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Propionate is a dominant inducer of bovine cytosolic phosphoenolpyruvate carboxykinase gene expression
Expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a critical control point for gluconeogenesis. Indirect evidence suggests that increased feed intake after calving and diet modification that increase rumen propionate production, the primary precursor of gluconeogenesis in ruminants, induces PCK1 mRNA. Our objective was to determine the direct effects of propionate on regulation of bovine PCK1 promoter activity and the relationship to hormones known to modulate glucose metabolism. The full length proximal promoter of bovine PCK1 from -1238 to +221 relative to transcription initiation and nested 5’ truncation deletions at -815, -409, -281 and -85 to +221 were ligated to pGL3 Firefly Luciferase Reporter Vector and transfected into rat hepatoma H4IIE cells. The pGL3-Basic (promoterless) and pGL3-Promoter (SV40 promoter driven) vectors served as negative and positive controls for the experiment. Renilla Luciferase Reporter Vector was cotransfected to normalize transfection efficiency. At 5 h after transfection, cells were exposed to either 2.5 mM propionate (PRO), 100 nM insulin (INS), 1 mM 8 Br-cAMP (cAMP), 5 µM dexamethasone (DEX), or the double and multiple combinations of PRO, INS, cAMP and DEX for 23 h. Promoter activity was expressed as the ratio of firefly luciferase to renilla luciferase and data were analyzed using the Mixed Procedure of SAS 9.3. All bovine PCK1 promoter constructs were capable of driving firefly luciferase expression. Propionate induced (P＜0.001) expression of all PCK1 promoter constructs compared with no treatment control. The induction by propionate was greatest for the -1238 to +221 promoter construct (up to 6-fold) and similar for the other four PCK1 promoter constructs (about 3-fold). Activity of the -1238 to +221 PCK1 promoter was not altered by cAMP and DEX alone but was induced (3-fold) by their combination (P＜0.01). Induction of the -1238 to +221 PCK1 promoter construct with cAMP and DEX, was repressed by 24% in response to INS and PRO prevented this effect (P＜0.0001). The data demonstrate an inductive effect of propionate on bovine PCK1 promoter activity that is dominant to the repressive effect of insulin. Furthermore, the effect of propionate to induce PCK1 appears to be independent of the actions of cAMP and dexamethasone to also induce bovine PCK1 transcription.
Keywords:
cytosolic phosphoenolpyruvate carboxykinase, propionate, hormones