Summary Metabolomic profiling of obese versus lean humans reveals a branched-chain amino acid (BCAA)-related metabolite signature that is suggestive of increased catabolism of BCAA and correlated with insulin resistance. To test its impact on metabolic homeostasis, we fed rats on high-fat (HF), HF with supplemented BCAA (HF/BCAA) or standard chow (SC) diets. Despite having reduced food intake and weight gain equivalent to the SC group, HF/BCAA rats were equally insulin resistant as HF rats. Pair-feeding of HF diet to match the HF/BCAA animals or BCAA addition to SC diet did not cause insulin resistance. Insulin resistance induced by HF/BCAA feeding was accompanied by chronic phosphorylation of mTOR, JNK, and IRS1(ser307), accumulation of multiple acylcarnitines in muscle, and was reversed by the mTOR inhibitor, rapamycin. Our findings show that in the context of a poor dietary pattern that includes high fat consumption, BCAA contributes to development of obesity-associated insulin resistance.

In preparation of the paper, there were several errors in the figure labeling, which were regretfully missed in the preparation and proofreading of the manuscript and which the authors would like to correct. None of these changes affects the data or the conclusions of the paper.(1) The heading of Figure 2H should read ''Glucose Infusion Rate,'' not ''Insulin Infusion Rate.'' (2) In the corresponding text on page 431 (right column, paragraph 2, line 13), the units for glucose infusion rate should be ''mg/kg/min,'' not ''mg/dl.'' (3) Likewise, on the y axis in Figure 2I, the units for glucose should read ''mg/kg/min'' rather than ''mg/dl.'' (4) On the y axis in Figures 3C, 4F, 4G, 4H, and 5D, the parenthetical reference to ''ARNT/Actin'' carried over from previous figures should simply be deleted. The correct specific genes or proteins measured in each panel are already indicated. (5) In Figure 5A, the correct units are ''mM,'' not ''mM/l.

No abstract

Lipid infusion or ingestion of a high-fat diet results in insulin resistance, but the mechanism underlying this phenomenon remains unclear. Here we show that, in rats fed a high-fat diet, whole-animal, muscle and liver insulin resistance is ameliorated following hepatic overexpression of malonyl-coenzyme A (CoA) decarboxylase (MCD), an enzyme that affects lipid partitioning. MCD overexpression decreased circulating free fatty acid (FFA) and liver triglyceride content. In skeletal muscle, levels of triglyceride and long-chain acyl-CoA (LC-CoA)-two candidate mediators of insulin resistance-were either increased or unchanged. Metabolic profiling of 36 acylcarnitine species by tandem mass spectrometry revealed a unique decrease in the concentration of one lipid-derived metabolite, beta-OH-butyrate, in muscle of MCD-overexpressing animals. The best explanation for our findings is that hepatic expression of MCD lowered circulating FFA levels, which led to lowering of muscle beta-OH-butyrate levels and improvement of insulin sensitivity.

Children with certain inherited metabolic disorders excrete diagnostic acylcarnitines which reflect unusual acyl-CoA intermediates accumulating at the metabolic block (Roe et at., 1986). These metabolites can be detected in urine, if their concentration exceeds about 50nmol/ml, by fast atom bombardment mass spectrometry (FAB-MS). By applying tandem mass spectrometry (MS/MS) it is possible to lower the detection limit to 1 nmot/ml in urine or blood (Millington et al., 1989). We investigated the potential of this new technique to identify metabolic disorders from small blood samples and from blood spots taken for existing neonatal screening tests (Guthrie cards). The preliminary results are presented here. These children were not receiving L-carnitine supplements at the time of sampling. A sample of umbilical cord blood from a normal child was employed as a control. The original Guthrie cards were recovered for children with subsequently confirmed diagnoses of MCAD deficiency and propionic acidaemia, recently diagnosed at Duke Medical Center. Controls were obtained from the North Carolina State laboratory. To determine recovery, normal blood was spiked appropriately with known concentrations of specific acylcarnitines and internal standards after spotting onto and recovery from Guthrie cards, as described by Millington et al. (1989). 321

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD ؉͞؊ mice yielded an abnormally low number of LCAD ؉͞؊ and ؊͞؊ offspring, indicating frequent gestational loss. LCAD ؊͞؊ mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD ؊͞؊ males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD ؊͞؊ mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.Mitochondrial fatty acid oxidation (FAO) is the primary means by which energy is derived from metabolism of fatty acids. This process is important during periods of fasting or prolonged strenuous activity, providing as much as 80 to 90% of fatty acid-derived energy for heart and liver function (1). Mitochondrial FAO also provides acetyl-CoA for hepatic ketogenesis and the energy required for nonshivering thermogenesis by brown adipose tissue (2). The initial step in mitochondrial FAO is the ␣-␤ dehydrogenation of the acyl-CoA ester by a family of four closely related, chain length-specific enzymes, the acyl-CoA dehydrogenases, which include verylong-chain, long-chain, medium-chain, and short-chain acylCoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively). These enzymes catalyze the same type of reaction but differ in specificity according to the chain length of their fatty acid (acyl-CoA) substrates.

North Carolina (NC) was the first US state to initiate universal tandem mass spectrometry (MS/MS) newborn screening. This began as a statewide pilot project in 1997 to determine the incidence and feasibility of screening for fatty acid oxidation, organic acid and selected amino acid disorders. The MS/MS analyses were done by a commercial laboratory and all follow-up and confirmatory testing was performed through the NC Newborn Screening (NBS) Program. In April 1999, the NC NBS Laboratory began the MS/MS analyses in-house. Between 28 July 1997 and 28 July 2005, 944,078 infants were screened and 219 diagnoses were confirmed on newborns with elevated screening results, for an overall incidence of 1:4,300. Ninety-nine infants were identified with fatty acid oxidation disorders, 58 with organic acidaemias and 62 with aminoacidopathies. Medium-chain acyl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase deficiency and disorders of phenylalanine metabolism were the most common disorders detected. Identification of affected infants has allowed retrospective testing of other family members, resulting in an additional 16 diagnoses. Seven neonates died from complications of their metabolic disorders/prematurity despite timely MS/MS screening. In addition, there were six infants who were not identified by elevated NBS results but who presented with symptoms later in infancy. The NC MS/MS NBS Program uses a two-tier system, categorizing results as either 'borderline' or 'diagnostic' elevated, for both the cutoffs and follow-up protocol. Infants with an initial borderline result had only a repeat screen. Infants with a diagnostic or two borderline results were referred for confirmatory testing. The positive predictive value of the NC MS/MS NBS for those infants requiring confirmatory testing was 53% for 2003 and 2004. The success of the NC MS/MS NBS Program in identifying infants with metabolic disorders was dependent on a comprehensive follow-up protocol integrating the public health laboratory and the academic metabolic centres.

A new method for quantifying specific amino acids in small volumes of plasma and whole blood has been developed. Based on isotope-dilution tandem mass spectrometry, the method takes only a few minutes to perform and requires minimal sample preparation. The accurate assay of both phenylalanine and tyrosine in dried blood spots used for neonatal screening for phenylketonuria in North Carolina successfully differentiated infants who had been classified as normal, affected, and falsely positive by current fluorometric methods. Because the mass-spectrometric method also recognizes other aminoacidemias simultaneously and is capable of automation, it represents a useful development toward a broad-spectrum neonatal screening method.