Differentiation-dependent expression of cathepsin D and importance of lysosomal proteolysis in the degradation of UCP1 in brown adipocytes

Abstract

The lysosomal protease cathepsin D increased markedly in brown adipocytes during differentiation in primary cultures. Differentiated cells had 20 times the amount of immunoreactive cathepsin D found in preadipocytes. Cathepsin D mRNA, as estimated by relative RT-PCR, was also present in higher amounts in differentiated brown fat cells. Cathepsin D expression was not influenced by repeated exposures of brown adipocytes to norepinephrine (NE). Cathepsin D levels were also unchanged when NE was withdrawn for 48 h after cells had been exposed to NE for 7 days. In contrast, exposure of the cells to NE for 7 days increased their UCP1 content by more than twofold, which returned to basal levels within 48 h of withholding NE. The half-life of UCP1 under basal conditions and in cells chronically exposed to NE was estimated from reductions in [35S]methionine-labelled immunoprecipitable UCP1 over 72 h. UCP1 t1/2 under basal conditions was 3.7+/-0.4 days, which was similar to the half-lives of labelled mitochondrial translation products (3.6+/-0.8 days). The turnover rates of both UCP1 and mitochondrial translation products were reduced by NE. The turnover rate of UCP1 in the presence or absence of NE cannot account solely for the rapid loss of UCP1 from brown adipocytes upon withdrawal of NE. This loss was reduced when cells were incubated with inhibitors of phosphatidylinositol 3-kinases (PI 3-kinase), previously shown to block formation of autophagic vacuoles. Thus, brown adipocytes acquire a large capacity for both uncoupled metabolism and for lysosomal proteolysis during differentiation. Withdrawal of NE, as often occurs in vivo from suppression of sympathetic nervous system activity, would not only terminate thermogenesis but also favor formation of autophagic vacuoles to rapidly reduce the cell content of UCP1-containing mitochondria.

Thermogenic activation represses autophagy in brown adipose tissue

“…In fact, this is consistent with early reports indicating repression of general proteolysis in brown adipocytes in response to NE, a finding suggestive of the involvement of autophagy. 30 Other biological processes induced during differentiation of brown adipocytes, but repressed by noradrenergic stimulation of already differentiated cells (e.g. PPARγ expression) have been reported previously.…”

Mitochondrial Turnover

“…the PINK1-Parkin pathway in Drosophila promotes selective turnover of membrane-bound RC subunits in a manner independent of conventional autophagy (23). Additionally, it has been proposed that the similarity of the turnover rate for UCP1 and other mitochondrial proteins is the result of a stimulatory effect of UCP1 on mitochondrial biogenesis (42) and parallel proteolytic rates, as was shown by Moazed and Desautels (43,44).…”

Uncoupling protein 1 expression and high-fat diets

“…This indicates that a certain proportion of increased Ucp1 protein can be attributed to mitochondrial biogenesis or reduced mitochondrial autophagy and thus an increase in all mitochondrial proteins, including Ucp1. Indeed, Ucp1 degradation is usually part of the general mitochondrial protein turnover mediated by autophagy and correlates with other mitochondrial translation products (39,50).…”