Liver Perilipin 5 Expression Worsens Hepatosteatosis But Not Insulin Resistance in High Fat-Fed Mice

Abstract

Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in oxidative tissues, including the fasted liver. However, its expression also increases in nonalcoholic fatty liver. To determine whether PLIN5 regulates metabolic phenotypes of hepatosteatosis under nutritional excess, liver targeted overexpression of PLIN5 was achieved using adenoviral vector (Ad-PLIN5) in male C57BL/6J mice fed high-fat diet. Mice treated with adenovirus expressing green fluorescent protein (GFP) (Ad-GFP) served as control. Ad-PLIN5 livers increased LD in the liver section, and liquid chromatography with tandem mass spectrometry revealed increases in lipid classes associated with LD, including triacylglycerol, cholesterol ester, and phospholipid classes, compared with Ad-GFP liver. Lipids commonly associated with hepatic lipotoxicity, diacylglycerol, and ceramides, were also increased in Ad-PLIN5 liver. The expression of genes in lipid metabolism regulated by peroxisome proliferator-activated receptor-α was reduced suggestive of slower mobilization of stored lipids in Ad-PLIN5 mice. However, the increase of hepatosteatosis by PLIN5 overexpression did not worsen glucose homeostasis. Rather, serum insulin levels were decreased, indicating better insulin sensitivity in Ad-PLIN5 mice. Moreover, genes associated with liver injury were unaltered in Ad-PLIN5 steatotic liver compared with Ad-GFP control. Phosphorylation of protein kinase B was increased in Ad-PLIN5-transduced AML12 hepatocyte despite of the promotion of fatty acid incorporation to triacylglycerol as well. Collectively, our data indicates that the increase in liver PLIN5 during hepatosteatosis drives further lipid accumulation but does not adversely affect hepatic health or insulin sensitivity.

Figure 1.

Adenovirus-mediated expression of PLIN5 in liver of mice on HFD. A, Quantitative PCR of Plin1-Plin5 levels in livers of Ad-GFP- and Ad-PLIN5-treated mice on 6 week of HFD presented taking the average levels from Ad-GFP mice as 100% and normalized using β-actin as an internal control. ND, not detected. Data are mean ± SEM; n = 5–6 per group; *, P < .05. B and C, Western blotting of PLIN5 (B) and PLIN2 (C) in Ad-GFP and Ad-PLIN5 livers using GAPDH as an internal control. Representative pictures on the left and band density of PLIN5 (B) and PLIN2 (C) taking the average expression levels from Ad-GFP-treated mice as 1 on the right. Data are mean ± SEM; n = 5–6 per group; **, P < .01.

Figure 2.

Change in lipids and LD in HFD mice treated with Ad-PLIN5. A, Hepatic TG quantitated in livers of Ad-GFP- and Ad-PLIN5-treated mice after 6 week of HFD. Data are mean ± SEM; n = 5–6 per group; *, P < .05. B, Liver sections from Ad-GFP (top row)- and Ad-PLIN5 (bottom row)-treated mice immunostained with PLIN2 and immunostained with PLIN5 along with negative control (no primary antibodies). Scale bar, 10 μm. C, Quantitative PCR determined the expression of genes involved in major lipid metabolic pathways in livers of Ad-PLIN5 male mice on HFD for 6 weeks. Levels are presented taking the levels of Ad-GFP on the corresponding duration of HFD as 100%. Expression levels were normalized using β-actin as an internal control. Data are mean ± SEM; n = 5–6; *, P < .05; **, P < .01. Pnpla2, ATGL; Lipe, hormone-sensitive lipase (aka HSL); Dgat1 and Dgat2, DG acyltransferases 1 and 2; Cpt1a, carnitine palmitoyltransferase 1α; Fasn, FA synthase; Scd1, stearoyl-CoA desaturase-1; Acot2, acyl-CoA thioesterase 2; Ppargc1a, PPAR-γ coactivator 1α.

Figure 3.

Changes in the hepatic lipidome of HFD mice treated with Ad-PLIN5. Lipids extracted from the livers of 6 weeks HFD mice treated with Ad-GFP or Ad-PLIN5 and harvested at fed ad libitum in late morning as in Materials and Methods were analyzed by Q-Exactive LC-MS/MS as in Materials and Methods. A, The representative total ion chromatogram of lipids in Ad-GFP liver in full MS. B–D, Relative intensity of neutral lipids (B), phospholipids (C), and Cers (D) determined as in Materials and Methods was compared between liver from Ad-GFP and Ad-PLIN5 mice. PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol. Data are mean ± SEM; n = 3; *, P < .05; **, P < .01; n.s., not significant.

Figure 4.

Profiles of the TG lipid class in the livers of HFD mice overexpressing PLIN5. A, Relative abundance (peak area) of the 100 most abundant TG species identified in the liver of Ad-GFP (white)- and Ad-PLIN5 (black)-treated mice (6-week HFD). TGs are grouped based on their total number of carbons (c) and unsaturation (number of double bonds). Data are mean ± SEM; n = 3; *, P < .05. B, Correlation between the unsaturation of C14 to C18 fatty acyl chains expressed as the number of double bonds/total carbon and abundance of fatty acyl chains expressed as an area ratio in Ad-PLIN5 over Ad-GFP liver of the TG species as appeared in A. C, Quantitative PCR determined the expression of genes involved in liver injury in livers of Ad-PLIN5 male mice fed HFD (6-week HFD). Levels are presented taking the levels of Ad-GFP on the corresponding duration of HFD as 100%. Expression levels were normalized using β-actin as an internal control. Data are mean ± SEM; n = 5–6; *, P < .05; **, P < .01. Afp, α-fetoprotein (AFP); Col1a2, collagen α1(II chain); H19, imprinted maternally expressed transcript (nonprotein coding).

Figure 5.

Glucose homeostasis of short- and long-term HFD mice treated with Ad-PLIN5. A–D, Mice were placed on a 6- or 16-week HFD, then treated with Ad-GFP (closed line) and Ad-PLIN5 (dashed line). Intraperitoneal glucose tolerance (A and C) and insulin tolerance (B and D) were evaluated in mice on the separate HFD time plans. Area under the curve (AUC) between time 0 and 60 minutes. E–G, Body weight (E), glucose in late morning fed ad libitum (F), and serum insulin (G) measured in Ad-GFP- and Ad-PLIN5-treated male C56BL/6J mice on NC, and 6- or 16-week HFD taken at harvest. Data are mean ± SEM; n = 3–6; *, P < .05; **, P < .01. H, Overnight incorporation of [³H]OAs into triglycerides (TG) in AML12 cells transduced with Ad-PLIN5 (PLIN5) and nontransduced cells (Cont). Data are mean ± SEM; n = 4; ***, P < .001. I, Representative Western blotting of phosphorylated protein kinase B (pAkt S473) and total protein kinase B (totAkt) in AML12 cells transduced with Ad-PLIN5 and Ad-GFP. Band density expressed taking the average expression levels of from Ad-GFP as 1 relative to totAkt.