Modulation of Hepatic Protein Kinase Cβ Expression in Metabolic Adaptation to a Lithogenic Diet

Abstract

Background & aims: Dietary factors are likely an important determinant of gallstone development, and difficulty in adapting to lithogenic diets may predispose individuals to gallstone formation. Identification of the critical early diet-dependent metabolic markers of adaptability is urgently needed to prevent gallstone development. We focus on the interaction between diet and genes, and the resulting potential to influence gallstone risk by dietary modification.

Methods: Expression levels of hepatic protein kinase C (PKC) isoforms were determined in lithogenic diet-fed mice, and the relationship of hepatic cholesterol content and PKCβ expression and the effect of hepatic PKCβ overexpression on intracellular signaling pathways were analyzed.

Results: Lithogenic diet feeding resulted in a striking induction of hepatic PKCβ and PKCδ mRNA and protein levels, which preceded the appearance of biliary cholesterol crystals. Unlike PKCβ deficiency, global PKCδ deficiency did not influence lithogenic diet-induced gallstone formation. Interestingly, a deficiency of apolipoprotein E abrogated the diet-induced hepatic PKCβ expression, whereas a deficiency of liver X receptor-α further potentiated the induction, suggesting a potential link between the degree of hepatic PKCβ induction and the intracellular cholesterol content. Furthermore, our results suggest that PKCβ is a physiologic repressor of ileum basal fibroblast growth factor 15 (FGF15) expression and activity of hepatic proto-oncogene serine/threonine-protein kinase Raf-1/mitogen-activated protein (MAP) kinase kinase/extracellular signal-regulated kinases 1/2 (Raf-1/MEK/ERK1/2) cascade proteins, and the complex interactions between these pathways may determine the degree of hepatic ERK1/2 activation, a potent suppressor of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase expression. We found that PKCβ regulated Raf-1 activity by modulating the inhibitory Raf-1^Ser259 phosphorylation.

Conclusions: Our results demonstrate a novel interaction between the hepatic PKCβ/Raf-1 regulatory axis and ileum PKCβ/FGF15/ERK axis, which could modulate the bile lithogenecity of dietary lipids. The data presented are consistent with a two-pronged mechanism by which intestine and liver PKCβ signaling converges on the liver ERK1/2 pathway to control the hepatic adaptive response to a lithogenic diet. Elucidating the impact and the underlying mechanism(s) of PKCβ action could help us understand how different types of dietary fat modify the risk of gallstone formation, information that could help to identify novel targets for therapeutic approaches to combat this disease.

Keywords: Akt, protein kinase B; ApoE, apolipoprotein E; Cyp7a1, cholesterol 7α-hydroxylase; Cyp8b1, sterol 12α-hydroxylase; ERK1/2, extracellular signal regulated kinase-1/2; FGF15, fibroblast growth factor 15; FXR, farnesoid X receptor; GSK-3, glycogen synthase kinase-3; Hepatic Cholesterol Metabolism; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein; LXR, liver X receptor; Lithogenic Diet; MEK, mitogen-activated protein (MAP) kinase kinase; MMLD, modified milk fat lithogenic diet; PKCβ, protein kinase C isoform β; Protein Kinase Cβ; Raf-1, Raf-1 hepatic proto-oncogene serine/threonine-protein kinase; SREBP, sterol response element-binding protein; Signal Transduction; WT, wild type.

Figure 1

Relative expression of selective protein kinase C (PKC) isoforms in the livers of male C57/BL6 WT mice (n = 6–8 mice per group) fed either a chow or lithogenic diet (MMLD) for the indicated periods. (A) Comparison of mRNA expression levels for the indicated PKC isoforms in the livers of mice fed MMLDL for 2 weeks. The data are expressed relative to corresponding level in chow fed livers. Results are expressed as mean ± standard deviation. **P < .01. (B) Protein levels of indicated PKC isoforms and β-actin were determined using immunoblotting of pooled liver extracts. Equal amounts of protein were loaded per well. (C) Polarizing light microscopy examination of cholesterol crystals in pooled gallbladder bile with the indicated genotype on feeding a lithogenic diet for 1 or 2 weeks.

Figure 2

Potential relationship between diet-induced hepatic protein kinase C isoform β (PKCβ) and increased hepatic cholesterol levels. (A) Thin-layer chromatography of total lipid extracts from livers of WT and PKCβ^−/− mice (n = 2) after a lithogenic diet feeding for 0 or 2 weeks. (B) Effect of feeding individual components of a lithogenic diet, diet containing high fat and cholesterol, or a lithogenic diet containing all three components for 2 weeks on hepatic PKCβ protein levels (n = 3). (C) Thin-layer chromatography of total lipid extracts from livers of two of the mice fed an indicated diet. The above results are representative of at least three separate experiments. Each value represents the mean ± standard deviation. *P < .05; **P < .01.

Figure 3

Effects of apolipoprotein E (ApoE) or liver X receptor-α (LXRα) deficiency on lithogenic diet-induced hepatic protein kinase C isoform β (PKCβ) expression. (A) ApoE^−/− deficiency attenuates diet-induced hepatic PKCβ. Livers from ApoE^−/− mice (n = 2) fed a lithogenic diet for 2 weeks were examined for PKCβ protein levels by immunoblotting. (B, C) LXRα deficiency potentiates diet-induced hepatic PKCβ expression; the induction is accompanied by an increase in hepatic cholesterol content. Livers from wild-type and LXR^−/− mice (n = 2 mice per diet group) fed modified milk fat lithogenic diet (MMLD) for 2 weeks were used to examine hepatic PKCβ protein levels and the lipid contents. (D) Effect of sterols (10 μg/mL cholesterol plus 2 μg/mL 25-hydroxycholesterol, 24 hours) treatment on endogenous PKCβ expression in mouse liver cell line H2.35 cultured in medium supplemented with lipoprotein-deficient serum. Equal amounts of cell extracts were probed for PKCβ expression by immunoblotting. This experiment was repeated twice with a similar outcome. *P < .05; **P < .001.

Figure 4

Lithogenic diet-fed PKCβ^−/−mice exhibit greater liver insulin-sensitivity than control mice. Wild-type (WT) and PKCβ^−/− mice (n = 2) fed a lithogenic diet for 0 or 2 weeks were either untreated or injected with insulin (0.9 U/kg body weight), and the livers were analyzed for the insulin-signaling proteins. Western blots are representative of two separate experiments. Fold change shows the band intensity ratio of PKCβ^−/− over WT. *P < .05, n = 4.

Figure 5

Effects of lithogenic diet feeding on ileum fibroblast growth factor 15 (FGF15) and protein kinase C isoform β (PKCβ) expression levels. (A) PKCβ deficiency significantly induces ileum FGF15 expression in response to lithogenic diet intake. FGF15 mRNA expression levels were analyzed in the ileum of WT and PKCβ^−/− mice fed a chow or lithogenic diet for 2 weeks. (B) The ilea from the animals were also used to examine PKCβ expression levels by immunoblotting. Levels of FGF15 and PKCβ in WT mice fed a chow diet were assigned a value of 1. Results are expressed as the mean ± standard deviation. *P < .05, **P < .01 (n = 5).

Figure 6

Protein kinase C isoform β (PKCβ) negatively regulates hepatic proto-oncogene serine/threonine-protein kinase Raf-1/mitogen-activated protein (MAP) kinase kinase/extracellular signal regulated kinase-1/2 (Raf-1/MEK/ERK1/2) signaling cascade in the liver. (A) Comparison of expression and phosphorylation levels of MAPKs in the pooled liver extracts of mice (n = 5) fed a lithogenic diet for the indicated periods. (B) Comparison of phosphorylation levels of ERK1/2 and p38^MAPK in the pooled liver extracts of wild-type (WT) and PKCβ^−/− mice (n = 5) fed a lithogenic diet for 2 weeks. (C) Overexpression of PKCβ reduces basal and FGF15-induced ERK1/2 phosphorylation in mouse hepatocytes. Primary mouse adipocytes were infected with either adenovirus overexpressing green fluorescent protein or adenovirus overexpressing protein kinase C isoform β at an multiplicity of infection of 0 and 100; after 24 hours the PKCβ and phospho-ERKs levels were measured by immunoblotting using anti-phospho-ERK1/2. The above Western blots are representative of two separate experiments. (D) Comparison of hepatic gene expression between lithogenic diet-fed WT and PKCβ^−/− mice. Levels for each gene in WT mice were assigned a value of 1. Each value represents the mean ± standard deviation (n = 5 mice/group). *P < .05; **P < .01.

Figure 7

Proposed model for the mechanism by which protein kinase C isoform β (PKCβ) activation in the liver and ileum contributes to adaptive response for proper handling of a large supply of dietary fat, cholesterol, and bile acids to prevent hepatic overaccumulation of toxic cholesterol. Cholesterol homeostasis is achieved through regulation of cholesterol uptake, cholesterol biosynthesis, cholesterol conversion to bile acids, and excretion of bile acids. The data presented in this study are consistent with a two-pronged mechanism by which PKCβ deficiency contributes to dysfunctional cholesterol homeostasis through disturbing the fibroblast growth factor 15/extracellular signal regulated kinase (FGF15/ERK) and Raf-1 hepatic proto-oncogene serine/threonine-protein kinase/mitogen-activated protein (MAP) kinase kinase/extracellular signal regulated kinase-1/2 (Raf-1/MEK/ERK1/2) regulatory axes.