Dietary epicatechin improves survival and delays skeletal muscle degeneration in aged mice

We recently reported that epicatechin, a bioactive compound that occurs naturally in various common foods, promoted general health and survival of obese diabetic mice. It remains to be determined whether epicatechin extends health span and delays the process of aging. In the present study, epicatechin or its analogue epigallocatechin gallate (EGCG) (0.25% w/v in drinking water) was administered to 20-mo-old male C57BL mice fed a standard chow. The goal was to determine the antiaging effect. The results showed that supplementation with epicatechin for 37 wk strikingly increased the survival rate from 39 to 69%, whereas EGCG had no significant effect. Consistently, epicatechin improved physical activity, delayed degeneration of skeletal muscle (quadriceps), and shifted the profiles of the serum metabolites and skeletal muscle general mRNA expressions in aging mice toward the profiles observed in young mice. In particular, we found that dietary epicatechin significantly reversed age-altered mRNA and protein expressions of extracellular matrix and peroxisome proliferator–activated receptor pathways in skeletal muscle, and reversed the age-induced declines of the nicotinate and nicotinamide pathway both in serum and skeletal muscle. The present study provides evidence that epicatechin supplementation can exert an antiaging effect, including an increase in survival, an attenuation of the aging-related deterioration of skeletal muscles, and a protection against the aging-related decline in nicotinate and nicotinamide metabolism.—Si, H., Wang, X., Zhang, L., Parnell, L. D., Ahmed, B., LeRoith, T., Ansah, T.-A., Zhang, L., Li, J., Ordovás, J. M., Si, H., Liu, D., Lai, C.-Q. Dietary epicatechin improves survival and delays skeletal muscle degeneration in aged mice. FASEB J. 33, 965–977 (2019)

Studies on the role of Ca²⁺ in the pancreatic acinar cell

Plasma membrane-enriched preparations obtained from cultured human skin fibroblasts by differential centrifugation and sucrose density centrifugation techniques were found to contain a Ca²⁺-stimulated ATPase activity. This enzyme was Mg²⁺-dependent and was stimulated by calmodulin and thus was similar to the (Mg²⁺ + Ca²⁺)-ATPase activity observed in other tissues. The specific activity of the (Mg²⁺ + Ca²⁺)-ATPase present was 4-5 fold higher than that present in crude membrane preparations and 80- 100 fold higher than that present in homogenates. The (Mg²⁺ + Ca²⁺)-ATPase activity of both crude membrane and plasma membrane-enriched preparations of cultured fibroblasts from Cystic Fibrosis (CF) patients was significantly reduced when compared to that activity observed in age-matched controls (p < 0.01 in the crude membrane preparations; p < 0.05 in the purified plasma membrane preparations). Reciprocal plots indicated that it is the maximal activation of the enzyme (V[sub Ca²⁺]) and not the affinity of the enzyme system (K[sub diss]) for calcium that is altered in CF strains. In order to determine if this decrease in (Mg²⁺ + Ca²⁺)-ATPase activity in CF was related to a decrease in the ability of these cells to transport calcium, inside-out (10) vesicles were prepared from red cells obtained from CF patients and age-matched controls. Ca²⁺-uptake activity was found to be significantly reduced in the preparations derived from CF patients. This reduction was apparent at all time points studied (10 seconds - 120 minutes) and at all free calcium concentrations used (10-150 μm). Reciprocal plots of the data revealed that the K[sub diss] for calcium of the calcium pump, was similar in control and CF samples but that the V[sub Ca²⁺] was significantly reduced (p < 0.001) in the CF preparations. Calmodulin prepared from red cell hemolysates of controls was found to stimulate Ca²⁺-transport activity to a similar extent in both CF and control samples; it did not, though, return Ca²⁺-uptake activity in CF preparations to control levels. An alteration in calcium transport activity in CF may have a number of implications that may explain some of the manifestations of the disease.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Studies on the role of Ca²⁺ in the pancreatic acinar cell

Calcium ions play an important role in the stimulation of enzyme secretion from the exocrine pancreas. It has been proposed that a secretagogue-induced rise in cytoplasmic free calcium triggers enzyme release from pancreatic acinar cells. The mechanism by which Ca²⁺ can regulate enzyme secretion was investigated using Isolated intact pancreatic acinar cells and plasma membrane-enriched preparations obtained from these cells. The viability of the cells isolated was greater than 95% as evaluated by the exclusion of trypan blue dye. Secretin, pancreozymin and carbachol produced a dose-dependent release of amylase in the acinar cells. The role of the calcium binding protein, calmodulin in enzyme secretion was studied indirectly by determining the effects of calmodulin antagonists on the secretory process. Trifluoperazine inhibited carbachol-stimulated amylase release (ED₅₀ of 10μM). Chlorpromazine at a concentration of 10μM inhibited both carbachol- and pancreozymin-stimulated amylase release to a significant degree without affecting secretin stimulated (non-calcium mediated) release. Propranolol did not show this selective inhibition. These studies indicate the possibility that phenothiazines inhibit calcium-mediated amylase release from pancreatic acinar cells by acting on a calmodulin-regulated step in the stimulus-secretion coupling process. A high ATP hydrolytic activity in the presence of either Mg or Ca was localized in acinar cell preparations enriched in plasma membranes. Kinetic analysis revealed that the enzyme had a significantly higher affinity (p < 0.05) for Ca²⁺ (K[sub d] of 1.73 μM) than Mg²⁺ (K[sub d] of 2.98 μM) but a similar maximal rate of activity. Further studies were carried out in order to determine if this ATPase activity represented one or more enzymes. A comparison of substrate requirements revealed very similar profiles for the Mg²⁺- and Ca²⁺-stimulated activities with ATP and GTP being the most effectivefollowed by ADP. The pH curves for both activities were identical. In addition, combinations of saturating concentrations of Mg²⁺ or Ca²⁺ produced the same degree of maximal activity. These data tend to indicate that the ATPase activity may be due to one enzyme with activation sites for both ions. Arrhenius plots though, revealed a difference in the transition temperatures. In addition, when SDS polyacrylamide gel electrophoresis was used to identify the phosphoprotein intermediates of the ATPase, one phosphoprotein intermediate (approx M.W. of 115,000) was observed in the presence of Ca²⁺ alone (endogenous Mg "r =1.5-2 μM) but upon the addition of both Ca²⁺ and Mg²⁺ , a second phosphoprotein intermediate was revealed (M.W. 130,000). Both of these phosphoproteins were hydroxylamine-sensitive, indicating that they were acyl phosphates. The presence of two acyl phosphoproteins would indicate possible separate intermediate reaction systems for the Mg²⁺ and Ca²⁺ stimulated ATPase activity. Calmodulin is known to regulate a number of enzyme systems including Ca²⁺ -transport ATPases. The effect of exogenous calmodulin on the Ca²⁺ -stimulated component of this ATPase activity was therefore investigated. In the presence of endogenous Mg²⁺ , significant stimulation by calmodulin of Ca²⁺ -ATPase activity was observed. This effect was dose-dependent with a for calmodulin of approximately 0.7 μM. Calmodulin increased the Ca²⁺ -sensitivity of this enzyme system; Mg²⁺ appeared to be required for this effect. This calmodulin stimulation was inhibited by trifluoperazine (ED₅₀ of 30μM), chlorpromazine (ED₅₀ of 55 (μM) and compound 48/80. Using an 125 ¹²⁵I-labeled calmodulin gel overlay technique, it was shown that calmodulin binds in a Ca²⁺ -dependent fashion to 133,000 and 230,000 dalton proteins present in the plasma membrane-enriched fraction. Further studies revealed that under conditions that favour Ca²⁺ -dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000 and a 19,000 dalton protein. The Ca²⁺ -ATPase was also stimulated by acidic phospholipids. Ca²⁺ -flux measurements in plasma membrane vesicles indicated the presence of two processes: there were significantly lower levels of 5 Ca²⁺ associated with vesicles 'loaded' in the presence of ATP than those 'loaded' in the absence of ATP indicating an energy-dependent efflux system. As well, a very rapid ATP-dependent Ca²⁺ -uptake system, that was Mg²⁺ -dependent was observed in sealed vesicles. In order to relate the (Mg²⁺ + Ca²⁺) ATPase activity and the Ca²⁺ fluxes observed to a possible functional role in the acinar cell, the orientation of the catalytic site of the enzyme was determined. The degree of ATPase activity observed when intact viable acinar cells were incubated with ATP and either Mg²⁺ or Ca²⁺ was similar to that observed in broken membranes. This provides evidence that this ATPase activity may be externally-oriented. The role of this system in acinar cell function remains to be elucidated.Pharmaceutical Sciences, Faculty ofGraduat