Training status diverges muscle diacylglycerol accumulation during free fatty acid elevation.

How endurance training alters muscle lipid metabolism while preserving insulin sensitivity remains unclear. Because acute free fatty acid (FFA) elevation by lipid infusion reduces insulin sensitivity, we hypothesized that training status would alter accumulation of muscle triacylglycerol (TAG), diacylglycerol (DAG), ceramide, and acylcarnitine during acute FFA elevation. Trained (n = 15) and sedentary (n = 13) participants matched for age, sex, and BMI received either a 6-h infusion of lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) during a hyperinsulinemic euglycemic clamp. Muscle biopsies were taken at 0, 120, and 360 min after infusion initiation to measure intramyocellular concentrations of TAG, DAG, ceramides, and acylcarnitines by liquid chromatography-tandem mass spectrometry. Trained participants had a higher Vo2 max and insulin sensitivity than sedentary participants. The lipid infusion produced a comparable elevation of FFA (594 ± 90 µmol/l in trained, 721 ± 30 µmol/l in sedentary, P = 0.4) and a decline in insulin sensitivity (-44.7% trained vs. -47.2% sedentary, P = 0.89). In both groups, lipid infusion increased the linoleic and linolenic acid content of TAG without changing total TAG. In the sedentary group, lipid infusion increased total, oleic, and linoleic acid and linolenic acid content of DAG. Regardless of training status, lipid infusion did not alter total ceramide, saturated ceramide, palmitoyl-carnitine, or oleoyl-carnitine. We conclude that during acute FFA elevation, trained adults have a similar decline in insulin sensitivity with less accumulation of muscle DAG than sedentary adults, suggesting that lipid-induced insulin resistance can occur without elevation of total muscle DAG.

Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice.

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 GYS3ß 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 GYS3ß 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.

A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans.

RATIONALE: Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre-existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U-(13)C]palmitate into intramyocellular ceramides. METHODS: We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and (13)C-isotopic enrichment of 16:0-ceramide. Chromatographic separation was performed using ultra-performance liquid chromatography. The analysis was performed on a triple quadrupole mass spectrometer using a positive ion electrospray ionization source with selected reaction monitoring (SRM). RESULTS: The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H](+). The [(13)C(16)]16:0-ceramide was monitored as [M+16+H](+). By monitoring two different transitions of the [(13)C(16)]16:0-ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U-(13)C]palmitate infusion. CONCLUSIONS: LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo.

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS.

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of (13)C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of (13)C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.

Effects of insulin deprivation and treatment on homocysteine metabolism in people with type 1 diabetes.

CONTEXT: Abnormal homocysteine metabolism may contribute to increased cardiovascular death in type 1 diabetes (T1DM). Amino acid metabolism is altered in T1DM. In vitro, insulin reduces hepatic catabolism of homocysteine by inhibiting liver transsulfuration. It remains to be determined whether methionine-homocysteine metabolism is altered in T1DM. OBJECTIVE: We sought to determine whether insulin deficiency during insulin deprivation or high plasma insulin concentration after insulin treatment alters homocysteine metabolism in T1DM. DESIGN: This was an acute interventional study with paired and comparative controls. SETTING: The study was conducted at a general clinical research center. PATIENTS AND INTERVENTION: We used stable isotope tracers to measure methionine-homocysteine kinetics in six patients with T1DM during insulin deprivation (I-) and also during insulin treatment (I+) and compared them with nondiabetic controls (n = 6). MAIN OUTCOME MEASURES: Homocysteine kinetics (transmethylation, transsulfuration, and remethylation) were from plasma isotopic enrichment of methionine and homocysteine and (13)CO(2). RESULTS: T1DM (I-) had lower rates of homocysteine-methionine remethylation (P < 0.05 vs. control and I+). In contrast, transsulfuration rates were higher in I- than controls and I+ (P < 0.05). Insulin treatment normalized transsulfuration and remethylation (P < 0.05 vs. I- and P > 0.8 vs. control). Plasma homocysteine concentrations were lower in T1DM (P < 0.05 vs. control during both I- and I+), which may be explained by increased homocysteine transsulfuration. Thus, significant alterations of methionine-homocysteine metabolism occur during insulin deprivation in humans with T1DM. CONCLUSIONS: Insulin plays a key role in the regulation of methionine-homocysteine metabolism in humans, and altered homocysteine may occur during insulin deficiency in type 1 diabetic patients.

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.

Plasma sphingolipid changes with autopsy-confirmed Lewy Body or Alzheimer's pathology.

INTRODUCTION: The clinical and pathological phenotypes of Dementia with Lewy Bodies (DLB) and Alzheimer's disease (AD) often overlap. We examined whether plasma lipids differed among individuals with autopsy-confirmed Lewy Body pathology or AD pathology. METHODS: We identified four groups with available plasma two years prior to death: high (n=12) and intermediate likelihood DLB (n=14) based on the third report of the DLB consortium; dementia with Alzheimer's pathology (AD; n=18); and cognitively normal with normal aging pathology (n=21). Lipids were measured using ESI/MS/MS. RESULTS: There were overall group differences in plasma ceramides C16:0, C18:1, C20:0 and C24:1 and monohexosylceramides C18:1 and C24:1. These lipids did not differ between the high likelihood DLB and AD groups, but both groups had higher levels than normals. Plasma fatty acid levels did not differ by group. DISCUSSION: Plasma ceramides and monohexosylceramides are elevated in people with dementia with either high likelihood DLB or AD pathology.

Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death.

A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression. However, the exact role of AMPK in tumor development is still controversial. Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8+ T cells, resulting in superior tumor suppression in vivo. While AMPK expression is dispensable for T cell development, genetic deletion of AMPK promotes T cell death during in vitro activation and in vivo tumor development. Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function. Altogether, our data suggest a novel mechanism by which AMPK regulates protein phosphatase activity in control of survival and function of CD8+ T cells, thereby enhancing their role in tumor immunosurveillance.

Identification of altered metabolic pathways in plasma and CSF in mild cognitive impairment and Alzheimer's disease using metabolomics.

Alzheimer's Disease (AD) currently affects more than 5 million Americans, with numbers expected to grow dramatically as the population ages. The pathophysiological changes in AD patients begin decades before the onset of dementia, highlighting the urgent need for the development of early diagnostic methods. Compelling data demonstrate that increased levels of amyloid-beta compromise multiple cellular pathways; thus, the investigation of changes in various cellular networks is essential to advance our understanding of early disease mechanisms and to identify novel therapeutic targets. We applied a liquid chromatography/mass spectrometry-based non-targeted metabolomics approach to determine global metabolic changes in plasma and cerebrospinal fluid (CSF) from the same individuals with different AD severity. Metabolic profiling detected a total of significantly altered 342 plasma and 351 CSF metabolites, of which 22% were identified. Based on the changes of >150 metabolites, we found 23 altered canonical pathways in plasma and 20 in CSF in mild cognitive impairment (MCI) vs. cognitively normal (CN) individuals with a false discovery rate <0.05. The number of affected pathways increased with disease severity in both fluids. Lysine metabolism in plasma and the Krebs cycle in CSF were significantly affected in MCI vs. CN. Cholesterol and sphingolipids transport was altered in both CSF and plasma of AD vs. CN. Other 30 canonical pathways significantly disturbed in MCI and AD patients included energy metabolism, Krebs cycle, mitochondrial function, neurotransmitter and amino acid metabolism, and lipid biosynthesis. Pathways in plasma that discriminated between all groups included polyamine, lysine, tryptophan metabolism, and aminoacyl-tRNA biosynthesis; and in CSF involved cortisone and prostaglandin 2 biosynthesis and metabolism. Our data suggest metabolomics could advance our understanding of the early disease mechanisms shared in progression from CN to MCI and to AD.

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.

In vivo measurement of synthesis rate of multiple plasma proteins in humans.

Advances in quantitative proteomics have facilitated the measurement of large-scale protein quantification, which represents net changes in protein synthesis and breakdown. However, measuring the rate of protein synthesis is the only way to determine the translational rate of gene transcripts. Here, we report a technique to measure the rate of incorporation of amino acids from ingested protein labeled with stable isotope into individual plasma proteins. This approach involves three steps: 1) production of stable isotope-labeled milk whey protein, oral administration of this intrinsically labeled protein, and subsequent collection of blood samples; 2) fractionation of the plasma and separation of the individual plasma proteins by a combination of anion exchange high-pressure liquid chromatography and gel electrophoresis; and 3) identification of individual plasma proteins by tandem mass spectrometry and measurement of stable isotopic enrichment of these proteins by gas chromatography-mass spectrometry. This method allowed the measurement of the fractional synthesis rate (FSR) of 29 different plasma proteins by using the same precursor pool. We noted a 30-fold difference in FSR of different plasma proteins with a wide range of physiological functions. This approach offers a tremendous opportunity to study the regulation of plasma proteins in humans in many physiological and pathological states.