Short, Kevin R.

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.

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.

Insulin resistance is associated with skeletal muscle mitochondrial (mito) dysfunction in many conditions including type 2 diabetes (T2D). Poor glycemic control in T2D is associated with muscle mito gene transcript alterations. It is unknown whether muscle mito changes are related to reduced insulin action or vice versa. We obtained vastus lateralis muscle biopsies from 7 type 1 diabetic patients (T1D) following 8–12 hrs of insulin deprivation (I−) and replacement (I+) and measured skeletal muscle mito ATP production (MATP) and gene transcripts. I− and I+ changed glucose and glucagon levels (glucose=291±15mg/dl in I− vs 97±3 in I+ and glucagon=79.8±15.3 pg/mL in I− vs 47.3±5.9 in I+, p<0.04). Whole body O 2 consumption increased during I− by 15% (p<0.05) but maximal MATP decreased by 21.3–27.9% (p<0.05). Gene transcript profiling in skeletal muscle analyzed using Affymetrix GeneChip arrays showed alterations in 2355 transcripts. Pathway analysis showed down‐regulation of oxidative phosphorylation and arachidonic acid metabolism, and up‐regulation of VEGF and actin cytoskeleton signaling in I−. We conclude that the reduced insulin action that occurred during I− in T1D resulted in substantial changes in gene transcript levels of muscle mito genes and reduced maximal MATP, supporting that insulin is key regulator of muscle mito function by exerting its effect at various levels. Supported by NIHRO1DK41973.

We report a technique to simultaneously measure fractional synthesis rates (FSR) of several muscle proteins that facilitate further understanding of translational regulation, a key step involved in gene expression in a tissue. Skeletal muscle samples were collected from five rats following bolus injection of [ring 13 C 6 ] phenylalanine. Muscle proteins were fractionated by differential centrifugation followed by separation of individual proteins by high resolution two‐dimensional gel electrophoresis. We identified 60 mitochondrial and 30 non‐mitochondrial proteins by liquid chromatography tandem mass spectrometry. Isotope enrichment of these 90 proteins was measured by gas chromatography tandem mass spectrometry, and FSR was calculated using the muscle tissue fluid isotopic enrichment as the precursor. Approximately 25 fold differences in the FSR of muscle proteins were observed, (e.g. FSR of structural protein such as myosin heavy chain‐1 = 0.19 %/h ± 0.03 and tropomysin‐4 = 0.16 ± 03 and that of mitochondrial proteins such as cytochrome c oxidase Va = 0.64 ± 0.07 and dihydrolipoamide dehydrogenase = 4.3 %/h ± 0.53). The current approach, based on in‐vivo metabolic labeling, to simultaneously measure FSR of multiple muscle proteins offers tremendous opportunity to study the regulation of skeletal muscle mitochondrial and non‐mitochondrial protein synthesis in various physiologic and pathologic states. Funding: NIH RO1 DK41973

Triiodothyronine (T3) increases O2 and nutrient flux through mitochondria (Mito) of many tissues, but it is unclear whether ATP synthesis is increased, particularly in different types of skeletal muscle, because variable changes in uncoupling proteins (UCP) and enzymes have been reported. Thus Mito ATP production was measured in oxidative and glycolytic muscles, as well as in liver and heart, in rats administered T3 for 14 days. Relative to saline-treated controls, T3 rats had 80, 168, and 62% higher ATP production in soleus muscle, liver, and heart, respectively, as well as higher activities of citrate synthase (CS; 63, 90, 25%) and cytochrome c oxidase (COX; 119, 225, 52%) in the same tissues (all P < 0.01). In plantaris muscle of T3 rats, CS was only slightly higher (17%, P < 0.05) than in controls, and ATP production and COX were unaffected. mRNA levels of COX I and III were 33 and 47% higher in soleus of T3 rats ( P < 0.01), but there were no differences in plantaris. In contrast, UCP2 and -3 mRNAs were 2.5- to 14-fold higher, and protein levels were 3- to 10-fold higher in both plantaris and soleus of the T3 group. We conclude that T3 increases oxidative enzymes and Mito ATP production and Mito-encoded transcripts in oxidative but not glycolytic rodent tissues. Despite large increases in UCP expression, ATP production was enhanced in oxidative tissues and maintained in glycolytic muscle of hyperthyroid rats.

Phenylalanine (Phe) kinetics are increasingly used in studies of amino acid kinetics, because the metabolic fate of Phe is limited to incorporation into protein (protein synthesis, Sp) and catabolism via hydroxylation ( Q pt) to tyrosine (Tyr). Besides an infusion of labeled Phe to measure Phe flux ( Q p), a priming dose of Tyr and an independent Tyr tracer are used to measure Tyr flux ( Q t) and Q pt. Alternatively, Q t, Q pt, and Sp can be approximated by using equations, based on Phe and Tyr concentrations in body proteins, that eliminate the need for a Tyr tracer. To evaluate the accuracy of this approach, data were obtained from 12 type I diabetic patients and 24 nondiabetic control subjects who were studied with the full complement of tracers both with and without insulin infusion. Sp approximations closely matched measured values in both groups (mean difference <2%, all values <5%), but the agreement was poor for Q pt (error range = −8 to +43%) and Q t (error range −22 to +41%). Insulin status had no effect on these comparisons. The lower approximation error for Sp vs. Q pt is due to the small contribution (∼10%) of Q pt to Q p. Approximation error for Q pt( r > 0.99) can be explained by variability in the ratio of Tyr to Phe coming from protein breakdown, ( Q t − Q pt)/ Q p. Ideally, all fluxes should be directly measured, but these data suggest that whole body Sp can be approximated with an acceptably small margin of error. However, the same equations do not yield reliably accurate values for Q pt or Q t.

Thyroid hormone excess is associated with increased energy expenditure. The contributions of increases in spontaneous physical activity and nonexercise activity thermogenesis (NEAT) to this effect have not been defined. To address the hypothesis that hyperthyroidism is associated with increased spontaneous physical activity and NEAT, we rendered rats hyperthyroid by using continuous infusion of high-dose triiodothyronine for 14 days and measured the effects on physical activity and NEAT. On day 14, in the hyperthyroid group the mean ± SD triiodothyronine concentration was 755 ± 137 (range 574–919) ng/dl and in the control group 59 ± 0.5 (58–59) ng/dl. Over the 14-day treatment period, mean spontaneous physical activity increased in the hyperthyroid rats from 24 ± 7 to 36 ± 6 activity units (AU)/min; P < 0.001 but did not increase in the controls (23 ± 7 vs. 22 ± 4 AU/min). Also, over the 14-day period, daily NEAT increased in the hyperthyroid rats from 8.1 ± 2.8 to 19.7 ± 5.0 kcal/day ( P < 0.001) but did not increase in the controls (8.7 ± 3.5 cf 9.4 ± 1.7 kcal/day; not significant). In conclusion, hyperthyroidism is associated with increased spontaneous physical activity and NEAT.