Fructose breakdown uniquely controls global metabolism

University Of Kentucky, Lexington KY

Investigators

Abstract

ABSTRACT- PROJECT 4 The overconsumption of sugar-sweetened beverages is a well-established risk factor for the development of obesity, insulin resistance, and type 2 diabetes (T2D). A nutrient-induced decline in mitochondrial function is thought to be the underlying driver of these metabolic complications. Conversely, an improvement in mitochondrial function could prevent the development of insulin resistance and progression to T2D. We show that mice consuming 30% fructose (HFD+F), but not 30% glucose (HFD+G), drinks on an HFD develop mitochondrial damage. These effects are dependent on fructose metabolism via ketohexokinase (KHK), the rate- limiting enzyme of fructose metabolism. Thus, our preliminary experiments confirmed that KHK knockdown restored mitochondrial fragmentation, increased complex I and complex II protein abundance, and enhanced CPT1α mediated fatty acid oxidation (FAO). These effects are, in part, mediated via acetylation, a post- translational protein modification. Indeed, acetylation of CPT1α at lysine (K) 508 lowered CPT1a protein and decreased mitochondrial FAO. Similarly, overexpression (OE) of KHK in cultured hepatocytes increased global protein acetylation, while it decreased CPT1α protein. Lastly, all HFDs that promote obesity contain sugar, while fat oxidation and weight loss are induced by sugar-fee (ketogenic) HFDs. Based on these novel findings, we hypothesize that fructose metabolism via KHK drives global protein acetylation triggering ER stress and mitochondrial fragmentation, while acetylation of mitochondrial complex I and II proteins directly impairs electron transport chain activity and mitochondrial function. This hypothesis will be tested in three specific aims. Aim 1, will elucidate if KHK mediated protein acetylation impairs mitochondrial remodeling. We will determine (i) if AAV- mediated OE of SIRT2, a major cytoplasmic deacetylase, can lower acetylation and ER stress in HFD+F-fed mice; ( ii ) elucidate whether SIRT2 OE is sufficient to restore mitochondrial fission/fusion defects; and ( iii if the inverse correlation between KHK and SIRT2 proteins, observed in our murine model, persists in ) probe human subjects, leveraging liver biopsies from our bariatric surgery biorepository. Aim 2 seeks to understand how fructose metabolism via KHK lowers mitochondrial function. This will involve (i) quantifying mitochondrial complex activity using Oroboros O2K Respirometer in HFD+F-fed mice treated with KHK or scramble siRNA; ( ) ii using immunoprecipitation and immunoblot techniques, we will ascertain if complex I (NDUFA2) and complex II (SDHA) proteins are acetylated in KHK dependent manner; and ( ) utilizing CRISPR Cas9, we will introduce gain/loss of function mutations in NDUFA2 and SDHB to elucidate the impact of acetylation mimicking (KQ) iii and acetylation null (KR) mutations on their respective activities. Aim 3 will determine if a modest amount of fructose within a solid HFD can affect the development of obesity and mitochondrial dysfunction. We will (i) measure mitochondrial function, and hepatic acetylome in male and female mice on custom diets treated with KHK or control siRNA, and ( hepatic insulin sensitivity in ii ) perform hyperinsulinemic, euglycemic clamps to determine if KHK KD can improve mice fed our custom diets.