Effects of ketoacidosis on rat apolipoprotein A1 gene expression: a link with acidosis but not with ketones

Abstract

To determine if ketoacidosis contributes to reduced apolipoprotein A1 (apoA1) expression in insulindeficient diabetic rats, we examined the regulation of apoA1 gene expression in response to changes in ambient pH or ketone body concentrations. Hepatic apoAI mRNA levels were reduced 42% in diabetic rats relative to nondiabetic controls (means ..; 321·8 43·7 vs 438·7 58·8 arbitrary units; P<0·03). Neither endogenous apoA1 mRNA nor transcriptional activity of the rat apoA1 gene promoter (from 474 to 7) were altered by sodium butyrate or isobutyramide (0·3 mM to 10 mM) in Hep G2 or Caco-2 cells. Rat hepatic and intestinal apoA1 mRNA levels, and plasma apoA1 concentration, were not altered 24 h after isobutyramide administration (500 mg/kg by gavage). When the effect of altering ambient pH within a wide range commonly encountered in vivo was studied, acidosis (pH 6·7), relative to alkalosis (pH 7·9), decreased apoAI mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase mRNA by 47% in Hep G2 cells (P<0·025) and by 24% in Caco-2 cells (P<0·017). Acidosis did not alter cytomegalo virus (CMV)--galactosidase activity, or the activity of the simian virus (SV40) early-region promoter, in either cell line transfected with the respective constructs. The lowering of ambient pH was associated with a graded reduction in apoAI promoter activity. At pH 6·7, apoAI promoter activity was reduced by 75% compared with promoter activity at pH 7·9. These observations indicate that acidosis, but not ketosis, contributes to the reduction in apoA1 expression during diabetic ketoacidosis by down-regulating apoAI promoter activity.

Regulation of apoA1 gene expression with acidosis: requirement for a transcriptional repressor

“…We have recently shown that apoA 1 mRNA content and apoA 1 promoter activity are suppressed by acidosis in both Caco-2 intestinal cells and HepG2 hepatocytes (Haas et al 2000). To confirm and extend our previous findings, apoA 1 protein content in HepG2 cells was measured by western blotting.…”

“…Quantitation of the results with a densitometer showed that acidosis reduced apoA 1 protein concentrations in HepG2 cells nearly 20%, from 100 2·3% to 80·3 5·4% (P<0·01). In this cell line, apoA 1 mRNA levels are also reduced by acidosis, by 20% (Haas et al 2000). This suggests that the steady-state apoA 1 protein concentration is likely to be directly related to the decrease in apoA 1 mRNA levels.…”

“…Expression of the apoA 1 gene is repressed at the transcriptional level by acidosis (Haas et al 2000). Acidosis repressed apoA 1 mRNA levels by 25-30%, and apoA 1 promoter activity by more than 50%, relative to alkalosis.…”

“…To determine whether the changes in apoA 1 promoter activity with acidosis (Haas et al 2000) that we had observed could be reproduced with various chemicals known to reduce intracellular pH directly or indirectly, the effect of various cation pump inhibitors and ionophores on apoA 1 promoter activity was evaluated. Changes in intracellular Na + and K + concentration results in changes in intracellular pH as a result of the activity of a Na + /H + antiporter (Aronson 1985, Krapf et al 1991.…”

“…Acidosis repressed apoA 1 mRNA levels by 25-30%, and apoA 1 promoter activity by more than 50%, relative to alkalosis. Repression due to acidosis is observed in HepG2 liver cells, and in Caco-2 cells (Haas et al 2000) -a cell line with the characteristics of an epithelial cell from the small intestine and which expresses the apoA 1 gene. In order to understand the mechanism(s) by which acid-base balance may regulate apoA 1 gene expression, we mapped the cis-element responsible for repression by acidosis.…”