Gregory C. Henderson

Identification of de novo synthesized and relatively older proteins: accelerated oxidative damage to de novo synthesized apolipoprotein A-1 in type 1 diabetes.

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 patients risk for vascular disease.

Coingestion of whey protein and casein in a mixed meal: demonstration of a more sustained anabolic effect of casein.

When consumed separately, whey protein (WP) is more rapidly absorbed into circulation than casein (Cas), which prompted the concept of rapid and slow dietary protein. It is unclear whether these proteins have similar metabolic fates when coingested as in milk. We determined the rate of appearance across the splanchnic bed and the rate of disappearance across the leg of phenylalanine (Phe) from coingested, intrinsically labeled WP and Cas. Either [¹⁵N]Phe or [¹³C-ring C₆]Phe was infused in lactating cows, and the labeled WP and Cas from their milk were collected. To determine the fate of Phe derived from different protein sources, 18 healthy participants were studied after ingestion of one of the following: 1) [¹⁵N]WP, [¹³C]Cas, and lactose; 2) [¹³C]WP, [¹⁵N]Cas, and lactose; 3) lactose alone. At 80-120 min, the rates of appearance (R(a)) across the splanchnic bed of Phe from WP and Cas were similar [0.068 ± 0.010 vs. 0.070 ± 0.009%/min; not significant (ns)]. At time 220-260 min, Phe appearance from WP had slowed (0.039 ± 0.008%/min, P < 0.05) whereas Phe appearance from Cas was sustained (0.068 ± 0.013%/min). Similarly, accretion rates across the leg of Phe absorbed from WP and Cas were not different at 80-120 min (0.011 ± 0.002 vs. 0.012 ± 0.003%/min; ns), but they were significantly lower for WP (0.007 ± 0.002%/min) at 220-260 min than for Cas (0.013 ± 0.002%/min) at 220-260 min. Early after meal ingestion, amino acid absorption and retention across the leg were similar for WP and Cas, but as rates for WP waned, absorption and assimilation into skeletal muscle were better retained for Cas.

Combined training enhances skeletal muscle mitochondrial oxidative capacity independent of age.

CONTEXT Skeletal muscle from sedentary older adults exhibits reduced mitochondrial abundance and oxidative capacity. OBJECTIVE The primary objective was to determine whether 8 weeks of combined training (CT) has a more robust effect than endurance training (ET) or resistance training (RT) on mitochondrial physiology in healthy young (18-30 years) and older (≥ 65 years) adults. INTERVENTION Thirty-four young and 31 older adults were randomly assigned to 8 weeks of ET, RT, and control/CT. Control subjects completed 8 weeks of no exercise (control) followed by 8 weeks of CT. Body composition, skeletal muscle strength, and peak oxygen uptake were measured before and after the intervention. Vastus lateralis muscle biopsy samples were obtained before and 48 hours after the intervention. Mitochondrial physiology was evaluated by high-resolution respirometry and expression of mitochondrial proteins and transcription factors by quantitative PCR and immunoblotting. RESULTS ET and CT significantly increased oxidative capacity and expression of mitochondrial proteins and transcription factors. All training modalities improved body composition, cardiorespiratory fitness, and skeletal muscle strength. CT induced the most robust improvements in mitochondria-related outcomes and physical characteristics despite lower training volumes for the ET and RT components. Importantly, most of the adaptations to training occurred independent of age. CONCLUSION Collectively, these results demonstrate that both ET and CT increase muscle mitochondrial abundance and capacity although CT induced the most robust improvements in the outcomes measured. In conclusion, CT provides a robust exercise regimen to improve muscle mitochondrial outcomes and physical characteristics independent of age.

Differential Effect of Endurance Training on Mitochondrial Protein Damage, Degradation, and Acetylation in the Context of Aging

Acute aerobic exercise increases reactive oxygen species and could potentially damage proteins, but exercise training (ET) enhances mitochondrial respiration irrespective of age. Here, we report a differential impact of ET on protein quality in young and older participants. Using mass spectrometry we measured oxidative damage to skeletal muscle proteins before and after 8 weeks of ET and find that young but not older participants reduced oxidative damage to both total skeletal muscle and mitochondrial proteins. Young participants showed higher total and mitochondrial derived semitryptic peptides and 26S proteasome activity indicating increased protein degradation. ET however, increased the activity of the endogenous antioxidants in older participants. ET also increased skeletal muscle content of the mitochondrial deacetylase SIRT3 in both groups. A reduction in the acetylation of isocitrate dehydrogenase 2 was observed following ET that may counteract the effect of acute oxidative stress. In conclusion aging is associated with an inability to improve skeletal muscle and mitochondrial protein quality in response to ET by increasing degradation of damaged proteins. ET does however increase muscle and mitochondrial antioxidant capacity in older individuals, which provides increased buffering from the acute oxidative effects of exercise.

Effects of Adiposity and 30 Days of Caloric Restriction Upon Protein Metabolism in Moderately vs. Severely Obese Women

Protein metabolism adapts during caloric restriction (CR) to minimize protein loss, and it is unclear whether greater fat stores favorably affect this response. We sought to determine whether protein metabolism is related to degree of obesity and whether the response to CR is impacted by pre‐CR adiposity level. Whole body protein metabolism was studied in 12 obese women over a wide range of BMI (30–53 kg/m2) as inpatients using [1‐13C]leucine as a tracer following 5 days of a weight‐maintaining diet and then after 30 days of CR (1,400 kcal deficit with maintained protein intake). When expressed as total rates, per body weight (BW) or per fat‐free mass (FFM), leucine rate of appearance (Ra), and nonoxidative leucine disposal (NOLD) were significantly higher in the individuals with a greater degree of obesity (P < 0.05). Leucine oxidation (Rox) was also higher in more highly obese women when expressed as a total rate (P < 0.05) but not if expressed per BW or FFM. CR reduced BW, FFM, and fat mass (P < 0.001), and declines were relatively similar between individuals. CR reduced Ra (P < 0.001), NOLD (P < 0.01), and Rox (P < 0.05), and the relative decline was not affected by differences in fat mass. CR‐induced declines were significant even when Ra and NOLD were normalized to BW or FFM. We conclude that fat mass, like FFM, is a key determinant of protein turnover. However, during CR, higher fat mass does not favorably alter the response of protein metabolism and does not mitigate the loss of FFM.