. C. Insulin-stimulated AKT phosphorylation in C2C12 myotubes treated with plasma lipids extracted from WT or KO mice. Values of bar plot were determined by phospho-AKT ELISA and corresponding immunoblotting results are shown as insets. D. MCP1 in conditional medium of adipose explants treated with plasma lipids of WT or KO mice. E. SCD-1 promoter activities in hepatocytes treated with fatty acids. All fatty acids were used at 300 µM final concentration except C16:1n7 2x is 600 µM. AA: arachidonic acid; asterisk: significantly different from controls; a: significantly different from AA-treated cells; b: significantly different from palmitate-treated cells. F. Immunobloting of Flag-tagged SCD-1 in hepatocytes treated with control, palmitate and palmitoleate. Tubulin was used as loading control. G. AKT phosphorylation in C2C12 myotubes treated with fatty acids and insulin. a: significantly different from cells treated with insulin; b: significantly different from cells treated with both insulin and palmitate. The bottom panel is the corresponding immunoblotting of total and phosphorylated AKT in cells treated with pooled lipids. H. Glucose uptake in C2C12 myotubes treated with insulin or palmitoleate. Top panel is immunoblotting of C2C12 cell lysates treated with insulin or palmitoleate using anti-Glut1 or Glut4 antibodies. Asterisk, p < 0.05. Error bars represent the SEM.

Figure 4. Systemic palmitoleate metabolism

A. Free fatty acid and triglyceride flux among ad- ipose, muscle, and liver tissues. Means and SEM of palmitoleate in free fatty acids (FFA) and triglyc- eride (TG) in plasma and adipose, muscle, and liver tissues of WT and FABP. B. Means and SEM of palmitoleate in triglyceride (TG) of WT and FABP^−/− mice. C. Model of free fatty acid and triglyceride flux among adipose, muscle and liver tissues. Muscle fatty acids, are mainly derived from liver in the form of VLDL-associated TG and from adipose tissue in the form of FFAs. DNL, de novo lipogenesis; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase. D. Percentile suppression of palmitoleate by HFD in TG fraction of muscle tissue in WT or DK mice. E. Total palmitoleate in liver of WT or DK mice. F. Triglycerides in liver of DK mice injected with control or SCD-1 adenoviruses. Asterisk, p < 0.05.

Figure 5. Lipid-mediated regulation of SCD-1 promoter activity in vivo

A. Schematics of SCD-1 -1500bp promoter. PUF, polyunsaturated fatty acid response element. B. Regulation of WT and mutant SCD-1 promoter activities by fatty acids. AA: arachidonic acid. C. Regulation of wild-type and mutant SCD-1 promoter activities by plasma lipids. Plasma lipids extracted from WT or FABP^−/− (KO) mice were used to treat FAO cells Infected with WT or mutant SCD-1 promoter-carrying adenoviruses and luciferase assays performed as described in Experimental Procedures. D. In vivo regulation of WT and mutant SCD-1 promoter activities. Luciferase assays were performed on liver tissues of WT or KO mice injected with WT or mutant SCD-1 reporter-carrying adenoviruses. E. Liver gene expression in mice infused with vehicle, TG:palmitate or TG:palmitoleate. Asterisk, p < 0.05.

Fig 6. Regulation of lipogenic genes in adipose and liver of WT and FABP^−/− mice

A. Lipogenic gene expression in adipose tissue of WT and FABP^−/− (KO) mice. Gene expression patterns in epididymal fat pad were determined by quantitative real-time PCR. B. Regulation of lipogenic gene promoter activites by FABPs in differentiated adipocytes. Differentiated FABP^−/− adiopocytes that expressed SCD-1 or FAS promoter luciferase constructs were infected with GFP, aP2 or both aP2 and mal1 adenoviruses and luciferase activities were determined by dual-glow luciferase system. a: significantly different from GFP-adenovirus-infected cells; b: significantly different from aP2-adenovirus-infected cells. C. Regulation of lipogenic gene expression in adipose tissue of mice treated with aP2 inhibitor. Gene expressions in epididymal fat pads from mice treated with vehicle or aP2 inhibitor were determined with quantitative real-time PCR. D. Differential regulation of lipogenic gene expression in adipose and liver tissues. Levels of the mRNAs in liver and epididymal fat pad from mice fed with HFD were determined and WT liver and KO adipose were set as 1. ACC1, acetyl-CoA carboxylase1; DGAT1, acyl-CoA:diacylglycerol acyltransferase 1. Asterisk, p < 0.05.

Fig 7. Regulation of insulin signaling and glucose metabolism by palmitoleate

A. Basal and insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and -2 (IRS-2) and AKT in liver of mice infused with vehicle (Veh), TG:palmitoleate (C16:1n7) or TG:palmitate (C16). Representative blots are shown and quantifications on the right are averaged results of three mice in each treatment group. B. Basal and insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), AKT and GSK in muscle tissues of mice infused with vehicle (Veh), TG:palmitoleate (C16:1n7) or TG:palmitate (C16). Representative blots are shown and quantifications on the right are averaged results of three mice in each treatment group. C. Basal hepatic glucose production (bHGP) of mice infused with vehicle (Veh) or palmitoleate (C16:1n7). D. Glucose infusing rate (GIR) of mice infused with vehicle (Veh) or palmitoleate (C16:1n7) during hyperinsulinemic-euglycemic clamp. E. Hepatic glucose production (Clamp HGP) of mice infused with vehicle (Veh) or palmitoleate (C16:1n7) during hyperinsulinemic-euglycemic clamp. F. Glucose disposal rate (RD) mice infused with vehicle (Veh) or palmitoleate (C16:1n7) during hyperinsulinemic-euglycemic clamp. G. Regulation of systemic metabolic responses by adipose-derived lipid hormones (Lipokines). In parallel with a variety of adipokines, specific lipids released from adipocytes in response to physiological stimuli act at remote sites including liver and muscle and regulate systemic lipid and carbohydrate metabolism. Lipid chaperones negatively regulate one of these lipokines, C16:1n7 (palmitoleate), and in the absence of these FABPs, strong flux of palmitoleate from adipose tissue to liver and muscle results in improved metabolic responses.