Role of Beta-Hydroxy-Beta-Methylbutyrate (HMB) in Leucine Stimulation of Mitochondrial Biogenesis and Fatty Acid Oxidation

Mitochondrial dysfunction and the resulting oxidative stress is widely recognized as a contributing factor to the development of numerous pathophysiologies including obesity, diabetes, cardiovascular disease, sarcopenia, liver disease, dementia, amongst others. Mitochondrial dysfunction results in a reduced mitochondrial number and oxidative capacity, causing an increase in free radical production and consequently oxidative stress. As such, the characterization of compounds that can upregulate mitochondrial biogenesis and function could provide the foundation for the development of therapeutic nutraceuticals that promote mitochondrial health, and consequently reduce oxidative stress. Leucine is well recognized to stimulate muscle protein synthesis, and we have recently demonstrated that leucine increases mitochondrial biogenesis and fatty acid oxidation (FAO) in muscle cells, although the mechanism of these effects is not clear. However, it is likely that that the leucine metabolites [alpha]-ketoisocaproic acid (KIC) and [beta]-hydroxy-[beta]-methylbutyrate (HMB) play a role. Once ingested, dietary leucine is transaminated by branched-chain aminotransferase (BCAT) to the [alpha]-keto analogue KIC. KIC is then metabolized into either isovaleryl-CoA via the branched chain [alpha]-ketoacid dehydrogenase (BCKD) enzyme, or HMB by the cytosolic enzyme KIC-dioxygenase (KICD). We investigated the roles of intact leucine versus KIC and HMB on markers of mitochondrial abundance and function in murine myotubes. All three compounds induced comparable increases in FAO. Both leucine and HMB increased myotube mitochondrial biogenesis, assessed fluorometrically via NAO binding. Consistent with this, HMB and leucine both stimulated expression of mitochondrial regulatory and component genes, which suggests that HMB mediates these effects of leucine. To confirm this, we transfected murine myoblasts with BCAT, BCKD, or KICD siRNA and investigated the role of intact leucine versus HMB on myoblast mitochondrial abundance. Both HMB and leucine increased mitochondrial mass, while the knockdown of BCAT and KICD abated the leucine-stimulation of mitochondrial biogenesis. Consistent with this, BCAT siRNA transfected cells displayed reduced expression of key mitochondrial genes. This suggests that the leucine effects on muscle mitochondrial biogenesis are in fact mediated by the metabolite HMB.