Thermogenic Capacity and Redox Regulation of Uncoupling Protein 1 in Non-shivering Thermogenesis
Abstract
Deep-water diving exposes divers to cold environments with high thermal conductivity, placing divers at risk of performance impairment and mortal endangerment. Extrinsic mechanisms for thermal insulation are essential in combatting body heat loss, but intrinsic thermogenesis is also necessary for body temperature maintenance. Recent discoveries have revealed that brown adipose tissue, capableof non-shivering thermogenesis, is maintained in adulthood and presents a potential solution to heat loss during diving. Non-shivering thermogenesis involves partial mitochondrial membrane uncoupling by short circuiting oxidative phosphorylation, regulated and facilitated by an integral membrane transporter, uncoupling protein 1 (UCP1). While the environmental factors that determine adult brown adipose tissue development and maintenance remain unknown, the utility of this tissue depends on its thermogenic capacity. This proposal seeks to evaluate the thermogenic capacity of UCP1 within brown adipocytes. This will be accomplished through a strategic technical approach pursuant to three objectives. Our first objective is to measure the proton flux through human UCP1 under physiological membrane potentials to define the thermogenic capacity of individual UCP1 proteins. Our approach involves the construction of a protein fusion construct, where the vectoral proton conductance of UCP1 is aligned antiparallel to bacteriorhodopsin, a light-stimulated proton pump. Reconstitution of this construct into mitochondrial-mimetic unilamellar lipid vesicles allows for light-controlledpH gradients to be rapidly and continuously generated. An attached pH sensor based on yellow-fluorescent protein provides a rapid pH readout to directly measure proton conductance, thus establishing the maximal thermogenic capacity of an individual UCP1. Under physiological conditions UCP1 is necessarily regulated by adenosine triphosphate (ATP) and free fatty acid levels, through mechanisms that remain unclear.Objective two is to elucidate how free fatty acids and ATP regulate UCP1 activity. To this end, isothermal titration calorimetry and proton conductance measurements will be performed to elucidate the specificity, affinity, and mechanism of regulation of UCP1. These studies will develop a molecular level understanding of UCP1 regulation, informing rational regulation for on-demand thermogenesis. Evidence from murine model studies suggest that reactive oxygen species may be important in regulating UCP1 function through post-translational modification of a specific cysteine residue on the matrix face. This is particularly relevant given the hyperbaric oxygen levels necessary to maintain diver air supply and counterpressure. The human variant, conspicuously, contains a second cysteine predicted to be exposed on the mitochondrial matrix, and thus may render murine model studies problematic. Objectng the effect of these modifications on conductance and regulation. In parallel, the effect of oxygen tension on both murine and human brown adipocyte UCP1 expression and thermogenesis will be evaluated These studies will represent the first evaluation of hyperbaric oxygen concentrations on thermogenesis function both in vivo and in vitro.It is anticipated that the results of the above studies will provide rigorous benchmarks for UCP1 thermogenic capacity and regulatory status under various metabolic conditions, includthose specific to deep-water diving. With the thermogenic capacity of brown adipose tissue quantified at both the single protein and cellular level, the potential role of this tissues in improving thermogenesis can be critically appraised.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 06, 2021
- Source ID
- N000142112360
Entities
People
- Brandon Greene
Organizations
- Office of Naval Research
- United States Navy
- University of California, Santa Barbara