Nair, K. Sreekumaran

Insulin resistance increase with age but it is uncertain whether it is related to age or other factors. We measured insulin sensitivity by hyperinsulinemic euglycemic clamp in 48 men and women −12 each of young lean (YL) and obese (YO) as well as old lean (OL) and obese (OO) groups. OL (69.9±1.4 years), OO (71.0±1.5 years), YL (22.8±1.0 years) and YO (24.2±0.6 years) were matched for BMI. Older groups had lower peak VO 2 (32.4±1.3 ml/kg FFM) than young (46.9±1.0)(p<0.01) but no differences noted among the two older and younger groups. Similar glucose levels (90.4 to 91.4 mg/dL) were maintained during the glucose clamp. The glucose infusion rate (GIR) at steady state, was similar in the YO (38.7±5.4 mmol/kgFFM/min) and OO (43.0±2.4) where as YL (55.5±3.7) and OL (54.2±3.4) were higher (p<0.02) than obese groups. GIR was significantly correlated to fasting insulin levels (R 2 =0.32, p<0.001). Energy expenditure per FFM, was not different amongst the 4 groups. Adiponectin levels were higher in the older groups (12526±1109 ng/mL) than in the younger (8640±392, p<0.01). A trend toward lower adiponectin levels was seen in OO (10394 ± 1187 ng/ml) than OL (14657 ±1705, p=0.05). A similar trend was not seen comparing the YO to YL. However, the OL had a significantly higher adiponectin level than the YL (9023.8 ± 595 ng/mL, p<0.01). GIR was not significantly correlated to age (R 2 =0.00005, p=NS) nor to peak VO2 (R 2 =0.007, p=NS) but was significantly correlated to truncal fat (R 2 =0.17, p<0.01) and BMI (R 2 =0.25, p<0.001). The age‐related decline in insulin sensitivity is not accounted for by fitness levels or age per se. The results demonstrated that insulin resistance in aging is explained by change in body composition rather than age.

Skeletal muscle protein synthesis rates decline with age. PURPOSE: We sought to determine if DHEA supplementation in elderly men (EM) and elderly women (EW) and low dose testosterone (T) supplementation in EM would raise protein synthesis rates. METHODS: 87 EM (with low bioavailable T and DHEAS) and 57 EW (with low DHEAS) were studied before and after 1 year in which EM received either T (5 mg/d), DHEA (75 mg/d), or placebo (P) and EW received either DHEA (50 mg/d) or P. To assess age effects, 30 young men and 32 young women were studied on a single occasion as controls. [ 15 N]phenylalanine and [ 2 H 4 ]tyrosine tracers were infused and isotopic enrichment was measured in plasma and v. lateralis muscle. Whole body amino acid kinetics were normalized to fat free mass, and mixed muscle protein fractional synthesis rate (FSR) was measured using plasma phenylalanine as the precursor. RESULTS: Despite induction of elevated androgen levels, no significant differences were seen between treated groups and the P group in either EM or EW. Whole body protein synthesis and muscle FSR were lower in elderly than in young (P < 0.05) and were higher in women than men (P < 0.05), with no age‐by‐gender interaction. CONCLUSIONS: Neither DHEA nor T supplementation for one year had any significant impact on age‐related decline in protein synthesis, while both age and gender do impact protein synthesis rates. NIH grants P01 AG14283 and UL1‐RR‐024150‐01.

Triiodothyronine (T3) has a major impact on the regulation of energetics and mitochondrial (mt) function. We determined whether chronic T3 administration alters the age‐related decline in mt function. Ten young (Y, 8 mo) and ten old (O, 27 mo) F344/BN rats were randomized to receive T3 (200 mg/d) or placebo for 14d via subcutaneous osmotic pump. Because OT3 rats decreased food consumption by 39%, we added ten pair fed rats (OPF). T3 treatment caused a significant weight loss (Y, −11.97%; O, −17.49%), while energy expenditure increased in both Y (50.86%) and O (54.40%), but decreased in PF (17.55%). Fat and muscle loss were similar in Y and O in response to T3 (fat, −52.53%(Y) vs −51.69% (O); muscle, −21.50% (Y) vs −21.03% (O) in soleus and −16.70% (Y) vs −17.35% (O) in plantaris), whereas these changes were minor with OPF (fat, −10.4%; soleus −1.69% ; plantaris 7.69%). While mt ATP production and citrate synthase activity in heart and soleus muscle were lower in O, these measures increased equally in Y and O in response to T3. In the soleus, mt DNA abundance increased in Y (54.39%) and O (23.36%) and UCP3 expression increased 10‐fold in both age groups. Gene transcripts levels of PGC1α, COX3&4 and NADH4, UCP2& 3 increased in soleus of T3 rats. In conclusion, T3 similarly increased energy expenditure, weight loss and mt biogenesis in both Y and O rats, despite differences in food intake. NIH grant PO1 AG09531 and Mayo Foundation

Age‐related mitochondrial dysfunction is paralleled by reductions in gene transcripts and expression of proteins involved in cellular ATP production. Short‐term endurance exercise has been shown to enhance muscle protein synthesis in the elderly. We determined the impact of long‐term endurance exercise on gene expression and protein abundance of numerous proteins involved in cellular energetics. Vastus lateralis tissue was biopsied from 11 young sedentary (YS), 10 old sedentary (OS), 11 young trained (YT), and 11 old trained (OT) subjects. Trained subjects vigorously exercised ¡Ý 1 hr/d, ¡Ý five d/wk for ¡Ý 4 years. Large‐scale measurement of relative protein abundance was performed using isobaric tags for relative and absolute quantitation (iTRAQ) reagents and tandem mass spectrometry. Gene transcript levels were measured by high‐density oligonucleotide microarrays. Ingenuity Pathway Analysis was used to determine canonical pathways that were altered by age. Of the 33 proteins involved in cellular ATP production 27 were less abundant in OS vs. YS, while only 4 of these proteins were less abundant in OT vs. YT. Genes involved in glycolysis and oxidative phosphorylation were underexpressed in OS vs. YS, with no effects of age in OT vs. YT. CONCLUSIONS: Long‐term vigorous endurance exercise ameliorates the detrimental effects of old age on muscle gene and protein expression in humans.

Aging is associated with a concomitant decline in non exercise activity (NEA) and mitochondrial ATP production rate (MAPR) in skeletal muscle. To determine whether caloric restriction (CR) could prevent the decline in MAPR and NEA we studied young (Y, 7 month‐old, n=8), old (O, 27 month‐old, n=7) and old caloric‐restricted F344/BN rats (OCR, 27 month‐old, 40% CR from 16 weeks of age, n=7). Energy expenditure (kcal/d/gBW) was lower in O (0.311±0.005) than Y (0.365±0.004) and was higher in OCR (0.341±0.008) (P<0.05). Muscle mass (mg/gBW) of O was lower than Y, and Y and OCR are similar (Y=0.96±0.18, O=0.71±0.16, OCR=1.03±0.04; P<0.05 OCR vs O). Citrate synthase activity and MAPR were lower in both O and OCR than Y (P<0.05). UCP3 protein level was higher in soleus and plantaris muscles from O rats than Y and OCR (p<0.005). CR lowered UCP3 expression in skeletal muscles to the level in Y rats (p<0.005 OCR vs O). CR also lowered expression of mRNA levels of NADH4 (p<0.005 OCR vs O). NEA levels were lower in O and OCR rats than in Y (p<0.05 Y vs O and OCR). In conclusion, the decline in mitochondrial functions during aging can be counteracted by CR but an age‐related decline in activity levels could not be prevented. Funded by NIH RO1 AG09531 and Mayo Foundation.

Leucine (Leu) is the most potent branched‐chain amino acid that selectively enhances protein synthesis via intracellular signaling pathways. Since protein synthesis is an energy consuming process, we hypothesized that Leu may enhance mitochondrial biogenesis and functions. We determined the effect of Leu at mitochondrial mRNA levels, enzyme activities, and ATP production rate (MAPR) in cultured human liver cell line THLE‐3. Cells were pre‐incubated in a low‐Leu (0.1mM), serum‐free media for 24 hours and gene transcript profiles were measured using Affymetrix HG‐U133A GeneChips at 0, 30, 60, 120 and 480 minutes after increasing the Leu concentrations from to 2.0 mM. Thirty mitochondrial gene transcripts, both mitochondrial DNA and nuclear‐encoded, were significantly increased at various time points with treatment of higher concentrations of Leu. The mRNA levels of 6 mitochondrial genes including two ATP synthase, cytochrome P450, cytochrome C oxidase III, and NADH dehydrogenase 4 were confirmed by real‐time PCR measurements. Western blot analysis showed that 8 hours of high concentrations of Leu increased protein levels of key mitochondrial proteins, citrate synthase and cytochrome c oxidase I, by 42% and 20% respectively. In addition, the enzyme activity of citrate synthase was significantly increased by 25%, and MAPR by 30–40% using various substrates after 8 hours of high Leu. In conclusion, Leu stimulated mitochondrial biogenesis and oxidative phosphorylation in liver cells. This work is supported by NIH grants T32 DK7352, and RO1 DK41972.

Whole body and mixed muscle protein synthesis rate declines with age in healthy people. In response to a 4‐month aerobic exercise program we showed that mixed muscle protein synthesis rate increased 22%. Several gene transcripts that encode mitochondrial (mito) proteins increased in response to exercise but it remains to be determined whether these are translated to protein synthesis. In this study we tested the hypothesis that mito protein synthesis also increased in response to aerobic exercise training. Healthy men and women (N = 42) from 30–74 y were studied before and after a 4‐month program of stationary bicycle training (3d/wk, up to 40min/d at 80% HRmax) or control activity. Muscle protein synthesis rate was measured using [ 15 N] phenylalanine as a tracer. Mito and sarcoplasmic (sarco) proteins were separated from muscle biopsies for analysis. At baseline there was a decline with age (p<0.05) in both mito (6%/decade, r= −0.32) and sarco (5.5%/decade, r= −0.42) protein synthesis rate. Muscle protein synthesis rates did not change in control subjects, but in the exercise group mito synthesis increased 27% (0.046 ± 0.003 %/h pre vs. 0.058 ± 0.005 post, p=0.017, N=25) and there was a trend for 10% higher sarco protein synthesis (0.036 ± 0.002 %/h pre vs. 0.040 ± 0.003 post, p=0.066, N=33). These effects of exercise did not vary with age. We conclude that despite a decline in mito protein synthesis and function with advancing age, the potential for mito biogenesis in response to aerobic exercise remains robust in older muscle, as demonstrated by enhanced mito protein synthesis, gene expression, and enzyme action. Support: NIH RO1DK41973, MO1RR00585.

Aging is associated with decreasing muscle mass and protein synthesis. Amino acid administration is reported to increase muscle protein synthesis and balance. We determined whether increasing dietary protein content from adequate (1.5g/kgFFM/d, AP) to high (3g/kgFFM/d, HP) had beneficial or adverse effects on whole body and muscle protein metabolism, renal function and insulin sensitivity in healthy young (24±1y) and older (70±2y) men and women. Subjects were randomized to a crossover design of 10 d of AP or HP protein diets, without changing caloric intake. Measurements of postabsoptive whole body leucine appearance rate (protein breakdown), oxidation, and non‐oxidative leucine disposal (protein synthesis) rates were performed using L‐[1‐ 13 C]‐leucine and fractional synthesis rate (FSR) of mixed muscle proteins was done using [ 15 N]‐lysine as tracers. Glomerular filtration rate (GFR) and insulin sensitivity were determined by iodothalamate and intravenous glucose tolerance test, respectively. HP diet increased protein breakdown in both young and old (p<0.05) and increased leucine oxidation in both age groups with greater increase in young (p<0.005). WBPS, muscle protein FSR and insulin sensitivity were unaffected by diet. GFR was lower (p<0.01) in older than in young during both diets. HP diet increased GFR in young (p<0.01) but decreased GFR in older people (p<0.01). In conclusion, increasing dietary protein content had no detectable beneficial effects on muscle or whole body protein metabolism in healthy older people and may cause deleterious effects on renal function. Support: NIH R01AG09531.