Impaired Ulk1 Ser555 Phosphorylation Promotes Amino Acid Reliance and Links Mitochondrial Inefficiency to Systemic Metabolic Inflexibility

Metabolic flexibility—the capacity and ability to switch between energetic substrates in response to nutrient availability—is essential for systemic energy balance and protection against metabolic disease. Loss of this adaptive capacity contributes to metabolic diseases marked by obesity, insulin resistance, and dyslipidemia, elevating the risk of type 2 diabetes and cardiovascular disease. Under nutrient stress, skeletal muscle typically adjusts substrate utilization from glucose to fatty acid oxidation, while the liver supports peripheral energy needs through glucose production and auxiliary amino acid metabolism. In insulin-resistant states, this inter-organ coordination deteriorates, resulting in reduced glucose utilization and increased catabolism of glucogenic amino acids such as alanine—leading to inefficient energy use and metabolic inflexibility. Unc-51-like autophagy activating kinase 1 (Ulk1) is a serine/threonine kinase best known for initiating autophagy, but also serves as a nutrient-sensing hub regulated by AMPK and mTORC1. AMPK activates Ulk1 via phosphorylation at serine 555 (S555) during energetic stress, while mTORC1 inhibits Ulk1 under nutrient-rich conditions. To investigate the specific role of Ulk1(S555), we used global Ulk1(S555A) knock-in mice, which prevent phosphorylation at this site, and examined responses to short-term caloric restriction (CR) and fasting. Ulk1(S555A) mice exhibited impaired glucose oxidative capacity in skeletal muscle and liver during nutrient stress, despite improved systemic glucose clearance. Metabolic cage analyses of WT and Ulk1(S555A) showed no significant differences in heat and energy expenditure, despite Ulk1(S555A) delayed substrate switching and reduced physical activity. U-14C-glucose tracing confirmed reduced glucose oxidation, while multi-omics analyses revealed a tissue-specific dependence on amino acids in response to nutrient stress. Ulk1(S555A) skeletal muscle mitochondria displayed increased basal oxidation of alanine and serine with blunted ADP-stimulated respiration, while Ulk1(S555A) hepatic mitochondria demonstrated an impairment of pyruvate entry into the TCA cycle. These findings identify Ulk1(S555) as a critical integrator of AMPK signaling, regulating substrate prioritization and metabolic communication between skeletal muscle and liver. Targeting this axis may offer new therapeutic avenues to restore metabolic flexibility in obesity, type 2 diabetes, and related disorders.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Human Nutrition, Foods, and Exercise

Department dc:contributor.department

Human Nutrition, Foods and Exercise

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2025

Author dc:creator

• Willoughby, Orion

Chair dc:contributor.committeechair

• Drake, Joshua Chadwick

Committee members dc:contributor.committeemember

• Gilbert, Elizabeth Ruth
• Warren, Junko
• Najt, Charles Paul
• Craige, Siobhan

• Caloric Restriction
• Fasting
• Nutrient Stress
• Bioenergetics
• Metabolism
• Nutrient Sensing
• Metabolic Flexibility
• Autophagy
• Mitophagy
• Skeletal Muscle
• Liver

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Dc Identifier Other

vt_gsexam:44884

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/139692