Cholesterol Depletion in Adipocytes Causes Caveolae Collapse Concomitant with Proteosomal Degradation of Cavin-2 in a Switch-Like Fashion

Caveolae, little caves of cell surfaces, are enriched in cholesterol, a certain level of which is required for their structural integrity. Here we show in adipocytes that cavin-2, a peripheral membrane protein and one of 3 cavin isoforms present in caveolae from non-muscle tissue, is degraded upon cholesterol depletion in a rapid fashion resulting in collapse of caveolae. We exposed 3T3-L1 adipocytes to the cholesterol depleting agent methyl-β-cyclodextrin, which results in a sudden and extensive degradation of cavin-2 by the proteasome and a concomitant movement of cavin-1 from the plasma membrane to the cytosol along with loss of caveolae. The recovery of cavin-2 at the plasma membrane is cholesterol-dependent and is required for the return of cavin-1 from the cytosol to the cell surface and caveolae restoration. Expression of shRNA directed against cavin-2 also results in a cytosolic distribution of cavin-1 and loss of caveolae. Taken together, these data demonstrate that cavin-2 functions as a cholesterol responsive component of caveolae that is required for cavin-1 localization to the plasma membrane, and caveolae structural integrity

Effect of sibutramine on cardiovascular outcomes in overweight and obese subjects.

BACKGROUND: The long-term effects of sibutramine treatment on the rates of cardiovascular events and cardiovascular death among subjects at high cardiovascular risk have not been established. METHODS: We enrolled in our study 10,744 overweight or obese subjects, 55 years of age or older, with preexisting cardiovascular disease, type 2 diabetes mellitus, or both to assess the cardiovascular consequences of weight management with and without sibutramine in subjects at high risk for cardiovascular events. All the subjects received sibutramine in addition to participating in a weight-management program during a 6-week, single-blind, lead-in period, after which 9804 subjects underwent random assignment in a double-blind fashion to sibutramine (4906 subjects) or placebo (4898 subjects). The primary end point was the time from randomization to the first occurrence of a primary outcome event (nonfatal myocardial infarction, nonfatal stroke, resuscitation after cardiac arrest, or cardiovascular death). RESULTS: The mean duration of treatment was 3.4 years. The mean weight loss during the lead-in period was 2.6 kg; after randomization, the subjects in the sibutramine group achieved and maintained further weight reduction (mean, 1.7 kg). The mean blood pressure decreased in both groups, with greater reductions in the placebo group than in the sibutramine group (mean difference, 1.2/1.4 mm Hg). The risk of a primary outcome event was 11.4% in the sibutramine group as compared with 10.0% in the placebo group (hazard ratio, 1.16; 95% confidence interval [CI], 1.03 to 1.31; P=0.02). The rates of nonfatal myocardial infarction and nonfatal stroke were 4.1% and 2.6% in the sibutramine group and 3.2% and 1.9% in the placebo group, respectively (hazard ratio for nonfatal myocardial infarction, 1.28; 95% CI, 1.04 to 1.57; P=0.02; hazard ratio for nonfatal stroke, 1.36; 95% CI, 1.04 to 1.77; P=0.03). The rates of cardiovascular death and death from any cause were not increased. CONCLUSIONS: Subjects with preexisting cardiovascular conditions who were receiving long-term sibutramine treatment had an increased risk of nonfatal myocardial infarction and nonfatal stroke but not of cardiovascular death or death from any cause. (Funded by Abbott; ClinicalTrials.gov number, NCT00234832.

Glycerol-3-phosphate acyltransferase 2 expression modulates cell roughness and membrane permeability: An atomic force microscopy study.

In mammalian cells, de novo glycerolipid synthesis begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferases (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions, and overexpressed in several types of cancers and cancer-derived human cell lines where its expression contributes to the tumor phenotype. Using gene silencing and atomic force microscopy, we studied the correlation between GPAT2 expression and cell surface topography, roughness and membrane permeability in MDA-MB-231 cells. In addition, we analyzed the glycerolipid composition by gas-liquid chromatography. GPAT2 expression altered the arachidonic acid content in glycerolipids, and the lack of GPAT2 seems to be partially compensated by the overexpression of another arachidonic-acid-metabolizing enzyme, AGPAT11. GPAT2 expressing cells exhibited a rougher topography and less membrane damage than GPAT2 silenced cells. Pore-like structures were present only in GPAT2 subexpressing cells, correlating with higher membrane damage evidenced by lactate dehydrogenase release. These GPAT2-induced changes are consistent with its proposed function as a tumor-promoting gene, and might be used as a phenotypic differentiation marker. AFM provides the basis for the identification and quantification of those changes, and demonstrates the utility of this technique in the study of cancer cell biology