Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes

Abstract

Hepatic glucokinase plays a key role in glucose metabolism as underlined by the anomalies associated with glucokinase mutations and the consequences of tissue-specific knock-out. In the liver, glucokinase transcription is absolutely dependent on the presence of insulin. The cis-elements and trans-acting factors that mediate the insulin effect are presently unknown; this is also the case for most insulin-responsive genes. We have shown previously that the hepatic expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) is activated by insulin. We show here in primary cultures of hepatocytes that the adenovirus-mediated transduction of a dominant negative form of SREBP-1c inhibits the insulin effect on endogenous glucokinase expression. Conversely, in the absence of insulin, the adenovirus-mediated transduction of a dominant positive form of SREBP-1c overcomes the insulin dependency of glucokinase expression. Hepatic fatty acid synthase and Spot-14 are insulin/glucose-dependent genes. For this latter class of genes, the dominant positive form of SREBP-1c obviates the necessity for the presence of insulin, whereas glucose potentiates the effect of SREBP-1c on their expression. In addition, the insulin dependency of lipid accumulation in cultured hepatocytes is overcome by the dominant positive form of SREBP-1c. We propose that SREBP-1c is a major mediator of insulin action on hepatic gene expression and a key regulator of hepatic glucose/lipid metabolism.

Figure 1

A dominant negative form of SREBP-1c decreases glucokinase gene expression in cultured hepatocytes of adult rats. Hepatocytes were cultured for 16 h after plating in the presence of 5 mM glucose. Cells were then incubated with 100 nM insulin and 5 mM glucose either without adenovirus or with null adenovirus (Ad null; 30 pfu per cell) or the SREBP-1c-DN adenovirus (3 or 30 pfu per cell). After 18 h, total RNAs were extracted and probed for the expression of glucokinase (GK), SREBP-1c DN and AGE genes, and 18S rRNA (18S). The Northern blot presented is representative of three experiments.

Figure 2

A dominant positive form of SREBP-1c abolishes the effect of the dominant negative form of SREBP-1c on glucokinase gene expression in cultured hepatocytes of adult rats. Hepatocytes were cultured for 16 h after plating in the presence of 5 mM glucose. Cells were then incubated with 100 nM insulin and 5 mM glucose either without adenovirus or with the SREBP-1c DN adenovirus (3 pfu per cell) in the absence or presence of increasing titers of the SREBP-1c-403 adenovirus (0, 0.3, 1, 3, or 10 pfu per cell). After 18 h, total RNAs were extracted and analyzed for the expression of GK and AGE genes and 18S rRNA. The Northern blot presented is representative of two experiments.

Figure 3

A dominant positive form of SREBP-1c induces glucokinase gene expression in cultured hepatocytes of adult rats in the absence of insulin. Hepatocytes were cultured for 16 h after plating in the presence of 5 mM glucose. Cells were then incubated with 5 mM glucose either without adenovirus and with or without 100 nM insulin, or with increasing titers of the SREBP1c-403 adenovirus (0, 0.3, 1, or 3 pfu per cell). After 18 h, total RNAs were extracted and analyzed for the expression of GK and AGE genes and 18S rRNA. The Northern blot presented is representative of three experiments.

Figure 4

Comparison of the expression of glucokinase and SREBP-1c genes in the liver of suckling and fed adult rats. Total RNAs were extracted and analyzed for the expression of GK and SREBP-1c genes and 18S rRNA. Each lane represents data obtained from the liver of one animal. This Northern blot is representative of data obtained with six different animals in each group.

Figure 5

Insulin-induced expression of SREBP-1c precedes the appearance of glucokinase mRNA. A dominant negative form of SREBP-1c abolishes the effect of insulin on glucokinase gene expression in cultured hepatocytes of suckling rats. (A and C) Cells were incubated with 5 mM glucose with the Ad null (3 pfu/cell) or (B and D) the SREBP-1c-DN adenovirus (3 pfu per cell). After 24 h, cells were incubated with 100 nM insulin and 5 mM glucose for 0, 6, 12, 24, 30, or 36 h. Total RNAs were extracted and analyzed for the concentration of GK, endogenous SREBP-1c and SREBP-1c DN mRNAs, and 18S rRNA. The Northern blot presented is representative of two experiments. (C and D) Graphs show the concentrations of GK and endogenous SREPB-1c mRNA, expressed as a ratio to the corresponding 18S signal and as the means ± SD of duplicate values obtained in two separate experiments.

Figure 6

The effect of the dominant positive form of SREBP-1c in the presence of a high glucose concentration on FAS and S14 gene expression and the determination of the glucose 6-phosphate concentrations in cultured hepatocytes of adult rats. After plating, hepatocytes were cultured for 16 h in the presence of 5 mM glucose. Cells were then incubated with 25 mM glucose in the absence of insulin either with the Ad null (A) or with SREBP-1c 403 (B). After 4, 6, 8, 10, 12, 14, 16, and 18 h, total RNA was extracted and analyzed for the expression of FAS and S14 genes and 18S rRNA and (C) the concentrations of glucose 6-phosphate were determined. The Northern blot presented is representative of three experiments. Glucose 6-phosphate concentrations are the means ± SD from values obtained from two separate experiments.

Figure 7

Comparison of the effects of the dominant positive form of SREBP-1c in the presence of a low or high glucose concentration on lipogenic gene expression in cultured hepatocytes of adult rats. After plating, hepatocytes were cultured for 16 h in the presence of 5 mM glucose. Cells were then incubated for 16 h with 5 or 25 mM glucose either with or without the SREBP-1c 403 adenovirus (0.3 and 1 pfu per cell). Total RNAs were extracted and analyzed for the expression of FAS, S14, and AGE genes and 18S rRNA. The Northern blot presented is representative of three experiments.

Figure 8

The effect of a dominant positive form of SREBP-1c on lipid accumulation in cultured hepatocytes of adult rats. Hepatocytes were cultured for 16 h after plating in the presence of 5 mM glucose. Cells were then incubated with 25 mM glucose either without adenovirus and with or without 100 nM insulin, or with the SREBP1c-403 adenovirus (3 or 30 pfu per cell). After 24 h, hepatocytes were stained for the presence of lipid droplets. Microscopic views of hepatocytes at a magnification of ×400 are shown.