No 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 Cathepsin D levels were also unchanged when NE was withdrawn for 48 h after cells had been exposed to NE for 7 days.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.

Heparin, traditionally given parenterally, is used to treat and prevent thrombosis. Our previous results suggest that orally administered unfractionated heparin (UFH) is absorbed and has antithrombotic effects. However, there is little evidence indicating the site and mechanism of heparin absorption. Our aim was to determine whether the stomach is an absorption site. Rat gastric mucosa was mounted in an Ussing chamber, and UFH was added to the mucosal buffer at pH 7.4. Potential difference (PD), resistance (R), and short circuit current (I sc ) across the mucosa were determined comparing the mucosal to the serosal side. Mucosal and serosal buffers and tissue were analyzed for chemical heparin and anticoagulant activity, antifactor Xa (anti-Xa) and antifactor IIa (anti-IIa) activity. The PD became more negative on UFH addition. Following a lag period, PD returned to the resting level. Changes in R followed those in PD, whereas I sc did not change. Heparin was found in the serosal and mucosal buffer and tissue. Heparin in the serosal buffer had anti-Xa and anti-IIa activity. Decreasing the pH of the mucosal buffer to 4.0, decreased the lag period for PD. Decreasing the concentration of UFH resulted in less pronounced changes in PD and less heparin in the serosal buffer. Changes in PD suggest that heparin moves across the mucosa. Presence of heparin in the serosal buffer and mucosal tissue, indicate that heparin crosses rat gastric mucosa. A stable I sc indicates passive diffusion contributes to heparin movement. The stomach could be a site for oral heparin absorption.

The objective of this study was to evaluate some of the mechanisms by which norepinephrine (NE) and insulin may influence protein degradation in mouse brown adipocytes differentiated in cultures. The effects of NE and insulin, alone or in combination, on three factors known to influence proteolysis (maintenance of cell ATP and 1-phosphatidylinositol 3-kinase (PI 3-kinase) and p70 ribosomal S6-kinase (p70 S6K) activities) were examined. It was proposed that NE affects proteolysis indirectly by decreasing cell ATP from activation of uncoupling protein-1 (UCP1)-dependent mitochondrial respiration. This was tested by comparing the effects of NE and fatty acids (which directly activate UCP1) on proteolysis in brown adipocytes, as well as in pre-adipocytes and 3T3-L1 adipocytes, which do not express UCP1. An inhibitory effect of insulin on proteolysis is observed in both pre-adipocytes and differentiated cells, whereas NE and exogenously added fatty acids inhibit proteolysis only in brown adipocytes. There is a linear relationship between reductions in cell ATP and proteolysis in response to increasing concentrations of NE or fatty acids. PI 3-kinase activity is required for proteolysis, because two selective inhibitors (wortmannin and LY294002) reduce proteolysis in both pre-adipocytes and differentiated cells. This effect is not additive to that of NE, which suggests they affect the same proteolytic pathway. In contrast to NE, insulin increases PI 3-kinase activity and phosphorylation of p70 S6K. Rapamycin, which prevented insulin-dependent increase in phosphorylation of p70 S6K, increases proteolysis in brown adipocytes and antagonizes the inhibitory effect of insulin on proteolysis, but not the inhibitory effect of NE. Thus, insulin inhibits proteolysis via rapamycin-sensitive activation of p70 S6K, whereas the effect of NE appears largely to be a function of decreasing cell ATP content.

This study demonstrates the design and microfabrication of single cylindrical submicron-sized pores in 1 lm-thick PMMA membranes, and their integration and assembly into all-polymeric electrophoretic detectors. Pore sizes vary from 200 to 600 nm. Fabrication includes electron beam lithography of the pore and mechanical microfabrication and assembly of the remaining detector system, using UV-curing glues and a silicon sacrificial substrate wafer. Initial electrophoretic translocation experiments have been performed for various potassium chloride (KCl) electrolyte concentrations between 0.1 and 1 M. Experiments prove that the detector system is hermetically sealed, that the pores are capable of sustaining an open pore current, and that they respond with a steady and low-noise signal. The same experiments have also been applied to analyze the pore metrology, and revealed that submicron pore sizes have been underestimated by roughly 150 nm.