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Background Tracing stable isotopes, such as 13 C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. However, extracting such information requires special care, such as a correction for naturally occurring isotopes, or overlapping mass spectra of various components of the cell culture medium. Developing a method for a correction of overlapping peaks is the primary objective of this study. Results Our computer program-MIDcor (free at https://github.com/seliv55/mid_correct) written in the R programming language, corrects the raw MS spectra both for the naturally occurring isotopes and for the overlapping of peaks corresponding to various substances. To this end, the mass spectra of unlabeled metabolites measured in two media are necessary: in a minimal medium containing only derivatized metabolites and chemicals for derivatization, and in a complete cell incubated medium. The MIDcor program calculates the difference ( D ) between the theoretical and experimentally measured spectra of metabolites containing only the naturally occurring isotopes. The result of comparison of D in the two media determines a way of deciphering the true spectra. (1) If D in the complete medium is greater than that in the minimal medium in at least one peak, then unchanged D is subtracted from the raw spectra of the labeled metabolite. (2) If D does not depend on the medium, then the spectrum probably overlaps with a derivatized fragment of the same metabolite, and D is modified proportionally to the metabolite labeling. The program automatically reaches a decision regarding the way of correction. For some metabolites/fragments in the case (2) D was found to decrease when the tested substance was 13 C labeled, and this isotopic effect also can be corrected automatically, if the user provides a measured spectrum of the substance in which the 13 C labeling is known a priori. Conclusion Using the developed program improves the reliability of stable isotope tracer data analysis.

Metabolic reprogramming is implicated in macrophage activation, but the underlying mechanisms are poorly understood. Here, we demonstrate that the NOTCH1 pathway dictates activation of M1 phenotypes in isolated mouse hepatic macrophages (HMacs) and in a murine macrophage cell line by coupling transcriptional upregulation of M1 genes with metabolic upregulation of mitochondrial oxidative phosphorylation and ROS (mtROS) to augment induction of M1 genes. Enhanced mitochondrial glucose oxidation was achieved by increased recruitment of the NOTCH1 intracellular domain (NICD1) to nuclear and mitochondrial genes that encode respiratory chain components and by NOTCH-dependent induction of pyruvate dehydrogenase phosphatase 1 (Pdp1) expression, pyruvate dehydrogenase activity, and glucose flux to the TCA cycle. As such, inhibition of the NOTCH pathway or Pdp1 knockdown abrogated glucose oxidation, mtROS, and M1 gene expression. Conditional NOTCH1 deficiency in the myeloid lineage attenuated HMac M1 activation and inflammation in a murine model of alcoholic steatohepatitis and markedly reduced lethality following endotoxin-mediated fulminant hepatitis in mice. In vivo monocyte tracking further demonstrated the requirement of NOTCH1 for the migration of blood monocytes into the liver and subsequent M1 differentiation. Together, these results reveal that NOTCH1 promotes reprogramming of mitochondrial metabolism for M1 macrophage activation.

The metabolism of [1, 2 13C2] glucose via the tricarboxylic acid (TCA) cycle yields a number of key glutamate mass isotopomers whose formation is a function of pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH). Analysis of the isotopomer distribution patterns was used to determine the relative flux of glucose entry into the TCA cycle through anaplerotic and oxidative pathways in the cerebral cortex of both uninjured and traumatically injured adult male rats. In the cerebral cortex of uninjured animals the PC/PDH ratio showed greater metabolism of glucose via pyruvate carboxylase, which is consistent with the notion that the majority of glucose taken up at rest is used as a substrate for anaplerotic processes and not as an energy source. While traumatic brain injury did not change the overall 13C enrichment of glutamate indicating a continued oxidation of glucose, the PC/PDH ratio was reduced in the injured cortex at 3.5 h after injury. This suggests that glucose metabolism is primarily directed through pathways associated with energy production in the early postinjury period. By 24 h, the anaplerotic flux decreased and the PC/PDH ratio increased in both the injured and non-injured cortex indicating a switch away from energy production to pathways associated with anabolic and/or regenerative processes.

Lipin Deficiency Impairs Diurnal Metabolic Fuel Switching Jun Xu 1 , W.N. Paul Lee 2 , Jack Phan 3 , Mohammed F. Saad 4 , Karen Reue 3 5 6 7 and Irwin J. Kurland 1 8 9 1 Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York 2 Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California 3 Department of Human Genetics, University of California, Los Angeles, California 4 Department of Preventative Medicine, State University of New York at Stony Brook, Stony Brook, New York 5 Department of Medicine, University of California, Los Angeles, California 6 Molecular Biology Institute, University of California, Los Angeles, California 7 VA Greater Los Angeles Healthcare System, Los Angeles, California 8 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 9 Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York Address correspondence and reprint requests to Irwin J. Kurland, MD, PHD, State University of New York at Stony Brook, HSC T-15 Room 060, Stony Brook, NY 11794-8154. E-mail: irwin.kurland{at}stonybrook.edu Abstract Fatty liver is a common feature of both obesity and lipodystrophy, reflecting compromised adipose tissue function. The lipin-deficient fatty liver dystrophy ( fld ) mouse is an exception, as there is lipodystrophy without a fatty liver. Using a combination of indirect calorimetry and stable-isotope flux phenotyping, we determined that fld mice exhibit abnormal fuel utilization throughout the diurnal cycle, with increased glucose oxidation near the end of the fasting period and increased fatty acid oxidation during the feeding period. The mechanisms underlying these alterations include a twofold increase compared with wild-type mice in tissue glycogen storage during the fed state, a 40% reduction in hepatic glucose production in the fasted state, and a 27-fold increase in de novo fatty acid synthesis in liver during the fed state. Thus, the inability to store energy in adipose tissue in the fld mouse leads to a compensatory increase in glycogen storage for use during the fasting period and reliance upon hepatic fatty acid synthesis to provide fuel for peripheral tissues during the fed state. The increase in hepatic fatty acid synthesis and peripheral utilization provides a potential mechanism to ameliorate fatty liver in the fld that would otherwise occur as a consequence of adipose tissue dysfunction. FNS, fraction of newly synthesized palmitate molecules HGP, hepatic glucose production PDH, pyruvate dehydrogenase PDK4, pyruvate dehydrogenase kinase isoform 4 PEPCK, phosphoenolpyruvate carboxykinase RQ, respiratory quotient TCA, tricarboxylic acid Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted August 4, 2006. Received February 23, 2006. DIABETES

Decreased Hepatic Futile Cycling Compensates for Increased Glucose Disposal in the Pten Heterodeficient Mouse Jun Xu 1 , Lori Gowen 2 , Christian Raphalides 2 , Katrina K. Hoyer 3 , Jason G. Weinger 3 , Mathilde Renard 3 , Joshua J. Troke 3 , Bhavapriya Vaitheesyaran 1 , W.N. Paul Lee 4 , Mohammed F. Saad 5 , Mark W. Sleeman 2 , Michael A. Teitell 3 6 and Irwin J. Kurland 1 7 1 Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York 2 Regeneron Pharmaceuticals, Tarrytown, New York 3 Department of Pathology, University of California Los Angeles, Los Angeles, California 4 Department of Pediatrics, Harbor-University of California Los Angeles Biomedical Institute, Torrance, California 5 Department of Preventive Medicine, State University of New York at Stony Brook, Stony Brook, New York 6 Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 7 Departments of Pharmacological Sciences and Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York Address correspondence and reprint requests to Irwin J. Kurland, SUNY at Stony Brook, HSC T-15 Room 060, Stony Brook, NY 11794-8154. E-mail: irwin.kurland{at}stonybrook.edu Abstract Despite altered regulation of insulin signaling, Pten +/− heterodeficient standard diet–fed mice, ∼4 months old, exhibit normal fasting glucose and insulin levels. We report here a stable isotope flux phenotyping study of this “silent” phenotype, in which tissue-specific insulin effects in whole-body Pten +/− -deficient mice were dissected in vivo. Flux phenotyping showed gain of function in Pten +/− mice, seen as increased peripheral glucose disposal, and compensation by a metabolic feedback mechanism that 1 ) decreases hepatic glucose recycling via suppression of glucokinase expression in the basal state to preserve hepatic glucose production and 2 ) increases hepatic responsiveness in the fasted-to-fed transition. In Pten +/− mice, hepatic gene expression of glucokinase was 10-fold less than wild-type ( Pten +/+ ) mice in the fasted state and reached Pten +/+ values in the fed state. Glucose-6-phosphatase expression was the same for Pten +/− and Pten +/+ mice in the fasted state, and its expression for Pten +/− was 25% of Pten +/+ in the fed state. This study demonstrates how intra- and interorgan flux compensations can preserve glucose homeostasis (despite a specific gene defect that accelerates glucose disposal) and how flux phenotyping can dissect these tissue-specific flux compensations in mice presenting with a “silent” phenotype. AUC, area under the curve G6PDH, glucose-6-phosphate dehydrogenase GC/MS, gas chromatography–mass spectrometry glucose-6-P, glucose-6-phosphate HGP, hepatic glucose production HR-dGTT, hepatic recycling deuterated glucose tolerance test HR-GTT, hepatic recycling glucose tolerance test ipGTT, intraperitoneal glucose tolerance test ITT, insulin tolerance test PI3-K, phosphatidylinositol 3-kinase PPAR, peroxisome proliferator–activated receptor PTEN, phosphatase and tensin homolog deleted on chromosome 10 TCA, trichloroacetic acid Footnotes Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted September 6, 2006. Received January 2, 2006. DIABETES

Background: Recent reports have described inherent problems with androgen immunoassays compared with mass spectrometry analyses. Methods: We developed a method for measuring serum testosterone (T) and 5α-dihydrotestosterone (DHT) simultaneously via liquid–liquid extraction followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) with positive-mode electrospray ionization. Results: The DHT and T calibrators showed a linear response from 0.069 nmol/L to 34.4 nmol/L and 69.3 nmol/L, respectively. T interference in the DHT assay and vice versa were negligible. Within- and between-run imprecision values were <5% for both analytes. Percent recoveries of T and DHT spiked into samples at concentrations spanning the calibration curve were 100%–113% and 98%–107%, respectively. The lower limit of quantification was 0.069 nmol/L for both steroids. Serum T concentrations measured by LC-MS/MS were different from those obtained by RIA, especially at lower T concentrations. Serum DHT concentrations measured by LC-MS/MS were markedly lower than those generated by RIA because of the nonselectivity of the RIA without chromatography. The reference intervals (mean ± 2 SDs) determined for T and DHT were 9.2–33.7 nmol/L and 0.47–2.65 nmol/L, respectively, for 113 healthy adult men and 0.33–2.02 nmol/L and 0.09–0.91 nmol/L, respectively, for 133 healthy premenopausal women. Conclusions: We have developed and validated a selective and precise method for simultaneous measurements of serum T and DHT that can be adopted for routine measurements of these androgens in health and disease in men and women.

Metabolic control analysis (MCA) provides a quantitative description of substrate flux in response to changes in system parameters of complex enzyme systems. Medical applications of the approach include the following: understanding the threshold effect in the manifestation of metabolic diseases; investigating the gene dose effect of aneuploidy in inducing phenotypic transformation in cancer; correlating the contributions of individual genes and phenotypic characteristics in metabolic disease (e.g., diabetes); identifying candidate enzymes in pathways suitable as targets for cancer therapy; and elucidating the function of \"silent\" genes by identifying metabolic features shared with genes of known pathways. MCA complements current studies of genomics and proteomics, providing a link between biochemistry and functional genomics that relates the expression of genes and gene products to cellular biochemical and physiological events. Thus, it is an important tool for the study of genotype–phenotype correlations. It allows genes to be ranked according to their importance in controlling and regulating cellular metabolic networks. We can expect that MCA will have an increasing impact on the choice of targets for intervention in drug discovery.

Background Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results The study of 13 C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ≤5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo , which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy.

Background: Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, significantly enhances lung maturation without affecting blood biochemical and metabolic profiles in the newborn period. However, whether this exposure to RGZ in neonatal life alters the adult metabolic phenotype is not known. Objective: To determine the effects of early postnatal administration of RGZ on the young adult metabolic phenotype. Methods: Newborn rat pups were administered either saline or RGZ for the first 7 days of life. At 11–14 weeks, glucose and insulin tolerance tests and deuterium labeling were performed. Blood and tissues were analyzed for various metabolic parameters. Results: Overall, there was no effect of early postnatal RGZ administration on young adult body weight, glucose and insulin tolerance, plasma cholesterol and triglyceride profiles, insulin, glucagon, cardiac troponin, fatty acid synthesis, or tissue adipogenic differentiation. Conclusions: Treatment with RGZ in early neonatal life does not alter later developmental metabolic programming or lead to an altered metabolic phenotype in the young adult, further re-enforcing the safety of PPARγ agonists as a novel lung-protective strategy.

To describe the uncommon presentation of hyperinsulinism in an 8-year-old boy. We describe the patient's clinical findings, results from biochemical and imaging studies, surgical approach, and outcome. The discussion encompasses a review of literature that provided the basis for the diagnostic and surgical approach applied to this patient's case. An obese 8.5-year-old boy initially presented with hypoglycemic seizures after initiation of dietary changes to treat obesity. Biochemical analysis indicated hyperinsulinism. Endoscopic ultrasonography showed no pancreatic lesions suggestive of insulinoma. Genetic studies identified no known mutations in the ABCC8, KCNJ11, GCK, or GLUD1 genes. Selective arterial calcium stimulation and hepatic venous sampling did not document a focal source for hyperinsulinism in the pancreas, and positron emission tomography with 18-fluoro-L-3,4-dihydroxyphenylalanine showed diffusely increased uptake in the pancreas. The patient ultimately required partial pancreatectomy because of continued hypoglycemia while taking diazoxide and octreotide. Intraoperative glucose monitoring directed the extent of surgical resection. A 45% pancreatectomy was performed, which resolved the hypoglycemia but led to impaired glucose tolerance after surgery. The unusual presentation of hyperinsulinism in childhood required a personalized approach to diagnosis and surgical management using intraoperative glucose monitoring that resulted in a conservative pancreatectomy.