Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects

Abstract

Increased adipose tissue lipogenesis is associated with enhanced insulin sensitivity. Mice overexpressing the Glut4 glucose transporter in adipocytes have elevated lipogenesis and increased glucose tolerance despite being obese with elevated circulating fatty acids. Lipidomic analysis of adipose tissue revealed the existence of branched fatty acid esters of hydroxy fatty acids (FAHFAs) that were elevated 16- to 18-fold in these mice. FAHFA isomers differ by the branched ester position on the hydroxy fatty acid (e.g., palmitic-acid-9-hydroxy-stearic-acid, 9-PAHSA). PAHSAs are synthesized in vivo and regulated by fasting and high-fat feeding. PAHSA levels correlate highly with insulin sensitivity and are reduced in adipose tissue and serum of insulin-resistant humans. PAHSA administration in mice lowers ambient glycemia and improves glucose tolerance while stimulating GLP-1 and insulin secretion. PAHSAs also reduce adipose tissue inflammation. In adipocytes, PAHSAs signal through GPR120 to enhance insulin-stimulated glucose uptake. Thus, FAHFAs are endogenous lipids with the potential to treat type 2 diabetes.

Figure 1. Discovery and characterization of a class of lipids (FAHFAs)

A) Comparative lipidomics of SQ white adipose tissue (WAT) from AG4OX and WT mice reveals the presence of a group of ions at m/z 509 (PAHPA), 535 (POHSA/OAHPA), 563 (OAHSA) and 537 (PAHSA) that are elevated 16–18-fold in AG4OX mice. B) Structural analysis of the 537 ion from AG4OX WAT by tandem MS demonstrates that it is composed of palmitic acid (m/z 255) and hydroxy stearic acid (m/z 299). Octadecanoic acid (m/z 281) results from the dehydration of hydroxy stearic acid. Fragmentation at high collision energies produces two ions at m/z 127 and 155, identifying carbon 9 as the position of the hydroxyl group on hydroxy-stearic acid, confirming the structure to be 9-PAHSA. C) Acyl chain carbon numbering scheme, molecular formula, mass and names of FAHFAs from the m/z 537 (PAHSA), m/z 509 (PAHPA), m/z 563 (OAHSA) m/z 535 (POHSA or OAHPA) ions. D) Constituent fatty acid and hydroxy-fatty acid components of FAHFAs. Quantification of 16 FAHFA family members identified in serum of WT and AG4OX mice. E) Total PAHSA levels in serum and tissues of WT and AG4OX mice. Inset, liver total PAHSA levels. n=3–5/group, *p<0.05 versus WT (t-test), ^†p<0.05 versus all other tissues within the same genotype (ANOVA). F) Total PAHSA levels in SQ-WAT, PG-WAT and serum of WT, AG4OX, ChREBP KO and AG4OX/ChREBP KO mice. n=3–5/group, *p<0.05 versus all other genotypes within same tissue or serum (ANOVA), # p<0.05 versus AG4OX and ChREBP-KO. Data are means±sem.

Figure 2. Identification and quantification of PAHSA isomers in mouse serum and tissues

A) Co-elution of PAHSA isomers from serum and SQ WAT of WT and AG4OX mice with synthetic standards for individual PAHSA isomers. The peak for 5-PAHSA is shown in red in the inset. B) Distribution and quantification of PAHSA isomers in serum and tissues of WT and AG4OX mice. ‘Ester position’ refers to the location of the ester bond in PAHSA isomers. n=3–5/group, *p<0.05 versus WT (t-test). C) Distribution and quantification of 13/12-, 9- and 5-PAHSA isomers in serum and tissues of WT female FVB mice. n=3–5/group, ^a,b,c Tissues with different letters are different from each other within the same isomer panel (p<0.05, ANOVA). D) Total PAHSA levels and E) PAHSA isomer levels in serum and tissues of WT mice in fed or fasted (16 h) states. *p<0.05 versus fed (t-test). ‘Ester position’ refers to the location of the ester bond in PAHSA isomers. n=3–5/group, *p<0.05, ^#<0.07 versus fed (t-test) ^a,b,c,dTissues with different letters are different from each other for the fed state (p<0.05, ANOVA). Data are means±sem. See also figure S1.

Figure 3. PAHSA isomer levels in tissues and food of mice on chow or HFD and biosynthetic activity of PAHSAs in liver, WAT and serum

A) Quantification of PAHSA isomers in serum, SQ WAT, PG WAT, BAT and liver of WT female FVB mice fed on chow or HFD for 9 weeks. ‘Ester position’ refers to the location of the ester bond in PAHSA isomers. n=3–6/group, *p<0.05 versus chow (t-test). B) Quantification of PAHSA isomers in mouse chow and HFD. n=3/group. C) 9-PAHSA levels in liver and PG-WAT lysates incubated with palmitoyl-CoA and 9-hydroxy stearic acid and heat-denatured Controls. n=3/group, *p<0.05 versus heat-denatured Controls (t-test). D) 9-palmitic-acid-hydroxy-heptadecanoic-acid (9-PAHHA) serum levels in mice 3h post gavage with 9-hydroxy-heptadecanoic-acid (9-HHA) or vehicle control. n=3/group, *p<0.05 versus vehicle (t-test). Data are means±sem. See also figure S2.

Figure 4. PAHSA levels are decreased in insulin-resistant humans and levels correlate with insulin sensitivity

A) Quantification of total PAHSAs and individual PAHSA isomers in serum of insulin-sensitive and insulin-resistant nondiabetic humans (see table S2 for metabolic characteristics). n=6–7/group. B) Correlation between insulin-sensitivity (clamp glucose infusion rate) and serum total PAHSA and individual PAHSA isomers. n=13. C) Quantification of total PAHSA and individual PAHSA isomers in SQ WAT of insulin-sensitive and insulin-resistant humans. n=6–7/group. D) Correlation between insulin-sensitivity (clamp glucose infusion rate) and SQ WAT total PAHSA and individual PAHSA isomers. n=13. E) Correlation between SQ WAT and serum 5-PAHSA levels. LBM: lean body mass. Individual p-values are shown on graphs, *p<0.05 versus insulin-sensitive (t-test, panels A and C). Data are means±sem for panels A and C. Correlations were determined by linear regression analysis for Panels B, D and E.

Figure 5. PAHSAs improve glucose tolerance and ambient glycemia in vivo and augment insulin and GLP-1 secretion

A) 4.5 hours after food removal, HFD-fed mice were gavaged with 5-PAHSA (upper panel), 9-PAHSA (lower panel) or vehicle control. 30 min later an oral glucose tolerance test (OGTT) was performed. n=12–14/group, mice were on HFD for 42–52 weeks. *p<0.05 versus vehicle at same time (t-test). Area under the curve (AUC) was calculated from-30 to 120 min. *p<0.05 versus vehicle (t-test). B) 2.5 hours after food removal, HFD-fed mice were gavaged with 5-PAHSA (upper panel), 9-PAHSA (lower panel) or vehicle control. Glycemia was measured immediately before (time 0) and at 2.5 hours (5-PAHSA) or 3 hours (9-PAHSA) after PAHSA gavage. n=12–14/group. *p<0.05 versus vehicle (t-test). C) 4.5 hours after food removal, aged, chow-fed mice (45-weeks old) were gavaged with 5-PAHSA 30 min prior to an OGTT. n=12–14/group. *p<0.05 versus vehicle at same time (t-test). Area under the curve (AUC) was calculated from−30 to 120 min. *p<0.05 versus vehicle (t-test). D) Serum insulin levels and E) serum GLP-1 levels 5 min post glucose challenge in chow-fed mice gavaged with 5-PAHSA or vehicle (glucose values shown in panel C). n=12–14/group, *p<0.05 versus vehicle (t-test). F) Insulin secretion from primary human islets from two independent donors. Islets were incubated with low (2.5 mM) or high (20 mM) glucose ex vivo in the presence of 5-PAHSA (20 μM) or Control (KRB buffer). Diluent for 5-PAHSA was methanol (0.25%) in panel F. n=100 islets/condition, *p<0.05 versus control 2.5 mM glucose (t-test), ^#p<0.05 versus all treatments at 2.5 mM glucose (t-test), ^$p<0.05 versus control and diluent at 20 mM glucose (t-test). G) Active GLP-1 secretion from STC-1 cells in response to 5-PAHSA (5-P), 9-PAHSA (9P), α-Linolenic Acid (ALA), GW9508 (GW) or vehicle control (CTL, DMSO). n=4/group, *p<0.05 versus vehicle (CTL) (t-test). Data are means±sem. See also table S3 and figure S3.

Figure 6. PAHSAs regulate glucose uptake and Glut4 translocation via GPR120

A) Insulin–stimulated glucose transport in 3T3-L1 adipocytes treated with 9-PAHSA (20μM) or vehicle (DMSO) control for 6 days. n=6/group, *p<0.001 versus vehicle (DMSO) at the same insulin concentration (ANOVA). B) Glucose transport in 3T3-L1 adipocytes treated for 48h with 9-PAHSA, 5-PAHSA, palmitic acid (PA), 9-hydroxy stearic acid (HSA) at 20μM or their respective vehicle controls (DMSO for 9- and 5-PAHSA. Ethanol for PA and HSA). n=6/group. ^a,b,cgroups with different letters are different from each other p<0.05 (ANOVA). C) Dose response of 9-PAHSA on GPR120 binding and receptor activation. n=3 wells/condition. D) Insulin (10nM)–stimulated glucose transport in 3T3-L1 adipocytes transfected with control siRNA (CTL) or GPR120 siRNA and treated with 5-PAHSA (10μM), 9-PAHSA (10μM) or vehicle (DMSO) control for 2 days. n=3/group, *p<0.05 versus control siRNA or GPR120 siRNA with DMSO without insulin (ANOVA), ^$p<0.05 versus all other conditions except each other (ANOVA). E) Glut4 plasma membrane translocation in 3T3-L1 adipocytes transfected with control siRNA or GPR120 siRNA and treated with 9-PAHSA in the presence or absence of insulin. Scale bar=50 micrometers. F) Quantification of Glut4 translocation in panel E. Bars show means of 6 independent experiments without siRNA knockdown and 3 with siRNA knockdown. Each experiment had an n>50 cells/condition.*p<0.05 versus everything else at same insulin concentration (ANOVA). All data are means±sem. See also figure S4.

Figure 7. 9-PAHSA inhibits LPS-induced dendritic cell maturation in vitro and pro-inflammatory cytokine production from adipose tissue macrophages in vivo

A) LPS induces dendritic cell (DC) maturation (increased percentage of CD11c⁺ cells expressing co-stimulatory molecules, CD80, CD86, CD40, and MHCII). This LPS effect is reduced in the presence of 9-PAHSA (40 μM) compared to vehicle (DMSO) control. Quantification of CD11c⁺ cells which are positive for co-stimulatory molecules from the panel above. n=3 mice/group. B) LPS–induced DC maturation is inhibited by increasing concentrations of 9-PAHSA. Red triangles represent vehicle for 9-PAHSA (DMSO) without LPS. MFI: median fluorescence intensity. n=3 mice/group, C) LPS–induced cytokine secretion from DC’s is inhibited by increasing concentrations of 9-PAHSA compared to vehicle for 9-PAHSA (DMSO, ‘-’) control. Red triangles represent vehicle for 9-PAHSA (DMSO, ‘-’) without LPS. n=3 mice/group. D) Flow cytometry representation of AT macrophages expressing TNFα and IL-1β. Mice fed on HFD or chow mice were gavaged for 3 days with 9-PAHSA (30mg/kg for chow mice and 45mg/kg for HFD mice) or vehicle control. PG-WAT was harvested on day 4 and the stromal vascular cells were incubated in vitro with PMA, ionomycin and brefeldin for 5 hours. AT macrophages (CD45⁺CD11b⁺F4/80⁺) were stained intracellularly for TNFα and IL-1β. E) Quantification of panel D percentage of AT macrophages expressing TNFα, IL-1β or both. n=5 mice/group. LPS concentration is 100ng/ml for all panels. *p<0.05 versus LPS-activated cells without PAHSA treatment (A-C) or control cells, same diet (E) by one-way (A-C) and two-way ANOVA (D). ^#p<0.05 versus all other groups by two-way ANOVA. Data are means±sem. See also figure S5.