Klaus, Katherine A.

Insulin and nutrients have profound effects on proteome homeostasis. Currently no reliable methods are available to measure postprandial protein turnover. A triple-tracer method was developed using phenylalanine stable isotope tracers to estimate appearance rates of ingested (R a meal ) and endogenous phenylalanine and the rate of phenylalanine disposal (R d ). This was compared with the “traditional” dual-tracer method, using one (1-CM)- and two (2-CM)-compartment models. For both methods, [ 13 C 6 ]phenylalanine was given orally, and [ 15 N]phenylalanine was constantly infused; the triple-tracer method added [ 2 H 5 ]phenylalanine, infused at rates to mimic meal [ 13 C 6 ]phenylalanine appearance. Additionally, incorporation of meal-derived phenylalanine into specific proteins was measured after purification by two-dimensional electrophoresis. The triple-tracer approach reduced modeling errors, allowing improved reconstruction of R a meal with a tracer-to-tracee ratio that was more constant and better estimated R d . The 2-CM better described phenylalanine kinetics and R d than 1-CM. Thus, the triple-tracer approach using 2-CM is superior for measuring non-steady-state postprandial protein turnover. This novel approach also allows measurement of postprandial synthesis rates of specific plasma proteins. We offer a valid non-steady-state model to measure postprandial protein turnover and synthesis of plasma proteins that can safely be applied in adults, children, and pregnant women.

Background Following pressure overload, compensatory concentric left ventricular remodeling ( CR ) variably transitions to eccentric remodeling ( ER ) and systolic dysfunction. Mechanisms responsible for this transition are incompletely understood. Here we leverage phenotypic variability in pressure overload–induced cardiac remodeling to test the hypothesis that altered mitochondrial homeostasis and calcium handling occur early in the transition from CR to ER , before overt systolic dysfunction. Methods and Results Sprague Dawley rats were subjected to ascending aortic banding, (n=68) or sham procedure (n=5). At 3 weeks post–ascending aortic banding, all rats showed CR (left ventricular volumes < sham). At 8 weeks post–ascending aortic banding, ejection fraction was increased or preserved but 3 geometric phenotypes were evident despite similar pressure overload severity: persistent CR , mild ER , and moderate ER with left ventricular volumes lower than, similar to, and higher than sham, respectively. Relative to sham, CR and mild ER phenotypes displayed increased phospholamban, S16 phosphorylation, reduced sodium‐calcium exchanger expression, and increased mitochondrial biogenesis/content and normal oxidative capacity, whereas moderate ER phenotype displayed decreased p‐phospholamban, S16, increased sodium‐calcium exchanger expression, similar degree of mitochondrial biogenesis/content, and impaired oxidative capacity with unique activation of mitochondrial autophagy and apoptosis markers ( BNIP 3 and Bax/Bcl‐2). Conclusions After pressure overload, mitochondrial biogenesis and function and calcium handling are enhanced in compensatory CR . The transition to mild ER is associated with decrease in mitochondrial biogenesis and content; however, the progression to moderate ER is associated with enhanced mitochondrial autophagy/apoptosis and impaired mitochondrial function and calcium handling, which precede the onset of overt systolic dysfunction.

The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca2+, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca2+ cycling, Ca2+ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/role-of-foxo3a-in-heart-failure/ .

Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.

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.

Abstract Context: A reduction in maximal mitochondrial ATP production rate (MAPR) and mitochondrial DNA (mtDNA) abundance occurs with age in association with muscle weakness and reduced endurance in elderly people. Branched chain amino acids (BCAA) have been extensively used to improve physical performance. Objective: The objective was to determine whether an 8-h infusion of BCAA enhances MAPR equally in healthy young and elderly adults. Methods: Using a crossover study design, we compared the effect BCAA vs. saline infusion in 12 young (23.0 ± 0.8 yr) and 12 elderly (70.7 ± 1.1 yr) participants matched for sex and body mass index. Skeletal muscle MAPR and mtDNA abundance were measured in muscle biopsy samples obtained before and at the end of the 8-h infusion. Results: In young participants, MAPR with the substrates glutamate plus malate (supplying electrons to complex I) and succinate plus rotenone (complex II) increased in response to BCAA infusion, relative to a decline in MAPR in response to the saline infusion. In contrast, MAPR was unaffected by BCAA infusion in the elderly participants. Moreover, mtDNA abundance was lower in the elderly compared with the young participants but was unaffected by the BCAA infusion. Insulin and C-peptide concentrations declined over time during the saline infusion, but these declines were prevented by the BCAA infusion. Conclusions: BCAA increased skeletal muscle MAPR in the young participants in comparison with saline, but this effect was not seen in the elderly participants indicating, that unlike in the young, BCAA does not increase muscle mitochondrial function in the elderly.

OBJECTIVE Transgenic expression of diacylglycerol acyltransferase-1 (DGAT1) in skeletal muscle leads to protection against fat-induced insulin resistance despite accumulation of intramuscular triglyceride, a phenomenon similar to what is known as the “athlete paradox.” The primary objective of this study is to determine how DGAT1 affects muscle fatty acid oxidation in relation to whole-body energy metabolism and insulin sensitivity. RESEARCH DESIGN AND METHODS We first quantified insulin sensitivity and the relative tissue contributions to the improved whole-body insulin sensitivity in muscle creatine kisase (MCK)-DGAT1 transgenic mice by hyperinsulinemic-euglycemic clamps. Metabolic consequences of DGAT1 overexpression in skeletal muscles were determined by quantifying triglyceride synthesis/storage (anabolic) and fatty acid oxidation (catabolic), in conjunction with gene expression levels of representative marker genes in fatty acid metabolism. Whole-body energy metabolism including food consumption, body weights, oxygen consumption, locomotor activity, and respiration exchange ratios were determined at steady states. RESULTS MCK-DGAT1 mice were protected against muscle lipoptoxicity, although they remain susceptible to hepatic lipotoxicity. While augmenting triglyceride synthesis, DGAT1 overexpression also led to increased muscle mitochondrial fatty acid oxidation efficiency, as compared with wild-type muscles. On a high-fat diet, MCK-DGAT1 mice displayed higher basal metabolic rates and 5–10% lower body weights compared with wild-type littermates, whereas food consumption was not different. CONCLUSIONS DGAT1 overexpression in skeletal muscle led to parallel increases in triglyceride synthesis and fatty acid oxidation. Seemingly paradoxical, this phenomenon is characteristic of insulin-sensitive myofibers and suggests that DGAT1 plays an active role in metabolic “remodeling” of skeletal muscle coupled with insulin sensitization.

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.

Abstract Context: Levels of dehydroepiandrosterone (DHEA) and its sulfate form (DHEAS) are inversely associated with cardiovascular mortality in men but not women. Very little evidence is available on the impact of DHEA administration on lipoprotein profile in women. DHEAS levels are very low/undetectable in hypoadrenal women. Objective: The objective of the study was to determine the impact of DHEA replacement on lipoprotein profile in hypoadrenal women. Design and Setting: A double-blind, randomized, placebo-controlled, cross-over design study was conducted at the Mayo Clinic. Participants: Thirty-three hypoadrenal Caucasian women (mean ± sd; age 50.3 ± 15.2 yr, body mass index 26.6 ± 4.4 kg/m2) took part in the study. Intervention: Study participants were assigned to receive either a placebo or 50 mg/d of DHEA for 3 months each. Lipid levels and lipoprotein profile were analyzed using the Lipo Science Lipoprotein nuclear magnetic resonance system. Main Outcome Measures: Changes in various lipoprotein sizes and levels were measured. Results: The DHEA period had higher plasma DHEAS levels than during placebo (&lt;0.3 ± 0.0 vs. 3.5 ± 1.3 nmol/liter, P &lt; 0.001). DHEA replacement significantly reduced total cholesterol (20.0 vs. −22, P = 0.02) and high-density lipoprotein (HDL) levels (2.0 vs. −6.0, P = 0.006) and tends to reduce triglyceride and total low-density lipoprotein levels. Although, DHEA replacement had no effect on low-density lipoprotein particle size, it significantly reduced larger HDL particles and to modest extent small HDL particles. Conclusions: Our study findings showed that oral DHEA administration in hypoadrenal women results in an unfavorable lipoprotein profile. The results warrant long-term studies to determine the impact of DHEA replacement on cardiovascular risk.