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Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyte Jak2 (JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyte Jak2 (JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.

We present a liquid chromatography--mass spectrometry (LC--MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water--methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 to 1000 m/z at 1 Hz and 100 000 resolution in negative ion mode on a stand alone orbitrap ('Exactive'). The median limit of detection, across 80 metabolite standards, was 5 ng/ mL with the linear range typically [greater than or equal to] 100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker's yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose [sup.13]C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling. 10.1021/ac902837x

Author(s): Amaia Zabala-Letona; Amaia Arruabarrena-Aristorena; Natalia Martn-Martn; Sonia Fernandez-Ruiz; James D. Sutherland; Michelle Clasquin; Julen Tomas-Cortazar; Jose Jimenez; Ines Torres; Phong Quang; Pilar Ximenez-Embun; Ruzica Bago; Aitziber Ugalde-Olano; Ana Loizaga-Iriarte; [...]

Author(s): Amaia Zabala-Letona [1, 2]; Amaia Arruabarrena-Aristorena [1]; Natalia Martn-Martn [1, 2]; Sonia Fernandez-Ruiz [1, 2]; James D. Sutherland [1]; Michelle Clasquin [3]; Julen Tomas-Cortazar [1]; Jose Jimenez [4]; Ines [...]

We present a liquid chromatography—mass spectrometry (LC—MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 mm, involves a water—methanol gradient on a C18 column with tributylamine as the ion pairing agent The MS portion involves full scans from 85 to 1000 m/z at 1 Hz and 100 000 resolution in negative ion mode on a stand alone orbitrap ("Exactive"). The median limit of detection, across 80 metaboilte standards, was 5 ng/ mL with the linear range typically ⩾ 100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker's yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose 13C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in an- notation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling. [ABSTRACT FROM AUTHOR]

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type 2 diabetes and metabolic syndrome. Emerging data also suggest that BCAA catabolism may play a role in heart failure progression. It is hypothesized that decreased catabolism, rather than increased consumption of BCAAs, is responsible for these correlations. Branched chain ketoacid (BCKA) dehydrogenase (BCKDH) kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDH E1a subunit. Using the BCKDK inhibitor molecule BT2, we demonstrate here a reduction of BCAA, BCKA and tissue p-BCKDH levels concomitant with improved glucose tolerance and reduced insulin levels in diet-induced obese mice after only one day of treatment. To investigate the mechanisms underlying this protection, we assessed plasma biomarkers and tissue gene expression after fasting and re-feeding in glucose intolerant high fat diet-fed animals. Remarkably, BT2 treated animals demonstrated reduced plasma glucose levels after re-feed, which was accompanied by dramatic reductions in plasma insulin levels, reduced activation of Akt in peripheral tissues, and a failure to suppress plasma free fatty acids and lipolytic machinery in adipose tissue. RNAseq was performed in liver to assess changes in gene expression profiles, and while over 400 genes were differentially regulated in vehicle treated re-fed mice compared with vehicle treated fasted mice, there were no differentially regulated genes in BT2 re-fed animals compared with BT2 fasted animals. These data suggest that activation of BCAA catabolism with the BCKDK inhibitor BT2 impairs the systemic response to feeding. Disclosure: E. Bollinger: Employee; Self; Pfizer Inc. C.P. Siddall: Employee; Self; Pfizer Inc. Employee; Spouse/Partner; Vertex Pharmaceuticals. T. Greizer: None. J.L. Libera: None. G. Hariri: None. E.E. Pashos: Employee; Self; Pfizer Inc. A. Shipstone: None. A. Hadjipanayis: None. Z. Sun: None. G. Xing: None. M.F. Clasquin: Employee; Spouse/Partner; Merck & Co., Inc. Employee; Self; Pfizer Inc. B.B. Zhang: Employee; Spouse/Partner; Janssen Pharmaceuticals, Inc. Employee; Self; Pfizer Inc. Other Relationship; Self; Eli Lilly and Company. R.A. Miller: Employee; Self; Pfizer Inc. R. Roth Flach: Employee; Self; Pfizer Inc. [ABSTRACT FROM AUTHOR]

Differential mobility spectrometry (DMS) is capable of separating molecules based on their size and shape. When coupled to mass spectrometry (MS), DMS reduces chemical background and enhances signal-to-noise (S/N) ratio. Flow injection analysis (FIA) is a technique used to introduce samples into the source of the DMS-MS platform. Here we describe the application of FIA-DMS-MS/MS for the analysis of urinary acylcarnitine species. More than 20 acylcarnitine species can be detected and quantified during a single FIA-DMS-MS/MS acquisition.