Mechanisms of Inflammation and Regeneration in the Eye

ORCID

https://orcid.org0000-0001-6667-9018

Date of Award

2-5-2025

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Fasting and caloric restriction are effective to treat patients with pre-diabetes or metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), but intensive lifestyle changes are difficult for many to implement and sustain. Therefore, by identifying and studying fasting-mediated signals, we hope to elicit and convey the therapeutic effects of fasting against MASLD without actual physiological fasting. We discovered a key pathway in the liver through unbiased transcriptomic screening in fasted mice, a novel glucose fasting-activated effector: the amino acid hydrolase, arginase 2 (ARG2). Our previously published data demonstrates that forced hepatocyte-specific Arg2 overexpression is sufficient to reduce peripheral insulin resistance and hepatic steatosis in pre-diabetic and MASLD models in mice. In this thesis, I am to investigate how ARG2 conveys its therapeutic effects and examine its efficacy to bring forth new therapies that mimic the actions of ARG2 to ultimately treat MASLD and MASLD-related metabolic disease. In the first part of my thesis, I define the structure-function relationship between ARG2 mitochondrial localization, arginine hydrolase activity, and the metabolic effects of ARG2. I generated and tested two ARG2 mutant constructs, one lacking the putative N-terminal mitochondrial targeting sequence (MTS, Arg21-22) and the other lacking its ureahydrolytic activity (Arg2H160F). Hepatocyte-specific overexpression of the mutant constructs in obese, diabetic (db/db) mice showed ARG2 attenuates hepatic steatosis independent of mitochondrial localization or ureahydrolase activity, and that enzymatic activity is dispensable for ARG2 to augment total body energy expenditure. Furthermore, ARG2-mediated increase in glucose-, insulin tolerance, and glucose appearance suppression during hyperinsulinemic-euglycemic clamping requires both mitochondrial localization and ureahydrolase activity. Seahorse respirometry in hepatocytes in vitro, and quantification of heavy-isotope-labeled glucose oxidation in vivo further revealed that both Arg21-22 and Arg2H160F mutants failed to induce ARG2-mediated increase in hepatic and systemic oxidative metabolism, respectively. These results further complement our previous work in ARG2 by providing a structure-based mechanism of ARG2 with respect to its metabolic effects and demonstrating that hepatic Arg2 is a prominent metabolic gene. In the second part of my thesis, I investigate the contribution of and necessity for hepatocyte ARG2 in prevention against MASLD progression to confer the metabolic effects in the pathogenesis of MASLD and its related metabolic complications. The results demonstrate that hepatocyte-specific Arg2 deletion impairs ureagenesis, TCA cycle, and mitochondrial function which has real physiological metabolic consequences. Hepatocyte-specific Arg2-deficiency drives obesity, liver steatosis, and insulin resistance in aging-associated metabolic decline and diet-induced mouse models of MASLD. Mechanistically, impaired oxidative metabolism and MASH in Arg2LKO mice is reversible through supplementation of NAD+ via nicotinamide mononucleotide or nicotinamide riboside. Translationally, Arg2-deficiency generates metabolite alterations in nitrogen flux, TCA cycle flux, and oxidative metabolism which is consistent with biomarkers perturbation that independently predict severe incident MASLD/MASH nearly a decade in advance from 106,606 healthy participants in the UK Biobank. Therefore, hepatocyte-specific Arg2-deficiency represents as a new paradigm to demonstrate the urea cycle’s hierarchical control over TCA cycle flux to regulate mitochondrial oxidative metabolism. Together, providing mechanistic insight into the long-observed association between urea cycle impairment and MASLD. We then identified a readily available pharmacological reagent, ADI-PEG 20, and found arginine depletion via ADI-PEG 20 is viable and holds great therapeutic potential as a candidate for treatment against obesity and MASLD. Furthermore, ADI-PEG 20 treatment induced favorable metabolic effects that were independently abolished in mice with liver-specific Fgf21 and Becn1 deletion. This study reveals a novel role of arginine catabolism in the pathophysiology of MASLD which is dependent on liver-specific functions such as FGF21 and autophagy. Together, our findings suggest that hepatocyte arginine status is central and modifiable in treatments against MASLD and metabolic complications.

Language

English (en)

Chair and Committee

Brian DeBosch

Committee Members

Ting Wang; Brian Finck; Brian Van Tine; Gary Patti

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