Joseph W. Starnes

Rapamycin, But Not Resveratrol or Simvastatin, Extends Life Span of Genetically Heterogeneous Mice

Rapamycin was administered in food to genetically heterogeneous mice from the age of 9 months and produced significant increases in life span, including maximum life span, at each of three test sites. Median survival was extended by an average of 10% in males and 18% in females. Rapamycin attenuated age-associated decline in spontaneous activity in males but not in females. Causes of death were similar in control and rapamycin-treated mice. Resveratrol (at 300 and 1200 ppm food) and simvastatin (12 and 120 ppm) did not have significant effects on survival in male or female mice. Further evaluation of rapamycins effects on mice is likely to help delineate the role of the mammalian target of rapamycin complexes in the regulation of aging rate and age-dependent diseases and may help to guide a search for drugs that retard some or all of the diseases of aging.

Acute exercise can improve cardioprotection without increasing heat shock protein content

The aim of this study was to determine the effects of acute bouts of exercise on myocardial recovery after ischemia and heat shock protein expression. Adult female Sprague-Dawley rats were divided into five groups: 1) 1-day run (1DR; n = 6) and 2) 3-day run (3DR; n = 7), in which rats ran for 100 min at a speed of 20 m/min up a 6 degrees grade for 1 or 3 consecutive days; 3) 1-day cold run (1CR), in which rats ran the same as 1DR but with wet fur at 8 degrees C, which prevented an elevation of core temperature (n = 8); 4) heat shock sedentary (HS), in which rats had their core temperatures raised to 42 degrees C one time for 15 min (n = 5); and 5) sedentary control (n=15). Cardiac function was analyzed 24 h after the last treatment using an isolated, working heart model. Nonpaced hearts were initially perfused under normoxic conditions, then underwent 17 min of global, normothermic (37 degrees C) ischemia, and, finally, were allowed to recover for 30 min under normoxic conditions. The concentration of the 72-kDa heat shock protein (HSP 72) was measured in each left ventricle. Compared with that in the sedentary group, recovery of cardiac output x systolic pressure (CO x SP) was enhanced (P < 0.05) in all treatment groups when the postischemic value was covaried with the preischemic value. No differences in CO x SP were found (P > 0.05) between the following groups: 1DR vs. 3DR, 1DR vs. HS, and 1DR vs. 1CR. Heat shock protein concentration was significantly greater (P < 0.05) than that in the sedentary controls in HS, 1DR, and 3DR groups, but not for 1CR. The concentration of HSP 72 was not significantly correlated with postischemic CO x SP (R2 = 0.197, P > 0.05). We conclude that acute bouts of exercise can produce cardioprotective effects without an elevation of HSP 72.The aim of this study was to determine the effects of acute bouts of exercise on myocardial recovery after ischemia and heat shock protein expression. Adult female Sprague-Dawley rats were divided into five groups: 1) 1-day run (1DR; n = 6) and 2) 3-day run (3DR; n = 7), in which rats ran for 100 min at a speed of 20 m/min up a 6° grade for 1 or 3 consecutive days; 3) 1-day cold run (1CR), in which rats ran the same as 1DR but with wet fur at 8°C, which prevented an elevation of core temperature ( n = 8); 4) heat shock sedentary (HS), in which rats had their core temperatures raised to 42°C one time for 15 min ( n = 5); and 5) sedentary control ( n=15). Cardiac function was analyzed 24 h after the last treatment using an isolated, working heart model. Nonpaced hearts were initially perfused under normoxic conditions, then underwent 17 min of global, normothermic (37°C) ischemia, and, finally, were allowed to recover for 30 min under normoxic conditions. The concentration of the 72-kDa heat shock protein (HSP 72) was measured in each left ventricle. Compared with that in the sedentary group, recovery of cardiac output × systolic pressure (CO × SP) was enhanced ( P < 0.05) in all treatment groups when the postischemic value was covaried with the preischemic value. No differences in CO × SP were found ( P > 0.05) between the following groups: 1DR vs. 3DR, 1DR vs. HS, and 1DR vs. 1CR. Heat shock protein concentration was significantly greater ( P < 0.05) than that in the sedentary controls in HS, 1DR, and 3DR groups, but not for 1CR. The concentration of HSP 72 was not significantly correlated with postischemic CO × SP ( R 2 = 0.197, P > 0.05). We conclude that acute bouts of exercise can produce cardioprotective effects without an elevation of HSP 72.

Effect of exercise training on the ability of the rat heart to tolerate hydrogen peroxide

OBJECTIVE The purpose of this study was to determine whether exercise training could precondition the myocardium against hydrogen peroxide (H(2)O(2))-induced damage. METHODS Male Fischer 344 rats ran on a treadmill for 9 weeks (60 min/day, 22 m/min, 6 degrees grade, 5 days/week). Isolated perfused working hearts from exercise trained (ET, n=8) and sedentary (SED, n=10) animals were perfused with 150 microM H(2)O(2). RESULTS Pre-H(2)O(2) baseline values for cardiac external work (COxSP), coronary flow (CF), and lactate dehydrogenase (LDH) release were similar between groups. At 5 min of H(2)O(2), COxSP was unchanged from baseline but CF was increased 30% in SED and 46% in ET (P<0.05 vs. SED). COxSP began to decline similarly thereafter in both groups, dropping to 20% of baseline at 20 min. CF in ET remained higher than SED throughout (P<0.05). LDH leakage remained near baseline during the first 15 min of H(2)O(2) exposure, but was elevated (P<0.05) 72% in SED and 40% in ET after 20 min, and was 2.2-fold greater in SED than ET (P<0.05) after 25 min. Heat shock protein 70 was 2.1-fold greater in ET than SED (P<0.05), but ET did not change catalase and glutathione peroxidase. CONCLUSIONS The results of this study indicate that chronic moderate exercise will enhance coronary flow and attenuate the development of myocardial injury when exposed to H(2)O(2), but will not affect H(2)O(2)-induced decrease in pump function.

Habitual low-intensity exercise does not protect against myocardial dysfunction after ischemia in rats.

Background It is well established that participation in a chronic exercise program can reduce coronary heart disease (CHD) risk factors and improve myocardial tolerance to ischemia-reperfusion (I-R) injury. Low-intensity exercise programs are known to be effective in reducing CHD risk factors in humans and rats, but whether similar programs are of sufficient intensity to improve intrinsic tolerance to I-R injury has not been established. Thus, the purpose of this study is to determine whether low-intensity exercise provides self-protection to the heart against I-R injury. Methods Male, Sprague-Dawley rats were exercised on a treadmill at an intensity of 55–60% VO2max, 40 min/day, 5 days/week for 16 weeks. Reperfusion injury following 20 min of global ischemia was evaluated using the isolated perfused working heart model. Left ventricular content of the cytoprotective protein heat shock protein 70 (HSP70) was determined by Western blotting. Results The exercise program elevated HSP70 2.7-fold, but did not provide enhanced protection following 20 min of ischemia. Final post-ischemic recovery of cardiac external work was 63±9% of pre-ischemic value in the sedentary group (n=9) and 51±11% in the exercising group (n=9) (P>0.05). Post-ischemic lactate dehydrogenase release was also similar between groups and the magnitude of release was low, consistent with stunning. Conclusions Regular exercise at 55–60% VO2max is below the threshold intensity necessary to induce intrinsic cardioprotection against I-R injury. Furthermore, elevated myocardial HSP70 is not necessarily a marker of improved protection against dysfunction associated with stunning. Eur J Cardiovasc Prev Rehabil 12: 169–174

Conjugated linoleic acid reduces adiposity and increases markers of browning and inflammation in white adipose tissue of mice

The objective of this study was to examine the mechanism by which conjugated linoleic acid (CLA) reduces body fat. Young male mice were fed three combinations of fatty acids at three doses (0.06%, 0.2%, and 0.6%, w/w) incorporated into AIN76 diets for 7 weeks. The types of fatty acids were linoleic acid (control), an equal mixture of trans-10, cis-12 (10,12) CLA plus linoleic acid, and an equal isomer mixture of 10,12 plus cis-9, trans-11 (9,11) CLA. Mice receiving the 0.2% and 0.6% dose of 10,12 CLA plus linoleic acid or the CLA isomer mixture had decreased white adipose tissue (WAT) and brown adipose tissue (BAT) mass and increased incorporation of CLA isomers in epididymal WAT and liver. Notably, in mice receiving 0.2% of both CLA treatments, the mRNA levels of genes associated with browning, including uncoupling protein 1 (UCP1), UCP1 protein levels, and cytochrome c oxidase activity, were increased in epididymal WAT. CLA-induced browning in WAT was accompanied by increases in mRNA levels of markers of inflammation. Muscle cytochrome c oxidase activity and BAT UCP1 protein levels were not affected by CLA treatment. These data suggest a linkage between decreased adiposity, browning in WAT, and low-grade inflammation due to consumption of 10,12 CLA.

Effects of Acute, Submaximal Exercise on Skeletal Muscle Vitamin E

Vitamin E is the major lipid soluble anti-oxidant and may play an important protective role against free radicals produced during exercise. The purpose of this study was to determine the effect of a submaximal exercise bout on vitamin E levels in selected tissues. Five week- old lean, female Zucker rats were randomly divided into sedentary and run groups. At least 4 days following a maximal VO2 test, the run group (n = 7) ran on a treadmill at 70.3 ± 1.5% VO2 max for 34-42 minutes. Duration was varied according to body weight to keep total work constant. Immediately post-exercise, animals were decapitated, exsanguinated and the quadriceps (red and white vastus lateralis), liver and heart quickly excised and stored under liquid nitrogen until analyzed. Lipids were extracted in heptane and alpha-tocopherol levels determined by reverse-phase HPLC with electrochemical detection. Quadriceps vitamin-E levels declined post-exercise 30% compared to sedentary controls. Specifically, in the red quadriceps from 37 ±...

Oxygen radical injury and loss of high-energy compounds in anoxic and reperfused rat heart: Prevention by exogenous fructose-1,6-bisphosphate

Isolated Langendorff-perfused rat hearts after 10 minutes preperfusion, were subjected to a substrate-free anoxic perfusion (20 minutes) followed by 20 minutes reperfusion with a glucose-containing oxygen-balanced medium. Under the same perfusion conditions, the effect of exogenous 5mM fructose-1,6-bisphosphate has been investigated. The xanthine dehydrogenase to xanthine oxidase ratio, concentrations of high-energy phosphates and of TBA-reactive material (TBARS) were determined at the end of each perfusion period in both control and fructose-1,6-bisphosphate-treated hearts. Results indicate that anoxia induces the irreversible transformation of xanthine dehydrogenase into oxidase as a consequence of the sharp decrease of the myocardial energy metabolism. This finding is supported by the protective effect exerted by exogenous fructose-1,6-bisphosphate which is able to maintain the correct xanthine dehydrogenase/oxidase ratio by preventing the depletion of phosphorylated compounds during anoxia. Moreover, in control hearts, the release of lactate dehydrogenase during reperfusion, is paralleled by a 50% increase in the concentration of tissue TBARS. On the contrary, in fructose-1,6-bisphosphate-treated hearts this concentration does not significantly change after reoxygenation, while a slight but significant increase of lactate dehydrogenase activity in the perfusates is observed. On the whole these data indicate a direct contribution of oxygen-derived free radicals to the worsening of post-anoxic hearts. A hypothesis on the mechanism of action of fructose-1,6-bisphosphate in anoxic and reperfused rat heart and its possible application in the clinical therapy of myocardial infarction are presented.

Exogenous fructose-1,6-bisphosphate is a metabolizable substrate for the isolated normoxic rat heart.

SummaryIsolated rat hearts were perfused by the recirculating Langendorff mode under normoxic conditions for 60 min. The Krebs-Ringer buffer was supplemented with 10mM glucose+12 IU/l insulin and either [U-14C]-fructose-1,6-bisphosphate (together with 5 mM cold fructose-1,6-bisphosphate) or [U-14C]-fructose (together with 5 mM cold fructose). At the end of perfusion, gaseous14CO2,14CO2 trapped in the perfusates,14C-lactate output and tissue14C-lactate were assayed in both groups of hearts. Analysis of high-energy compounds, glycogen, lactate, and pyruvate was also performed on the neutralized perchloric acid extracts of the freeze-clamped hearts. Data obtained from the14C catabolites, originating from the metabolism of the radiolabeled substrates, indicated that the isolated normoxic rat heart metabolizes an 8.5 times higher amount of fructose-1,6-bisphosphate (7.07 μmoles/min/g d. w.) than of fructose (0.83 μmoles/min/g d.w.). CrP, CrP/Cr, glycogen, and total lactate in both tissue and perfusate were significantly higher in fructose-1,6-bisphosphate-perfused hearts. The overall indication is that fructose-1,6-bisphosphate can be taken up in its intact form by myocytes and successively metabolized to support their energy demand, and that its effects on myocardial performance and metabolism should be attributed to the molecule itself rather than to its eventual degradation products.

Bioenergetics in the aging fischer 344 rat: Effects of exercise and food restriction

The capacity for energy production was evaluated in male, Fischer 344 rats as they advanced from adulthood through senescence. At 10 months of age, the animals were divided into three groups: sedentary, fed ad libitum (S); exercised by treadmill running, fed ad libitum (E); and sedentary, caloric restricted by alternate day feeding (R). Activities of selected enzymes, ADP-stimulated respiration and levels of cytochromes, were determined in homogenates of liver and gastrocnemius muscle prepared from young controls (10-month old S) and 18-, 24-, and 30-month old animals. In liver, age-linked decrements were found in the activities of 3-hydroxyacyl-CoA dehydrogenase (S, E, and R) and citrate synthase (S), and in cytochrome c content (S and E), whereas substrate-catalysed oxidations were unaffected. In the gastrocnemius muscle (S, E, and R), respiration, activities of enzymes of the Krebs cycle and glycolysis, and cytochrome content were decreased after the age of 18 months. Oxidative capacity was increased in muscle through exercise (about 40%) and in liver by food restriction (about 20%). Body and soleus muscle mass declined similarly in all groups (about 14% from 30 to 18 months of age), whereas the loss of weight in the gastrocnemius muscle was much greater (34%). The data indicate that energy metabolism in the senescent animal is competent to meet its needs and age-related declines in energy metabolism are secondary to the aging process.

Myocardial injury after hypoxia in immature, adult and aged rats

We evaluated the abilities of isolated perfused hearts from immature (IM) (2.5–3 months), ADULT (11–13 months) and OLD (24–26 months) Fischer 344 rats to tolerate and recover from oxygen deprivation. Hearts were perfused at 60 mmHg for a 30-minute prehypoxic period with oxygenated buffer supplemented with 10 mM glucose (+insulin) and 2 mM acetate, then 30 minutes with substrate-free, hypoxic buffer gassed with 95% N2:5% CO2, and finally reoxygenated for an additional 45 minutes with the same buffer used during the prehypoxic period. During prehypoxia, all groups were similar in ventricular mechanical function, glycogen content, high-energy phosphates (HEP), reduced glutathione (GSH), Ca++ content, and mitochondrial state 3 rates. At the end of the hypoxic period, glycogen levels were similar and almost completely depleted in all groups, HEP were lower (p<0.05) in ADULT vs other groups, mitochondrial state 3 rates were decreased (24%, p<0.05) only in ADULT, and GSH was depleted by 34% in IM vs only 13% in OLD (p<0.05). After 45 minutes of reoxygenation, IM and OLD had recovered 48% and 45% of their respective prehypoxic function which was two-fold greater than the 23% recovery by ADULT. Loss of cytosolic enzymes, an indicator of sarcolemmal damage, was estimated by measuring lactate dehydrogenase (LDH) release. LDH release and Ca++ content during reoxygenation in IM were only about half of that observed in ADULT or OLD. We conclude that immature and aged hearts tolerate and recover from hypoxia better than hearts from adults, and that the sarcolemmal membranes of immature rat hearts are less susceptible to damage from hypoxic stress than those of either older group.