Impact of Long-Term Poor and Good Glycemic Control on Metabolomics Alterations in Type 1 Diabetic People.

CONTEXT: Poor glycemic control in individuals with type 1 diabetes (T1D) is associated with both micro- and macrovascular complications, but good glycemic control does not fully prevent the risk of these complications. OBJECTIVE: The objective of the study was to determine whether T1D with good glycemic control have persistent abnormalities of metabolites and pathways that exist in T1D with poor glycemic control. DESIGN: We compared plasma metabolites in T1D with poor (glycated hemoglobin ≥ 8.5%, T1D[-] and good (glycated hemoglobin < 6.5%, T1D[+]) glycemic control with nondiabetic controls (ND). SETTING: The study was conducted at the clinical research unit. PATIENTS OR OTHER PARTICIPANTS: T1D with poor (n = 14), T1D(-) and good, T1D(+) (n = 15) glycemic control and matched (for age, sex, and body mass index) ND participants were included in the study. INTERVENTION(S): There were no intervention. MAIN OUTCOME MEASURE(S): Comparison of qualitative and quantitative profiling of metabolome was performed. RESULTS: In T1D(-), 347 known metabolites belonging to 38 metabolic pathways involved in cholesterol, vitamin D, tRNA, amino acids (AAs), bile acids, urea, tricarboxylic acid cycle, immune response, and eicosanoids were different from ND. In T1D(+),154 known metabolites belonging to 26 pathways including glycolysis, gluconeogenesis, bile acids, tRNA biosynthesis, AAs, branch-chain AAs, retinol, and vitamin D metabolism remained altered from ND. Targeted measurements of AA metabolites, trichloroacetic acid, and free fatty acids showed directional changes similar to the untargeted metabolomics approach. CONCLUSIONS: Comprehensive metabolomic profiling identified extensive metabolomic abnormalities in T1D with poor glycemic control. Chronic good glycemic control failed to normalize many of these perturbations, suggesting a potential role for these persistent abnormalities in many complications in T1D.

Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins.

Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impacts the sensitivity of the measurement. We therefore applied a high-resolution orbitrap mass spectrometer to the analysis of [ring-(13)C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high-resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension. At higher enrichment levels, enrichment measurements from the orbitrap platform showed significant correlation (R (2) > 0.5) with those of the triple quadrupole platform. Together, these results indicate that high-resolution MS platforms such as the orbitrap are not only as capable of performing isotope enrichment measurements as the more commonly preferred triple quadrupole instruments, but offer unparalleled advantages in terms of mass accuracy and sensitivity in the presence of similar-mass contaminants.

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet - A pilot study.

BACKGROUND & AIMS: Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. METHODS: To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. RESULTS: FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. CONCLUSIONS: Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake.

Comparison of different mass spectrometry techniques in the measurement of L-[ring-(13)C6]phenylalanine incorporation into mixed muscle proteins.

Precise measurement of low enrichment of stable isotope labeled amino-acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L-[ring-(13)C(6)]phenylalanine and a bolus dose of L-[ring-(13)C(6)]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC-combustion-isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring-(13)C(6)]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R(2)= 0.9962 and R(2) = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra-assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter-assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L-[ring-(13)C(6)]phenylalanine tracer enrichment in low abundance and in small quantity samples.

Sparing of muscle mass and function by passive loading in an experimental intensive care unit model.

The response to mechanical stimuli, i.e., tensegrity, plays an important role in regulating cell physiological and pathophysiological function, and the mechanical silencing observed in intensive care unit (ICU) patients leads to a severe and specific muscle wasting condition. This study aims to unravel the underlying mechanisms and the effects of passive mechanical loading on skeletal muscle mass and function at the gene, protein and cellular levels. A unique experimental rat ICU model has been used allowing long-term (weeks) time-resolved analyses of the effects of standardized unilateral passive mechanical loading on skeletal muscle size and function and underlying mechanisms. Results show that passive mechanical loading alleviated the muscle wasting and the loss of force-generation associated with the ICU intervention, resulting in a doubling of the functional capacity of the loaded versus the unloaded muscles after a 2-week ICU intervention. We demonstrate that the improved maintenance of muscle mass and function is probably a consequence of a reduced oxidative stress revealed by lower levels of carbonylated proteins, and a reduced loss of the molecular motor protein myosin. A complex temporal gene expression pattern, delineated by microarray analysis, was observed with loading-induced changes in transcript levels of sarcomeric proteins, muscle developmental processes, stress response, extracellular matrix/cell adhesion proteins and metabolism. Thus, the results from this study show that passive mechanical loading alleviates the severe negative consequences on muscle size and function associated with the mechanical silencing in ICU patients, strongly supporting early and intense physical therapy in immobilized ICU patients.

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.

Concordance of changes in metabolic pathways based on plasma metabolomics and skeletal muscle transcriptomics in type 1 diabetes.

Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry-based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome-based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment.

Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms.

The muscle wasting and impaired muscle function in critically ill intensive care unit (ICU) patients delay recovery from the primary disease, and have debilitating consequences that can persist for years after hospital discharge. It is likely that, in addition to pernicious effects of the primary disease, the basic life support procedures of long-term ICU treatment contribute directly to the progressive impairment of muscle function. This study aims at improving our understanding of the mechanisms underlying muscle wasting in ICU patients by using a unique experimental rat ICU model where animals are mechanically ventilated, sedated and pharmacologically paralysed for duration varying between 6 h and 14 days. Results show that the ICU intervention induces a phenotype resembling the severe muscle wasting and paralysis associated with the acute quadriplegic myopathy (AQM) observed in ICU patients, i.e. a preferential loss of myosin, transcriptional down-regulation of myosin synthesis, muscle atrophy and a dramatic decrease in muscle fibre force generation capacity. Detailed analyses of protein degradation pathways show that the ubiquitin proteasome pathway is highly involved in this process. A sequential change in localisation of muscle-specific RING finger proteins 1/2 (MuRF1/2) observed during the experimental period is suggested to play an instrumental role in both transcriptional regulation and protein degradation. We propose that, for those critically ill patients who develop AQM, complete mechanical silencing, due to pharmacological paralysis or sedation, is a critical factor underlying the preferential loss of the molecular motor protein myosin that leads to impaired muscle function or persisting paralysis.

Impact of type 1 diabetes and insulin treatment on plasma levels and fractional synthesis rate of retinol-binding protein 4.

CONTEXT: Retinol binding protein 4 (RBP4) levels are elevated in insulin-resistant states and reduced in type 1 diabetes (T1D), but it is unknown whether changes in insulin levels and glycemic control alter RBP4 levels. In vivo synthesis rates of RBP4 and their relationship to RBP4 levels remain to be determined. OBJECTIVE: The aim of the study was to determine whether the synthesis rate of RBP4 is altered in people with T1D during both insulin deficiency and insulin treatment. DESIGN: Seven T1D participants were studied on two occasions, during 8 h of insulin deprivation and during insulin treatment, and compared with nondiabetic (ND) controls. MAIN OUTCOME MEASURES: We measured in vivo fractional synthesis rate of RBP4 using [ring-(13)C(6)]phenylalanine as a tracer and RBP4 concentration in plasma by nephelometric assay and Western blot analyses. RESULTS: Plasma RBP4 levels were lower (P < 0.01) in insulin-treated T1D than in ND but were not different between insulin-deprived T1D and ND participants. Synthesis rates of RBP4 in ND (2.46 +/- 0.29%/h) were higher than in insulin-treated T1D (1.45 +/- 0.21) (P = 0.02), but there was no difference between ND and insulin-deprived T1D (2.24 +/- 0.24). Glucose levels were not different between ND and insulin-treated T1D, but insulin levels were higher in insulin-treated T1D (82.8 +/- 2 pmol/liter) than in ND (28.7 +/- 6) and insulin-deprived T1D (4.6 +/- 1.6) (P < 0.01). CONCLUSIONS: Insulin treatment that achieved normoglycemia but relative hyperinsulinemia was associated with lower RBP4 synthesis and levels in T1D. Short-term insulin deprivation and hyperglycemia had no effect on RBP4 levels and synthesis rates in T1D.

Higher muscle protein synthesis in women than men across the lifespan, and failure of androgen administration to amend age-related decrements.

We investigated age and sex effects and determined whether androgen replacement in elderly individuals (> or = 60 yr) could augment protein synthesis. Thirty young men and 32 young women (18-31 yr) were studied once, whereas 87 elderly men were studied before and after 1 yr of treatment with 5 mg/day testosterone (T), 75 mg/day dehydroepiandrosterone (DHEA), or placebo (P); and 57 elderly women were studied before and after 1 yr of treatment with 50 mg/day DHEA or P. [(15)N]Phenylalanine and [(2)H(4)]tyrosine tracers were infused, with measurements in plasma and vastus lateralis muscle. Whole-body protein synthesis per fat-free mass and muscle protein fractional synthesis rate (FSR) were lower in elderly than in young individuals (P<0.001), not significantly affected by hormone treatments, and higher in women than in men (P<0.0001), with no sex x age interaction. In regression analyses, peak O2 consumption (VO2peak), resting energy expenditure (REE), and sex were independently associated with muscle FSR, as were VO2peak, REE, and interactions of sex with insulin-like growth factor-II and insulin for whole-body protein synthesis. Women maintain higher protein synthesis than men across the lifespan as rates decline in both sexes, and neither full replacement of DHEA (in elderly men and women) nor partial replacement of bioavailable T (in elderly men) is able to amend the age-related declines.

Dehydroepiandrosterone replacement therapy in hypoadrenal women: protein anabolism and skeletal muscle function.

OBJECTIVE: To determine whether dehydroepiandrosterone (DHEA) replacement therapy in hypoadrenal women improves performance, muscle protein accretion, and mitochondrial functions. PARTICIPANTS AND METHODS: Thirty-three hypoadrenal women were enrolled in the study from May 1, 2002, through May 31, 2003. Twenty-eight completed a 12-week, prospective, randomized, placebo-controlled, crossover study with either daily placebo or 50 mg of DHEA with a 2-week washout period and then crossed over to the other treatment. Body composition, physical performance, whole-body and muscle protein metabolism, and mitochondrial functions were determined. RESULTS: Administration of DHEA significantly increased plasma levels of DHEA sulfate, testosterone, and androstenedione but did not change body composition, muscle strength, peak aerobic capacity, and whole-body protein turnover or synthesis rates of mitochondrial, sarcoplasmic, or mixed muscle proteins. Muscle mitochondrial oxidative enzymes and messenger RNA (mRNA) levels of genes encoding mitochondrial proteins and nuclear transcription factors did not change after DHEA administration. However, mRNA levels of muscle myosin heavy chain 1 (P=.004), which determines muscle fiber type, and those of insulinlike growth factor binding proteins 4 and 5 significantly decreased (P=.02 and P=.03, respectively). CONCLUSION: Three months of DHEA administration increased DHEA sulfate and androgen levels but had no effect on physical performance, body composition, protein metabolism, or muscle mitochondrial biogenesis in hypoadrenal women. However, lowering of mRNA levels of binding proteins of insulinlike growth factor 1 and myosin heavy chain 1 suggests potential effects of longterm treatment with DHEA on muscle fiber type.

In vivo measurement of synthesis rate of individual skeletal muscle mitochondrial proteins.

Skeletal muscle mitochondrial dysfunction occurs in many conditions including aging and insulin resistance, but the molecular pathways of the mitochondrial dysfunction remain unclear. Presently, no methodologies are available to measure synthesis rates of individual mitochondrial proteins, which limits our ability to fully understand the translational regulation of gene transcripts. Here, we report a methodology to measure synthesis rates of multiple muscle mitochondrial proteins, which, along with large-scale measurements of mitochondrial gene transcripts and protein concentrations, will enable us to determine whether mitochondrial alteration is due to transcriptional or translational changes. The methodology involves in vivo labeling of muscle proteins with l-[ring-(13)C(6)]phenylalanine, protein purification by two-dimensional gel electrophoresis of muscle mitochondrial fraction, and protein identification and stable isotope abundance measurements by tandem mass spectrometry. Synthesis rates of 68 mitochondrial and 23 nonmitochondrial proteins from skeletal muscle mitochondrial fraction showed a 10-fold range, with the lowest rate for a structural protein such as myosin heavy chain (0.16 +/- 0.04%/h) and the highest for a mitochondrial protein such as dihydrolipoamide branched chain transacylase E2 (1.5 +/- 0.42%/h). This method offers an opportunity to better define the translational regulation of proteins in skeletal muscle or other tissues.

Proteasome production in human muscle during nutritional inhibition of myofibrillar protein degradation.

Protein undernutrition inhibits adenosine triphosphate (ATP)-dependent muscle protein degradation-a hallmark of the proteasome system. Here we report decreased myofibrillar protein degradation during dietary protein restriction without a concomitant decrease in proteasome gene expression, proteasome protein abundance, or proteasome in vivo fractional synthesis rate. Healthy human subjects consuming the average minimum adult protein requirement (0.71 g x kg(-1) fat-free mass x d(-1)) exhibited substantially lower (68%) excretion of 3-methylhistidine, an indicator of myofibrillar protein breakdown, when compared with subjects consuming an ample, American-style protein intake (1.67 g x kg(-1) fat-free mass x d(-1)). However, they displayed no difference in the expression of mRNA for proteasome subunits C2 or C3, in the content of C2 protein, or in the rate of incorporation of stable isotopically labeled l-[1-(13)C]-leucine into proteasome proteins. The results demonstrate that nutritional inhibition of myofibrillar protein degradation does not involve suppression in vivo of proteasome production in man. This suggests that other elements of the ubiquitin-proteasome system, such as ubiquitination pathways, are more important than proteasome abundance in the nutritional regulation of skeletal muscle mass.

Potential mechanisms of marked hyperoxaluria not due to primary hyperoxaluria I or II.

BACKGROUND: Hyperoxaluria may be idiopathic, secondary, or due to primary hyperoxaluria (PH). Hepatic alanine:glyoxylate aminotransferase (AGT) or glyoxylate/hydroxypyruvate reductase (GR/HPR) deficiency causes PHI or PHII, respectively. Hepatic glycolate oxidase (GO) is a candidate enzyme for a third form of inherited hyperoxaluria. METHODS: Six children were identified with marked hyperoxaluria, urolithiasis, and normal hepatic AGT (N = 5) and GR/HPR (N = 4). HPR was below normal and GR not measured in one. Of an affected sibling pair, only one underwent biopsy. GO mutation screening was performed, and dietary oxalate (Diet(ox)), enteric oxalate absorption (EOA) measured using [13C2] oxalate, renal clearance (GFR), fractional oxalate excretion (FE(ox)) in the children, and urine oxalate in first-degree relatives (FDR) to understand the etiology of the hyperoxaluria. RESULTS: Mean presenting age was 19.2 months and urine oxalate 1.3 +/- 0.5 mmol/1.73 m2/24 h (mean +/- SD). Two GO sequence changes (T754C, IVS3 - 49 C>G) were detected which were not linked to the hyperoxaluria. Diet(ox) was 42 +/- 31 mg/day. EOA was 9.4 +/- 3.6%, compared with 7.6 +/- 1.2% in age-matched controls (P = 0.33). GFR was 90 +/- 19 mL/min/1.73 m2 and FE(ox) 4.2 +/- 1.4. Aside from the two brothers, hyperoxaluria was not found in FDR. CONCLUSIONS: These patients illustrate a novel form of hyperoxaluria and urolithiasis, without excess Diet(ox), enteric hyper-absorption, or hepatic AGT, GR/HPR deficiency. Alterations in pathways of oxalate synthesis, in liver or kidney, or in renal tubular oxalate handling are possible explanations. The affected sibling pair suggests an inherited basis.

Aminoacyl-tRNA enrichment after a flood of labeled phenylalanine: insulin effect on muscle protein synthesis.

Muscle protein synthesis in dogs measured by flooding with L-[(2)H(5)]phenylalanine (70 mg/kg) was significantly stimulated by infusion of insulin with amino acids. The stimulation of muscle protein synthesis was similar when calculated from the enrichment of phenylalanyl-tRNA (61 +/- 10%, P < 0.001), plasma phenylalanine (61 +/- 10%, P < 0.001), or tissue fluid phenylalanine (54 +/- 10%, P < 0.001). The time course for changes in enrichment of L-[(2)H(5)]phenylalanine throughout the flooding period was determined for plasma, tissue fluid, and phenylalanyl-tRNA in the basal state and during the infusion of insulin with amino acids. Enrichments of plasma free phenylalanine and phenylalanyl-tRNA were equalized between 20 and 45 min, although the enrichment of phenylalanyl-tRNA was lower at early time points. Rates of muscle protein synthesis obtained with the flooding method and calculated from plasma phenylalanine enrichment were comparable to those calculated from phenylalanyl-tRNA and also to those obtained previously with a continuous infusion of phenylalanine with phenylalanyl-tRNA as precursor. This study confirms that, with a bolus injection of labeled phenylalanine, the enrichment of aminoacyl-tRNA, the true precursor pool for protein synthesis, can be assessed from more readily sampled plasma phenylalanine.