Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Abstract

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Keywords: diabetes mellitus; dyslipidemia; glucose.

Fig. 1.

Effect of hepatic GCGR knockdown on hepatic GCGR mRNA, body weight, and plasma glucose, glucagon and insulin levels in db/db mice. Mice were injected with GCGR siRNA and euthanized 11 days after injection. A: Hepatic Gcgr mRNA expression by real-time quantitative PCR in si-Control and si-GCGR. B: Body weight at 1, 2, 3, 4, 8, and 11 days after siRNA injection in si-Control and si-GCGR mice. Ambient glucose (C), fasting glucose (D), glucagon (E), and insulin levels (F) measured in si-Control and si-GCGR mice plasma samples. Values are expressed as means ± SEM (n = 7–8). The statistical significance of the differences was determined by Student's t-test, and a P value <0.05 was considered significant (*P < 0.05; **<0.01 vs. si-Control).

Fig. 2.

Plasma lipoprotein analysis of si-Control and si-GCGR-treated db/db mice. A: Quantification of plasma TC, VLDL-C, LDL-C, and HDL-C from si-Control and si-GCGR-injected mice. B: Western blot analysis of LDLR protein levels in liver tissues from si-Control and si-GCGR mice. C: Quantification of plasma PCSK9 levels in si-Control and si-GCGR mice. D: Representative sample of apoB-100 and apoB-48 levels shown in A. Ultracentrifugation-isolated apoB-containing lipoproteins in VLDL(left panel) and LDL(right panel) were resolved by SDS-PAGE and visualized by Coomassie blue staining. E: Quantification of plasma apoB levels in si-Control and si-GCGR mice. F: Lipoprotein separation by FPLC. Plasma samples from si-Control and si-GCGR-injected mice. The results given in figures A, C, and E are shown as means ± SEM (n = 8). The statistical significance of the differences was determined by Student's t-test, and a P value <0.05 was considered significant (*P < 0.05 vs. si-Control). TC, total cholesterol; VLDL-C, VLDL cholesterol; LDL-C, LDL cholesterol; HDL-C, HDL cholesterol.

Fig. 3.

Hepatic lipid content and mRNA expression of genes involved in lipid metabolism. A: Hepatic TG and cholesterol levels in the livers of si-Control and si-GCGR mice. Data are shown as the mean ± SEM (n = 8). The statistical significance of the differences was determined by Student's t-test, and a P value <0.05 was considered significant (*P < 0.05; **<0.01 vs. si-Control). B: Analysis of hepatic gene expression by real-time quantitative PCR in si-Control and si-GCGR mice (n = 8). Fold regulation indicates the ratio of expression levels in si-GCGR over si-Control. Livers were collected from mice in the fed condition. The statistical significance of the differences was determined by Student's t-test, and a P value <0.05 was considered significant.

Fig. 4.

Cholesterol and palmitate synthesis and TG production. Newly synthesized cholesterol (A) and palmitate (B) of si-Control and si-GCGR-injected mice. C: TG production in si-Control and si-GCGR mice. TG production was determined by measuring plasma TG concentrations at indicated times after P407 injection. Quantifications of plasma levels of TG (D), NEFA (E), and β hydroxybutylate (bHB) (F) from si-Control and si-GCGR mice. All values are expressed as means ± SEM (n = 6–8). The statistical significance of the differences was determined by Student's t-test, and a P value <0.05 was considered significant (*P < 0.05; **<0.01 vs. si-Control).