Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities.

Caveolae organelles and caveolin-1 protein expression are most abundant in adipocytes and endothelial cells. Our initial report on mice lacking caveolin-1 (Cav-1) demonstrated a loss of caveolae and perturbations in endothelial cell function. More recently, however, observation of the Cav-1-deficient cohorts into old age revealed significantly lower body weights, as compared with wild-type controls. These results suggest that Cav-1 null mice may have problems with lipid metabolism and/or adipocyte functioning. To test this hypothesis directly, we placed a cohort of wild-type and Cav-1 null mice on a high fat diet. Interestingly, despite being hyperphagic, Cav-1 null mice show overt resistance to diet-induced obesity. As predicted, adipocytes from Cav-1 null null mice lack caveolae membranes. Early on, a lack of caveolin-1 selectively affects only the female mammary gland fat pad and results in a near complete ablation of the hypo-dermal fat layer. There are also indications of generalized adipose tissue pathology. With increasing age, a systemic decompensation in lipid accumulation occurs resulting in dramatically smaller fat pads, histologically reduced adipocyte cell diameter, and a poorly differentiated/hypercellular white adipose parenchyma. To gain mechanistic insights into this phenotype, we show that, although serum insulin, glucose, and cholesterol levels are entirely normal, Cav-1 null mice have severely elevated triglyceride and free fatty acid levels, especially in the post-prandial state. However, this build-up of triglyceride-rich chylomicrons/very low density lipoproteins is not due to perturbed lipoprotein lipase activity, a major culprit of isolated hypertriglyceridemia. The lean body phenotype and metabolic defects observed in Cav-1 null mice are consistent with the previously proposed functions of caveolin-1 and caveolae in adipocytes. Our results show for the first time a clear role for caveolins in systemic lipid homeostasis in vivo and place caveolin-1/caveolae as major factors in hyperlipidemias and obesity.     

A new class of potent, slowly reversible dehydropeptidase inhibitors

Dehydrodipeptide analogs whose scissile carboxamide has been replaced with a PO(OH)CH2 group have been found to be potent inhibitors of the zinc protease dehydrodipeptidase 1 (DHP-1, renal dipeptidase, EC 3.4.13.11). The best of these inhibitors, compound 25 (Ki = 0.52 nM), is two hundred times more potent than cilastatin 2 which is used clinically as a component of the broad-spectrum antibiotic combination Primaxin. Compound 25 is a tight binding inhibitor exhibiting slow binding kinetics with a remarkably slow off rate from DHP-1 (half life greater than 8 hours). The kinetics of its binding are consistent with a simple on-off mechanism whereas the less active D-enantiomer 26 appears to bind in an initial loose complex with the enzyme which slowly rearranges to a tighter complex (Ki = 83 nM).     

Occurrence of human-associated yeasts in bivalve shellfish from Long Island Sound.

Candida parapsilosis, C. tropicalis, and Torulopsis glabrata were the human-associated yeasts most frequently isolated from quahogs, oysters, and mussels collected from four estuarine areas along the northern shore of Long Island Sound. Some inconsistency and seasonal variation in the occurrence of these and other yeast species were noted. In particular, C. albicans densities were greatest during colder months in the more heavily polluted waters. A total of 347 yeasts were isolated and cultured at 37 degrees C and, of these, 219 of 62% were human-associated forms. Generally, these yeasts in the animals sampled reflected the overall pollution status of the estuary from which they were taken. This study represents a clear demonstration of potentially pathogenic yeasts in a valuable marine resource.     

Normalized metabolic stress for 31P-MR spectroscopy studies of human skeletal muscle: MVC vs. muscle volume

Fowler, M. D., T. W. Ryschon, R. E. Wysong, C. A. Combs, andR. S. Balaban. Normalized metabolic stress for³¹P-MR spectroscopy studies ofhuman skeletal muscle: MVC vs. muscle volume. J. Appl.Physiol. 83(3): 875-883, 1997.A criticalrequirement of submaximal exercise tests is the comparability ofworkload and associated metabolic stress between subjects. In thisstudy, ³¹P-magnetic resonancespectroscopy was used to estimate metabolic strain in the soleus muscleduring dynamic, submaximal plantar flexion in which target torque was10 and 15% of a maximal voluntary contraction (MVC). In 10 healthy,normally active adults, (PCr + P_i)/PCr, where PCr isphosphocreatine, was highly correlated with power output normalized tothe volume of muscle in the plantar flexor compartment(r = 0.89, P < 0.001). The same variable was also correlated, although less strongly(r = 0.78, P < 0.001), with power normalized toplantar flexor cross-sectional area. These findings suggest thatcomparable levels of metabolic strain can be obtained in subjects ofdifferent size when the power output, or stress, for dynamic plantarflexion is selected as a function of plantar flexor muscle volume. Incontrast, selecting power output as a function of MVC resulted in apositive linear relationship between (PCr + P_i)/PCr and thetorque produced, indicating that metabolic strain was increasing ratherthan achieving constancy as a function of MVC. These findings providenew insight into the design of dynamic muscle contraction protocolsaimed at detecting metabolic differences between subjects of differentbody size but having similar blood flow capacity and mitochondrialvolume per unit of muscle.

     

Effects of Oxidation on Neurofibrillar Argyrophilia

The effect of oxidation on neurofibrillar argyrophilia was studied by subjecting nervous tissues containing both normal and degenerating fibers to the action of potassium permanganate, periodic acid, chromic acid, lead tetraacetate, and sodium bismuthate prior to silver impregnation. The argyrophilic response of normal fibers to such treatment was studied with the Nonidez silver nitrate block technic, the double impregnation method of Bielschowsky on both blocks and sections, and a silver proteinate procedure. The response of degenerating fibers was studied by the Cajal formula 6 block technic and the modified Bielschowsky procedure of Nauta and Ryan for sections. The experimental data indicated that such oxidation did not produce any differential staining effects between normal or degenerating fibers.     

Mechanism of Inhibition of beta-site amyloid precursor protein-cleaving enzyme (BACE) by a statine-based peptide

Inhibition of beta-site amyloid precursor protein-cleaving enzyme by a statine-based inhibitor has been studied using steady state and stopped-flow methods. A slow onset rate of inhibition has been observed under steady state conditions, and a K(i) of 22 nm has been derived using progress curves analysis. Simulation of stopped-flow protein fluorescence transients provided an estimate of the K(d) for initial inhibitor binding of 660 nm. A two-step inhibition mechanism is proposed, wherein slower "tightening up" of the initial encounter complex occurs. Two hypotheses have been proposed in the literature to address the nature of the slow step in the inhibition of aspartic proteases by peptidomimetic inhibitors: a conformational change related to the "flap" movement and displacement of a catalytic water. We compared substrate and inhibitor binding rates under pre-steady-state conditions. Both ligands are likely to cause flap movement, whereas no catalytic water replacement occurs during substrate binding. Our results suggest that both ligands bind to the enzyme at a rate significantly lower than the diffusion limit, but there are additional rate limitations involved in inhibitor binding, resulting in a k(on) of 3.5 x 10(4) m(-)1 s(-)1 for the inhibitor compared with 3.5 x 10(5) m(-)1 s(-)1 for the substrate. Even though specific intermediate formation steps might be different in the productive inhibitor and substrate binding to beta-site amyloid precursor protein-cleaving enzyme, a similar final optimized conformation is achieved in both cases, as judged by the comparable free energy changes (DeltaDeltaG of 2.01 versus 1.97 kcal/mol) going from the initial to the final enzyme-inhibitor or enzyme-substrate complexes.     

Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue

Several lines of evidence suggest that a functional relationship exists between caveolin-1 and insulin signaling. However, it remains unknown whether caveolin-1 is normally required for proper insulin receptor signaling in vivo. To address this issue, we examined the status of insulin receptor signaling in caveolin-1 (–/–)-deficient (Cav-1 null) mice. Here, we show that Cav-1 null mice placed on a high-fat diet for 9 mo develop postprandial hyperinsulinemia. An insulin tolerance test (ITT) revealed that young Cav-1 null mice on a normal chow diet are significantly unresponsive to insulin, compared with their wild-type counterparts. This insulin resistance is due to a primary defect in adipose tissue, as evidenced by drastically reduced insulin receptor protein levels (>90%), without any changes in insulin receptor mRNA levels. These data suggest that caveolin-1 acts as a molecular chaperone that is necessary for the proper stabilization of the insulin receptor in adipocytes in vivo. In support of this notion, we demonstrate that recombinant expression of caveolin-1 in Cav-1 null mouse embryo fibroblasts rescues insulin receptor protein expression. These data provide evidence that the lean body phenotype observed in the Cav-1 knockout mice is due, at least in part, to a defect in insulin-regulated lipogenesis. caveolae; caveolin; insulin signaling; protein stabilization; knockout mice