Abstract
The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.
Highlights
Gallstone disease is a major public health problem in all developed countries
The low incidence of gallstone formation in WT mice observed in our study is mainly attributed to the feeding of a semisynthetic and refined purified lithogenic diet (PLD) as compared with lithogenic diets used in other studies [35, 36]
Our observation that Protein kinase C (PKC) deficiency stimulates diet-induced gallstone formation provides direct evidence that this signaling kinase is important in maintaining cholesterol and bile acid homeostasis
Summary
Animals and Lithogenic Diet—PKC-deficient C57BL/6J mice have been described earlier [22, 23]. Analysis of lipids in lipoprotein fractions was performed after separating pooled plasma samples by fast-performance liquid chromatography (FPLC) with a Superose 6 10/300 GL high performance column (GE Healthcare Lifescience) by personnel in the Mouse Metabolic Phenotyping Center facility at the University of Cincinnati College of Medicine. Bile Acid Pool Size and Fecal Bile Acid Output Determination—Bile acid pool size was determined as the sum total bile acid content of the entire small intestine, gallbladder, and liver, which were homogenized and extracted together in ethanol [32]. Total bile acid mass was determined enzymatically. Fecal bile acid output was determined from stool quantitatively collected from individually housed mice for periods up to 72 h. Quots of dried feces were extracted into ethanol as described [32], and total bile acid mass was again measured enzymatically. Immunoreactive proteins were visualized via enhanced chemiluminescence (ECL Plus; Amersham Biosciences) and quantified via densitometry using the Molecular Analyst software (Bio-Rad)
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