Abstract Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of liver-specific gene expression, is regulated by two promoters (P1 and P2) which drive expression of two groups of HNF4α isoforms referred to as HNF4α1 and HNF4α7. HNF4α is a known regulator of gluconeogenesis and mutated in maturity onset diabetes of the young one (MODY1). Conventionally, it was thought that HNF4α1, but not HNF4α7, is expressed in the normal adult liver, while HNF4α1 is downregulated and HNF4α7 is upregulated in liver cancer. Now, research in our lab reveals a previously undescribed role for HNF4α7 in the normal adult mouse liver – one involved in the diurnal variations of lipid and carbohydrate metabolism. More specifically, HNF4α1 appears to be a major driver of gluconeogenesis while HNF4α7 is a driver of ketogenesis: we propose that alterations in the levels of the HNF4α isoforms during the day flip the molecular switch between the two. Our preliminary data also show that HNF4α7 is required for increased levels of circulating ketone bodies in female mice. AMP-Activated Protein Kinase (AMPK), an energy-sensing enzyme, has been shown to phosphorylate HNF4α1 in vitro, but effects in vivo and on HNF4α7 are not known. SIRT1 is a deacetylase that works with AMPK to regulate glucose and lipid metabolism. HNF4α1 is known to be acetylated and our preliminary data suggest that HNF4α7 but not HNF4α1 interacts with SIRT1. Here, we propose to use HNF4α1-expressing (α1HMZ) and HNF4α7-expressing exon swap mice (α7HMZ) to determine the physiological function of the HNF4α isoforms in the switch between gluconeogenesis and ketogenesis, and to characterize the impact of sex on those functions. In Aim 1, we will determine whether intermittent fasting and a ketogenic diet increase the levels of HNF4α7 in the liver, and whether the increase occurs in all hepatocytes, or just a subset. We will determine the consequences of HNF4α7 on gene expression. Kidney and intestines will also be explored. In Aim 2, we will determine whether the AMPK pathway acts in a differential fashion on the HNF4α isoforms to help flip the metabolic switch. Phosphorylation by AMPK and deacetylation by SIRT1 will be explored. Finally, in Aim 3, we will determine whether the estrogen pathway impacts the HNF4α isoforms in female mice and determine the consequences for the metabolic switch. Our compelling preliminary data that the HNF4α isoforms are involved in the switch between gluconeogenesis and ketogenesis shed new light on this basic metabolic process that occurs on a daily basis and under conditions of feeding and fasting. The results from this proposal will illuminate not only the molecular mechanism underlying the switch but also how that mechanism is impacted by sex. The proposed studies have the potential to impact our understanding of numerous metabolic diseases, including diabetes, obesity, fatty liver disease and cancer. Finally, given the fact that ketone bodies serve as a source of fuel for th...