Fetal Hepatic Sirt1, a Histone H3 Deacetylase, Is Decreased in a Nonhuman Primate Model of Maternal Obesity.

Abstract

Sirt1 is a known and highly conserved NAD+-dependent protein deacetylase and an important regulator of metabolism, senescence, cancer and longevity. We have previously reported in our nonhuman primate model of maternal obesity that fetal hepatic acetylation levels of histone H3 lysine 14 (H3K14ac) are increased with in utero maternal high fat diet (HFD) exposure compared with control diet, and that increased H3K14ac is accompanied by diminished histone deacetylase (HDAC) activity. However, the identity of the HDAC(s) mediating increased fetal H3K14ac had eluded us. We hypothesized that Sirt1 might in fact retain histone deacetylase in addition to protein deacetylase activity, and thereby modulate fetal epigenomic alterations in our model. Employing QPCR, Western blot and IP analyses from extracts of fetal liver under conditions of high fat or control diet exposure in utero, we observed that fetal hepatic Sirt1 transcription and translation is significantly reduced in offspring of high fat diet fed dams (p<0.001). In order to analyze Sirt1 activity, we transfected human wildtype and dominant negative mutant (H363Y) Sirt1 cDNA into Cos-1 cells. Sirt1 activity, as measured by a chromogenic protein and HDAC assay (BioMol), was greatly increased in the over-expressed recombinant Sirt1 Cos-1 cell line (p<0.001), while Sirt1 activity in cell lysates transfected with the dominant negative Sirt1 was inhibited (p<0.01). Resveratrol, a Sirt1 agonist, further increased Sirt1 protein and histone in vitro deacetylase activity in Cos-1 cells. Finally, to clearly delineate Sirt1-mediated histone deacetylation of H3K14ac, MOLDTI-TOF mass spectrometry with constructed histone peptides as substrate. Both the human recombinant Sirt1 and transfected Cos-1 cell lysate significantly and specifically the histone peptide deacetylation on mass spec. Taken together, these findings are consistent with fetal hepatic Sirt1 functioning as both a protein and histone deacetylase, with altered expression and activity following exposure to maternal HFD. The implications of these findings are to suggest that Sirt1 may function as a highly conserved regulator of metabolism and longevity, which is reprogrammable in fetal life. This research was supported by NIH Director New Innovator Pioneer Award DP2120OD001500-01 to K.A.T. and NICHD March of Dimes Reproductive Scientist Development Program to K.A.T. (poster)