Phosphorylation of AS160-serine 704 is not essential for exercise-increase in insulin-stimulated glucose uptake by skeletal muscles from female or male rats.

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle from rodents and humans of both sexes. We recently found that concurrent mutation of three key sites to prevent their phosphorylation (Ser588, Thr642, and Ser704) on Akt substrate of 160 kDa (AS160; also known as TBC1D4) reduced the magnitude of the enhancement of postexercise ISGU (PEX-ISGU) by muscle from male, but not female rats. However, we did not test the role of individual phosphorylation sites on PEX-ISGU. Accordingly, our current aim was to test whether AS160 Ser704 phosphorylation (pSer704) is required for elevated PEX-ISGU by muscle. AS160-knockout (AS160-KO) rats (female and male) were studied when either in sedentary or 3 h after acute exercise. Adeno-associated virus (AAV) vectors were used to enable muscle expression of wild-type AS160 (AAV-WT-AS160) or AS160 mutated Ser704 to alanine to prevent phosphorylation (AAV-1P-AS160). Paired epitrochlearis muscles from each rat were injected with AAV-WT-AS160 or AAV-1P-AS160. We discovered that regardless of sex 1) AS160 abundance in AS160-KO rats was similar in paired muscles expressing WT-AS160 versus 1P-AS160; 2) muscles from exercised versus sedentary rats had greater ISGU, and PEX-ISGU was slightly greater for muscles expressing 1P-AS160 versus contralateral muscles expressing WT-AS160; and 3) pAS160Thr642 was lower in muscles expressing 1P-AS160 versus paired muscles expressing WT-AS160. These results indicate that pAS160Ser704 was not essential for elevated PEX-ISGU by skeletal muscle from rats of either sex. Furthermore, elimination of the postexercise increase in pAS160Thr642 did not lessen the postexercise effect on ISGU.NEW & NOTEWORTHY The current study evaluated the role of Akt substrate of 160 kDa (AS160) phosphorylation on Ser704 in increased insulin-stimulated glucose uptake by skeletal muscle after exercise. Adeno-associated virus vectors were engineered to express either wild-type-AS160 or AS160 mutated so that it could not be phosphorylated on Ser704 in paired muscles from AS160-knockout rats. The results demonstrated that AS160 phosphorylation on Ser704 was not essential for exercise-induced elevation in insulin-stimulated glucose uptake by rats of either sex.

A novel genetic model provides a unique perspective on the relationship between postexercise glycogen concentration and increases in the abundance of key metabolic proteins after acute exercise.

Some acute exercise effects are influenced by postexercise (PEX) diet, and these diet-effects are attributed to differential glycogen resynthesis. However, this idea is challenging to test rigorously. Therefore, we devised a novel genetic model to modify muscle glycogen synthase 1 (GS1) expression in rat skeletal muscle with an adeno-associated virus (AAV) short hairpin RNA knockdown vector targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Muscles from exercised (2-hour-swim) and time-matched sedentary (Sed) rats were collected immediately postexercise (IPEX), 5-hours-PEX (5hPEX), or 9-hours-PEX (9hPEX). Rats in 5hPEX and 9hPEX experiments were refed (RF) or not-refed (NRF) chow. Muscles were analyzed for glycogen, abundance of metabolic proteins (pyruvate dehydrogenase kinase 4, PDK4; peroxisome proliferator-activated receptor γ coactivator-1α, PGC1α; hexokinase II, HKII; glucose transporter 4, GLUT4), AMP-activated protein kinase phosphorylation (pAMPK), and glycogen metabolism-related enzymes (glycogen phosphorylase, PYGM; glycogen debranching enzyme, AGL; glycogen branching enzyme, GBE1). shRNA-GS1 versus paired shRNA-Scr muscles had markedly lower GS1 abundance. IPEX versus Sed rats had lower glycogen and greater pAMPK, and neither of these IPEX-values differed for shRNA-GS1 versus paired shRNA-Scr muscles. IPEX versus Sed groups did not differ for abundance of metabolic proteins, regardless of GS1 knockdown. Glycogen in RF-rats was lower for shRNA-GS1 versus paired shRNA-Scr muscles at both 5hPEX and 9hPEX. HKII protein abundance was greater for 5hPEX versus Sed groups, regardless of GS1 knockdown or diet, and despite differing glycogen levels. At 9hPEX, shRNA-GS1 versus paired shRNA-Scr muscles had greater PDK4 and PGC1α abundance within each diet group. However, the magnitude of PDK4 or PGC1α changes was similar in each diet group regardless of GS1 knockdown although glycogen differed between paired muscles only in RF-rats. In summary, we established a novel genetic approach to investigate the relationship between muscle glycogen and other exercise effects. Our results suggest that exercise-effects on abundance of several metabolic proteins did not uniformly correspond to differences in postexercise glycogen.

AS160 expression, but not AS160 Serine-588, Threonine-642, and Serine-704 phosphorylation, is essential for elevated insulin-stimulated glucose uptake by skeletal muscle from female rats after acute exercise.

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle in both sexes. We recently found that muscle expression and phosphorylation of key sites of Akt substrate of 160 kDa (AS160; also called TBC1D4) are essential for the full-exercise effect on postexercise-ISGU (PEX-ISGU) in male rats. In striking contrast, AS160's role in increased PEX-ISGU has not been rigorously tested in females. Our rationale was to address this major knowledge gap. Wild-type (WT) and AS160-knockout (KO) rats were either sedentary or acutely exercised. Adeno-associated virus (AAV) vectors were engineered to express either WT-AS160 or AS160 mutated on key serine and threonine residues (Ser588, Thr642, and Ser704) to alanine to prevent their phosphorylation. AAV vectors were delivered to the muscle of AS160-KO rats to determine if WT-AS160 or phosphorylation-inactivated AS160 would influence PEX-ISGU. AS160-KO rats have lower skeletal muscle abundance of the GLUT4 glucose transporter protein. This GLUT4 deficit was rescued using AAV delivery of GLUT4 to determine if eliminating muscle GLUT4 deficiency would normalize PEX-ISGU. The novel results were as follows: (1) AS160 expression was required for greater PEX-ISGU; (2) rescuing muscle AS160 expression in AS160-KO rats restored elevated PEX-ISGU; (3) AS160's essential role for the postexercise increase in ISGU was not attributable to reduced muscle GLUT4 content; and (4) AS160 phosphorylation on Ser588, Thr642, and Ser704 was not essential for greater PEX-ISGU. In conclusion, these novel findings revealed that three phosphosites widely proposed to influence PEX-ISGU are not required for this important outcome in female rats.

Greater Phosphorylation of AMPK and Multiple AMPK Substrates in the Skeletal Muscle of 24-Month-Old Calorie Restricted Compared to Ad-Libitum Fed Male Rats.

AMP-activated protein kinase (AMPK), a highly conserved, heterotrimeric serine/threonine kinase with critical sensory and regulatory functions, is proposed to induce antiaging actions of caloric restriction (CR). Although earlier studies assessed CR's effects on AMPK in rodent skeletal muscle, the scope of these studies was narrow with a limited focus on older animals. This study's purpose was to fill important knowledge gaps related to CR's influence on AMPK in skeletal muscle of older animals. Therefore, using epitrochlearis muscles from 24-month-old ad-libitum fed (AL) and CR (consuming 65% of AL intake for 8 weeks), male Fischer-344 × Brown Norway F1 rats, we determined: (a) AMPK Thr172 phosphorylation (a key regulatory site) by immunoblot; (b) AMPKα1 and AMPKα2 activity (representing the 2 catalytic α-subunits of AMPK), and AMPKγ3 activity (representing AMPK complexes that include the skeletal muscle-selective regulatory γ3 subunit) using enzymatic assays; (c) phosphorylation of multiple protein substrates that are linked to CR-related effects (acetyl-CoA carboxylase [ACC], that regulates lipid oxidation; Beclin-1 and ULK1 that are autophagy regulatory proteins; Raptor, mTORC1 complex protein that regulates autophagy; TBC1D1 and TBC1D4 that regulate glucose uptake) by immunoblot; and (d) ATP and AMP concentrations (key AMPK regulators) by mass spectrometry. The results revealed significant CR-associated increases in the phosphorylation of AMPKThr172 and 4 AMPK substrates (ACC, Beclin-1, TBC1D1, and TBC1D4), without significant diet-related differences in ATP or AMP concentration or AMPKα1-, AMPKα2-, or AMPKγ3-associated activity. The enhanced phosphorylation of multiple AMPK substrates provides novel mechanistic insights linking AMPK to functionally important consequences of CR.

Exercise-Induced Improvement in Insulin-Stimulated Glucose Uptake by Rat Skeletal Muscle Is Absent in Male AS160-Knockout Rats, Partially Restored by Muscle Expression of Phosphomutated AS160, and Fully Restored by Muscle Expression of Wild-Type AS160.

One exercise session can elevate insulin-stimulated glucose uptake (ISGU) in skeletal muscle, but the mechanisms remain elusive. Circumstantial evidence suggests a role for Akt substrate of 160 kDa (AS160 or TBC1D4). We used genetic approaches to rigorously test this idea. The initial experiment evaluated the role of AS160 in postexercise increase in ISGU using muscles from male wild-type (WT) and AS160-knockout (KO) rats. The next experiment used AS160-KO rats with an adeno-associated virus (AAV) approach to determine if rescuing muscle AS160 deficiency could restore the ability of exercise to improve ISGU. The third experiment tested if eliminating the muscle GLUT4 deficit in AS160-KO rats via AAV-delivered GLUT4 would enable postexercise enhancement of ISGU. The final experiment used AS160-KO rats and AAV delivery of AS160 mutated to prevent phosphorylation of Ser588, Thr642, and Ser704 to evaluate their role in postexercise ISGU. We discovered the following: 1) AS160 expression was essential for postexercise increase in ISGU; 2) rescuing muscle AS160 expression of AS160-KO rats restored postexercise enhancement of ISGU; 3) restoring GLUT4 expression in AS160-KO muscle did not rescue the postexercise increase in ISGU; and 4) although AS160 phosphorylation on three key sites was not required for postexercise elevation in ISGU, it was essential for the full exercise effect.

The Effects of Exercise and Restriction of Sugar-Sweetened Beverages on Muscle Function and Autophagy Regulation in High-Fat High-Sucrose-Fed Obesity Mice.

BACKGROUND: Autophagy maintains muscle mass and healthy skeletal muscles. Several recent studies have associated sugar-sweetened beverage (SSB) consumption with diseases. We investigated whether muscle dysfunction due to obesity could be restored by SSB restriction (SR) alone or in combination with exercise (EX) training. METHODS: Obese mice were subjected to SR combined with treadmill EX. Intraperitoneal glucose tolerance test, grip strength test, hanging time test, and body composition analysis were performed. Triglyceride (TG) and total cholesterol (TC) serum concentrations and TG concentrations in quadriceps muscles were analyzed. Western blot and reverse transcription-quantitative polymerase chain reaction helped analyze autophagy-related protein and mRNA expression, respectively. RESULTS: SR alone had no significant effect on fasting blood glucose levels, glucose tolerance, and muscle function. However, it had effect on serum TC, serum TG, and BCL2 interacting protein 3 expression. SR+EX improved glucose tolerance and muscle function and increased serum TC utilization than SR alone. SR+EX reduced P62 levels, increased glucose transporter type 4 and peroxisome proliferator-activated receptor γ coactivator-1α protein expression, and improved grip strength relative to the high-fat and high-sucrose liquid (HFHS) group, and this was not observed in the HFHS+EX group. CONCLUSION: SR induced mitophagy-related protein expression in quadriceps, without affecting muscle function. And, the combination of SR and EX activated mitophagy-related proteins and improved muscle function.

Effects of Exercise and a High-Fat, High-Sucrose Restriction Diet on Metabolic Indicators, Nr4a3, and Mitochondria-Associated Protein Expression in the Gastrocnemius Muscles of Mice with Diet-Induced Obesity.

BACKGROUND: Exercise and high fat, high sucrose restriction diets are well known treatments for obesity. The aim of this study was to measure the effects of those lifestyle interventions on molecular transducers of exercise, such as Nr4a3, mitochondria-associated proteins, and muscle function. METHODS: We conducted 8 weeks of treadmill exercise and sucrose or fat restriction diets in obese mice. The mice were divided into eight groups: the normal diet (CON) group, normal diet with exercise (CONEX) group, high fat, high sucrose diet (HFHS) group, HFHS with exercise (HFHSEX) group, sucrose restriction (SR) group, SR with exercise (SREX) group, high fat, high sucrose restriction (ND) group, and ND with exercise (NDEX) group. RESULTS: The 8 weeks of exercise reduced body weight, improved lipid profiles (total cholesterol, triglycerides), and increased hanging time. The combination of exercise and a fat and sucrose restriction diet improved glucose tolerance and increased grip strength. The 8 weeks of intervention did not significantly affect the Nr4a3 protein level. The sucrose and fat restriction diet increased the phosphorylated protein kinase B (pAkt)/Akt ratio, and its level was lower in the HFHS group. Exercise increased the protein expression level of PGC-1α in obese conditions. Moreover, SR led reduced the phosphorylated AMP-activated protein kinase (pAMPK)/AMPK ratio and PGC-1α to the control level. CONCLUSION: The 8 weeks of exercise or a sucrose and fat restriction diet improved metabolic indicators and muscle function. SR reduced pAMPK/AMPK and PGC-1α to the control level. Nr4a3 protein expression was not significantly changed by either exercise or a fat and sucrose restriction diet.

Effects of exercise-induced beta-hydroxybutyrate on muscle function and cognitive function.

Recent studies have shown that exercise improves skeletal muscle and cognitive function by stimulating the secretion of numerous molecules. In particular, previous studies have suggested that exercise-induced beta-hydroxybutyrate (BHB) release might improve skeletal muscle and cognitive function, but to date these studies have been limited to cell and animal models. Therefore, we aimed to determine how an exercise-induced increase in BHB affects skeletal muscle and cognitive function at a cellular level, in an animal model, and in humans. The effects of BHB on skeletal muscle and cognitive function were determined by treating C2C12 and C6 cell lines with BHB, and by measuring the skeletal muscle and serum BHB concentrations in aged mice after endurance or resistance exercise. In addition, serum BHB concentration was measured before and after high-speed band exercise in elderly people, and its relationships with muscle and cognitive function were analyzed. We found that BHB increased cell viability and brain-derived neurotrophic factor expression level in C6 cells, and endurance exercise, but not resistance exercise, increased the muscle BHB concentration in aged mice. Furthermore, the BHB concentration was positively related to skeletal muscle and cognitive function. Exercise did not increase the serum BHB concentration in the elderly people and BHB did not correlate with cognitive function, but after excluding the five people with the highest preexisting serum concentrations of BHB, the BHB concentrations of the remaining participants were increased by exercise, and the concentration showed a tendency toward a positive correlation with cognitive function. Thus, the BHB released by skeletal muscle following endurance exercise may improve muscle and cognitive function in animals and humans.

Exercise reduces metabolic burden while altering the immune system in aged mice.

Although several evidence has suggested the impact of exercise on the prevention of aging phenotypes, few studies have been conducted on the mechanism by which exercise alters the immune-cell profile, thereby improving metabolism in senile obesity. In this study, we confirmed that 4-week treadmill exercise sufficiently improved metabolic function, including increased lean mass and decreased fat mass, in 88-week-old mice. The expression level of the senescence marker p16 in the white adipose tissue (WAT) was decreased after 4-weeks of exercise. Exercise induced changes in the profiles of immune-cell subsets, including natural killer (NK) cells, central memory CD8+ T cells, eosinophils, and neutrophils, in the stromal vascular fraction of WAT. In addition, it has been shown through transcriptome analysis of WAT that exercise can activate pathways involved in the interaction between WAT and immune cells, in particular NK cells, in aged mice. These results suggest that exercise has a profound effect on changes in immune-cell distribution and senescent-cell scavenging in WAT of aged mice, eventually affecting overall energy metabolism toward a more youthful state.

Effects of treadmill exercise on the regulation of tight junction proteins in aged mice.

Tight junction protein is representative regulator of gut permeability. Also, it has been noted for controlling inflammatory responses through tight junction. Therefore, in this study, we examined that whether tight junction protein is changed in aged mice, and to further, confirmed the effect of treadmill exercise on the tight junction protein. In in vitro study, doxorubicin that induces cell senescence was treated to Caco2 cells (colon cell) to mimic aging effect. After that, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), exercise mimic chemical that stimulates AMPK level, was also administered to Caco2 cells. In animal study, 2 months and 21 months C57BL/6 J mouse were treated with treadmill exercise for 4 weeks (YE = 5, OE = 5). Then, the tight junction protein expression level was examined by western blot. Also, serum lipopolysaccharide (LPS) and zonulin level were analyzed to identify gut permeability. In vitro studies showed that doxorubicin downregulates tight junction protein expression levels in Caco2 cell, and also AICAR treatment upregulates tight junction protein expression levels. In animal study, 4 weeks treadmill exercise upregulated claudin-1 (p < 0.05) and occludin (p < 0.01) protein expression level in 21 months old mice. Also, zonula occluden-1 (ZO-1) protein expression level was not significant difference among all mice group. In addition, old mice group had higher level of serum LPS compared to young mice group, but the level was downregulated in both 2 months and 21 months mice group after four weeks of treadmill exercise. Zonulin, which is known as degrading tight junction protein, is not significantly changed by both age and exercise. This study compared that tight junction protein expression level in old mice compared to its level in young mice, and also clarified that the effect of treadmill exercise on tight junction protein in both young and old mice.

Does exercise-induced apelin affect sarcopenia? A systematic review and meta-analysis.

PURPOSE/OBJECTIVE: There have been a number of studies on the role of the novel protein apelin, identified in 1998, in improving muscular function and structure in various human organs, as well as on how it is involved in pathological processes. The aim of this systematic review and meta-analysis was to determine the effect of exercise on serum apelin levels to provide up-to-date data for the development of an exercise intervention for older adults. METHODS: We searched for articles in PubMed, Ovid MEDLINE, and EMBASE from database inception to May 31, 2019. To conduct a meta-analysis of the primary outcome (serum apelin level), we analyzed intervention effect sizes of the differences between the exercise group and control groups for the primary outcome measure at post-treatment. The outcomes were analyzed using Hedge's statistic effect size (Zr) for weight mean difference (WMD) from various statistical results, including t, F, x2, and r. A heterogeneity test was conducted using Higgin's I2 statistic and Q statistics (p > 0.10) via a forest plot. A fixed-effect model was considered if Higgin's I2 was less than 50%. If heterogeneity was high (I2 > 50%), a random-effects model with a subgroup analysis or meta-regression was used. A meta-analysis using nine studies showed that exercise could increase serum apelin levels, which was beneficial for such metabolic diseases as diabetes. RESULTS: In the subgroup analysis, the 50-60-year-old group showed significant effects of exercise. However, the BMI (normal, overweight, and obesity) categories failed to show any difference in exercise-induced effect. CONCLUSION: Further studies are needed to clarify in greater detail the effect of resistance exercise on apelin levels, including data on frequency, type, intensity, and time of aerobic exercise, to compare their effects on sarcopenia and cognitive disorders.

Effect of a Single Bout of Exercise on Autophagy Regulation in Skeletal Muscle of High-Fat High-Sucrose Diet-Fed Mice.

BACKGROUND: Autophagy maintains metabolic homeostasis of muscles, and its impairment may cause muscle dysfunction. Exercise can improve muscle dysfunction induced by long-term high-fat diet. This study aimed to explore the association of autophagy with impaired muscle dysfunction in obese conditions and investigate its relationship with exercise-induced muscle function improvement. METHODS: Male C57BL/6 mice (n=24) were randomly assigned to four groups: low-fat diet+plain water feeding sedentary (CON) group, low-fat diet+plain water feeding exercise (CON+EX) group, high-fat high-sucrose (HFHS) diet-fed sedentary group, and HFHS diet-fed exercise (HFHS+EX) group, and subjected to a single bout of exhaustive exercise. RESULTS: HFHS diet resulted in shorter hanging time, reduced grip force, and lower exhaustion time and distance, and decreased lean mass per body weight. Moreover, in the soleus, which is chosen as a representative red (oxidative) muscle, LC3II/LC3I ratio, P62, and Bnip3 levels were altered following the HFHS diet, and were negatively correlated with muscle performance parameters; exercise significantly decreased the LC3II/LC3 ratio while P62 increased with HFHS diet. Autophagy-related protein changes were not found in the white (glycolytic) gastrocnemius. CONCLUSION: The study revealed that 20-week HFHS diet causes a significant increase in body weight and fat mass, along with a decrease in muscle function. Autophagy-related LC3 and P62 protein expression was negatively correlated with muscle function, and they were reduced when a single bout of exercise stimulated the soleus of obese mice. However, no change of autophagy-related proteins was seen in the gastrocnemius.

Potential role of exercise-induced glucose-6-phosphate isomerase in skeletal muscle function.

PURPOSE: Recent studies have shown that glucose-6-phosphate isomerase (GPI)-which is a glycolysis interconversion enzyme-reduces oxidative stress. However, these studies are limited to tumors such as fibrosarcoma, and there are no studies that have examined the effects of exercise on GPI expression in mice skeletal muscle. Furthermore, GPI acts in an autocrine manner thorough its receptor, autocrine motility factor receptor (AMFR); therefore, we investigated expression level changes of secreted GPI from skeletal muscle in in vitro study to examine the potential role of GPI on skeletal muscle. METHODS: First, we performed an in vitro study, to identify the condition that upregulates GPI levels in skeletal muscle cells; we treated C2C12 muscle cells with an exercise-mimicking chemical, AICAR. AICAR treatment upregulated GPI expression level in C2C12 cell and its secretomes. To confirm the direct effect of GPI on skeletal muscle cells, we treated C2C12 cells with GPI recombinant protein. RESULTS: We found that GPI improved the viability of C2C12 cells. In the in vivo study, the exercise-treated mice group showed upregulated GPI expression in skeletal muscle. Based on the in vitro study results, we speculated that expression level of GPI in skeletal muscle might be associated with muscle function. We analyzed the association between GPI expression level and the grip strength of the all mice group. The mice group's grip strengths were upregulated after 2 weeks of treadmill exercise, and GPI expression level positively correlated with the grip strength. CONCLUSION: These results suggested that the exercise-induced GPI expression in skeletal muscle might have a positive effect on skeletal muscle function.

Exercise, the Gut Microbiome, and Frailty.

The gut microbiome is deeply associated with both skeletal muscle and brain function. In particular, gut microbiome dysbiosis may accelerate age-related diseases by affecting these systems. Although there is increasing evidence of the correlations between the gut microbiome and skeletal muscle and brain, it remains unclear whether changes in the gut microbiome due to exercise training can lead to healthy aging. This review covers the current status of gut microbiome-related research and future directions related to aging (e.g., physical frailty and cognitive dysfunction) as well as the effect of exercise training on both. We reviewed relevant literature including original articles and reviews identified from searches of the PubMed, Google Scholar, SCOPUS, EBSCOHost, ScienceDirect, Cochrane Library, and EMBASE databases using the following terms: 'gut microbiome', 'exercise', 'physical frailty', and 'cognitive dysfunction'. We identified a strong positive correlation between cognitive dysfunction or physical frailty and the gut microbiome. Furthermore, exercise had a significant effect on the composition of the gut microbiome. These results suggest that exercise training can prevent physical frailty or cognitive dysfunction by altering the gut microbiome. However, the exact mechanism by which these effects occur is not yet clear. Further studies are needed to determine whether exercise training can prevent age-related diseases by balancing the gut microbiome.

Angiogenesis: focusing on the effects of exercise in aging and cancer.

PURPOSE: Although it is known that exercise induces angiogenesis, a clear mechanism has remained elusive due to various experimental limitations. This review presents the current status of angiogenesis-related experiments and future directions of experimentation in relation to exercise, aging, and cancer. METHODS: We conducted a PubMed search of the available literature to identify reported exercise related changes of angiogenic factors obtained in vitro using C2C12 cells and endothelial cells, and in vivo using animal experiments and in clinical studies. RESULTS: Exercise induced angiogenesis under normal conditions. Aging decreased angiogenic factors and increased during exercise. On the other hand, in cancer, the results indicate that angiogenic factors tend to increase in general, and that the effects of exercise need to be studied more. The exact mechanism remains unclear. CONCLUSION: The effect of exercise on angiogenesis appears positive. Both resistance and aerobic exercise have positive effects, but many evidences suggest that the effects are more pronounced with aerobic exercise. Further research on the precise mechanism(s) is necessary. It is expected that these studies will include models of aging and cancer.