Morse, Dawn M.

There is evidence that the anabolic response to exercise is blunted in skeletal muscle in older adults. Older adults also exhibit reduced mitochondrial content and lower oxidative capacity. Since mitochondria provide chemical energy for muscle protein synthesis, it is possible that age‐related changes to mitochondrial physiology may contribute to anabolic resistance. We evaluated this possibility by measuring mitochondrial function and anabolic responses to exercise in 12 young (19–34 yrs) and 12 older (67–83 yrs) adults. Serial vastus lateralis biopsies were collected during an infusion of 13 C 6 phenylalanine to measure muscle protein synthesis at baseline and 16 hours after a single bout of unaccustomed resistance exercise (unilateral knee extension, 8 sets, 10 repetitions at 70% of 1‐repetition maximum). Real‐time PCR and RNA sequencing were used to evaluate the transcriptional response to acute exercise in muscle tissue. Respiratory capacity of isolated mitochondria was measured by high‐resolution respirometry. Mitochondrial respiratory capacity was significantly lower in older adults (4.67±0.47 pmol/s/ug) compared to young (6.60±0.70 pmol/s/ug, p=0.024). Postabsorptive protein synthesis rates were similar in young and old for mixed muscle (Y: 0.067±0.005 %/hr, O: 0.104±0.030 %/hr, p=0.13), mitochondrial (Y: 0.076±0.014 %/hr, O: 0.089±0.017 %/hr, p=0.58), and myofibrillar fractions (Y: 0.073±0.005 %/hr, O: 0.072±0.015 %/hr, p=0.94). Exercise increased protein synthesis similarly in young and old for mixed muscle (Fold changes Y: 1.45±0.16, O: 1.46±0.18, p=0.98), mitochondrial (Y: 0.90±0.28, O: 1.74±0.41, p=0.11), and myofibrillar (Y: 1.35±0.13, O: 1.37±0.18, p=0.93). These data demonstrate older adults have lower mitochondrial respiratory capacity yet exhibit similar anabolic responses to resistance exercise compared to young. Protein synthesis does not appear limited by the decreased mitochondrial capacity with age.

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

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.

Altered protein homeostasis may explain the increased risk of degenerative and cardiometabolic disorders with aging. Here, we investigated the impact of chronological age and obesity on the accumulation (ratio of old to de novo synthesized proteins) of haptoglobin, fibrinogen, and apolipoprotein A‐1 (ApoA‐1) in 7 each of elderly lean (69.8 ± 5.0 years, 23.2 ± 1.8 kg/m 2 ), elderly obese (72.6 ± 5.6 years, 30.6 ± 2.3 kg/m 2 ), young lean (23.1 ± 3.2 years, 23.4 ± 1.9 kg/m 2 ), and young obese (25.2 ± 2.4 years, 30.6 ± 2.4 kg/m 2 ). Proteins were labeled with [ 13 C 6 ]‐phenylalanine, separated by 2‐dimentional gel electrophoresis, and identified by mass spectrometry. Insulin sensitivity was lower in the obese groups, while percent body fat was higher, and lean mass was lower, in the elderly. Haptoglobin accumulation was greater in the obese compared to lean (0.512 ± 0.229 vs. 0.272 ± 0.114; p = 0.008) across all ages, with no age effect. Fibrinogen trended toward higher accumulation in the obese compared to lean, with no age effect, but did not reach statistical significance (1.263 ± 2.108 vs. 0.598 ± 0.197; p = 0.094). ApoA‐1 accumulation showed no obesity or age variation. Associations remained unchanged when controlled for gender, age, and BMI. Thus, adiposity, and not chronological age, promotes protein accumulation, potentially contributing to degenerative and cardiometabolic disease risk. Supported by NIH grant RO1‐AG09531.

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

OBJECTIVE The accumulation of old and damaged proteins likely contributes to complications of diabetes, but currently no methodology is available to measure the relative age of a specific protein alongside assessment of posttranslational modifications (PTM). To accomplish our goal of studying the impact of insulin deficiency and hyperglycemia in type 1 diabetes upon accumulation of old damaged isoforms of plasma apolipoprotein A-1 (ApoA-1), we sought to develop a novel methodology, which is reported here and can also be applied to other specific proteins. RESEARCH DESIGN AND METHODS To label newly synthesized proteins, [ring-13C6]phenylalanine was intravenously infused for 8 h in type 1 diabetic participants (n = 7) during both insulin treatment and 8 h of insulin deprivation and in nondiabetic participants (n = 7). ApoA-1 isoforms were purified by two-dimensional gel electrophoresis (2DGE) and assessment of protein identity, PTM, and [ring-13C6]phenylalanine isotopic enrichment (IE) was performed by tandem mass spectrometry. RESULTS Five isoforms of plasma ApoA-1 were identified by 2DGE including ApoA-1 precursor (pro-ApoA-1) that contained the relatively highest IE, whereas the older forms contained higher degrees of damage (carbonylation, deamidation) and far less IE. In type 1 diabetes, the relative ratio of IE of [ring-13C6]phenylalanine in an older isoform versus pro-ApoA-1 was higher during insulin deprivation, indicating that de novo synthesized pro-ApoA-1 more rapidly accumulated damage, converting to mature ApoA-1. CONCLUSIONS We developed a mass spectrometry–based methodology to identify the relative age of protein isoforms. The results demonstrated accelerated oxidative damage to plasma ApoA-1, thus offering a potential mechanism underlying the impact of poor glycemic control in type 1 diabetic patients that affects a patient's risk for vascular disease.

It remains to be determined whether systemic insulin replacement normalizes synthesis rates of different plasma proteins and whether there are differential effects on various plasma proteins. We tested a hypothesis that insulin deprivation differentially affects individual plasma protein synthesis and that systemic insulin treatment may not normalize synthesis of all plasma proteins. We measured synthesis rates of 41 plasma proteins in seven each of type 1 diabetic (T1DM) and nondiabetic participants (ND) using [ ring-13C6]phenylalanine as a tracer. T1DM were studied while on chronic insulin treatment and during 8 h insulin deprivation. Insulin treatment normalized glucose levels, but plasma insulin levels were higher during insulin treatment than during insulin deprivation in T1DM and ND. Individual plasma proteins were purified by affinity chromatography and two-dimensional gel electrophoresis. Only 41 protein gel spots from over 300 were chosen based on their protein homogeneity. Insulin deprivation and hyperglycemia either significantly increased ( n = 12) or decreased ( n = 12) synthesis rates of 24 of 41 plasma proteins in T1DM compared with ND. Insulin treatment normalized synthesis rates of 13 of these 24 proteins, which were altered during insulin deprivation. However, insulin treatment significantly altered the synthesis of 14 additional proteins. In conclusion, acute insulin deprivation caused both a decrease and increase in synthesis rates of many plasma proteins with various functions. Moreover, chronic systemic insulin treatment not only did not normalize synthesis of all plasma proteins but also altered synthesis of several additional proteins that were unaltered during insulin deprivation.