Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus

Abstract

We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured by mass spectrometry (lipidomics). In vitro studies were undertaken in 3T3-L1 adipocytes. rHDL infusion inhibited fasting-induced lipolysis (P = 0.03), fatty acid oxidation (P < 0.01), and circulating glycerol (P = 0.04). In vitro, HDL inhibited adipocyte lipolysis in part via activation of AMPK, providing a possible mechanistic link for the apparent reductions in lipolysis observed in vivo. In contrast, circulating NEFA increased after rHDL infusion (P < 0.01). Lipidomic analyses implicated phospholipase hydrolysis of rHDL-associated phosphatidylcholine as the cause, rather than lipolysis of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing circulating NEFA.

Figures

Fig. 1.

Fatty acid (palmitate) tracer kinetics during rHDL and placebo infusion. A: Percent change (compared with baseline, 0 hr; actual baseline values: rHDL Ra, 2.87 ± 0.17 μM; Placebo Ra, 2.66 ± 0.12 μM; rHDL Rd, 2.96 ± 0.21 μM; Placebo Rd, 2.67 ± 0.15 μM) in rates of plasma palmitate tracer appearance (Ra, circles) and disappearance (Rd, triangles) during 4 hr rHDL (solid lines) and placebo (dotted lines) infusion. B: Rates of palmitate tracer oxidation (μmol/min) during 4 hr rHDL (solid lines) and placebo (dotted lines) infusion. *P < 0.05 between groups as measured by repeated measures-ANOVA, n = 13. rHDL, reconstituted HDL.

Fig. 2.

Measures of basic lipolysis markers in plasma of patients during rHDL and placebo infusion. Percent change (compared with baseline, 0 hr) in levels of plasma (A) glycerol (actual baseline values; rHDL, 46 ± 11 μM; Placebo, 44 ± 9 μM); (B) NEFA (actual baseline values; rHDL, 388 ± 76 μM; Placebo, 384 ± 39 μM); and (C) TG (actual baseline values; rHDL, 1.86 ± 0.36 mM; Placebo, 1.56 ± 0.23 mM) during 4 hr rHDL (solid lines) and placebo (dotted lines) infusion. *P < 0.05 between groups as measured by repeated measures-ANOVA, n = 13. rHDL, reconstituted HDL; TG, triglyceride.

Fig. 3.

In vitro AMPK signaling and lipolysis after HDL treatment in 3T3-L1 adipocytes. Differentiated and lipid-loaded 3T3-L1 adipocytes were treated for 4 hrs with AICAR (2 mM), PF (1 mM), ISO (10 μM), and HDL (50 μg/ml) before cells and media were harvested. Cell lysates were analyzed for (A) AMPK phosphorylation and protein level and (B) its downstream target ACC phosphorylation and β-actin protein expression. Media was analyzed for the amount of (C) glycerol and (D) NEFA released by adipocytes into the media, expressed as fold change from control (amount of release under basal/control conditions; glycerol, 223 ± 32 μM and NEFA 310 ± 44 μM). *P < 0.05 from control as measured by one-way ANOVA, n = 5. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, AMP-activated protein kinase; ISO, isoproteronol; PF, phenformin.

Fig. 4.

Plasma lipidomics analysis for total amounts of selected plasma lipids pre- and post-rHDL and placebo infusions. Total amounts of plasma (A) DAG, (B) ceramides, (C) cholesterol, (D) cholesterol ester, (E) PC, (F) LPC, and (G) total NEFA in patients before and after infusion of rHDL (black bars) and placebo (white bars). #P < 0.05 between placebo and rHDL post measurements, *P < 0.05 from premeasurement within each infusion as measured by one-way ANOVA, n = 13. DAG, diacylglycerol; rHDL, reconstituted HDL; LPC, lysophosphatidylcholine; PC, phosphatidylcholine.