bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simultaneous 2H and 13C Metabolic Flux Analysis of Liver Metabolism Using NMR and GC-MS—Methods Validation and New Applications
Authors: DEJA STANISLAW , FLETCHER JUSTIN A. , KUCEJOVA BLANKA , FU XIAORONG , YOUNG JAMEY , BURGESS SHAWN C. 
Published: June 2018

Hepatic metabolism is critically altered by the pathology of many diseases, including obesity, diabetes and NAFLD. Quantitative estimation of gluconeogenic flux and TCA cycle turnover are clinically useful parameters as they represent major biosynthetic and energetic pathways that are perturbed by these diseases. NMR spectroscopy is an important technique used to study hepatic fluxes since it can precisely distinguish between 2H and 13C metabolic tracers, and provide position-specific enrichment. However, the throughput of NMR for flux analysis is hindered by its low sensitivity, making microscale experiments that have limited biological mass (e.g., primary hepatocytes, tissue biopsies or mouse blood analysis) often infeasible. Thus, we investigate an alternative approach based on GC-MS measurements of glucose mass isotopomers and mathematical modeling of metabolic fluxes. In contrast with NMR, MS approaches are very sensitive, but cannot directly distinguish nuclei or positional enrichment information. Hence, we validated a GC-MS method in isolated perfused mouse liver and in vivo infusions in rats against NMR measurements. A medium-scale metabolic network containing atom transitions for both hydrogen and carbon atoms in the gluconeogenic pathway and TCA cycle was able to deconvolute 2H and 13C labeling information and estimate metabolic fluxes. Although some fluxes were significantly different between the NMR and GC-MS, there was excellent correlation between the two approaches. Finally, we applied GC-MS based simultaneous 2H and 13C approach in primary mouse hepatocytes cultured in 60 mm dishes, an experiment not feasible using standard NMR equipment. This opens future possibilities for drug screening and translational flux studies.

Disclosure
S. Deja: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. J. Young: None. S.C. Burgess: None.
A Novel Mechanism by Foxo1 Phosphorylation Mediates Glucagon Signaling in Control of Glucose Homeostasis
Authors: WU YUXIN , YAN HUI , PAN QUAN , ZHENG HONGTING , DAHANAYAKA SUDATH A. , SUN YUXIANG , ZHOU AIMIN , ZHANG LIN , GUO SHAODONG 
Published: June 2018

Glucagon increases hepatic glucose production (HGP) to maintain blood glucose homeostasis. The forkhead transcription factor Foxo1 promotes HGP through increasing expression of genes encoding the rate-limiting metabolic enzymes for gluconeogenesis. We previously established that insulin suppresses Foxo1 by Akt-mediated phosphorylation at Ser256 in human hepatocytes. Here we demonstrated glucagon promotes Foxo1 nuclear translocation and stability via protein kinase A (PKA)-dependent phosphorylation of Foxo1 at Ser276, serving as a novel mechanism of glucagon action in control of glucose homeostasis. In vitro protein kinase assay showed a direct phosphorylation of Foxo1 at Ser276 by PKA. Replacing Foxo1-Ser276 with alanine (S276A) or aspartate (S276D) reduced or increased Foxo1 stability in human hepatocytes, respectively. To establish the in vivo function of Foxo1-Ser276 phosphorylation in glucose metabolism, we generated Foxo1-S273A and Foxo1-S273D knock-in mice, revealing the novel mechanism by which glucagon, via PKA-dependent phosphorylation of Foxo1 at Ser276, promotes Foxo1 nuclear localization, stability, and HGP. Thus, Foxo1-Ser276 is a potential target site for the control of Foxo1 bioactivity and associated metabolic diseases.

Disclosure
Y. Wu: None. H. Yan: None. Q. Pan: None. H. Zheng: None. S.A. Dahanayaka: None. Y. Sun: None. A. Zhou: None. L. Zhang: None. S. Guo: None.
Protection of Progression of Nonalcoholic Steatohepatitis in Diet-Induced Obese Mice by a Low-Iron Diet
Authors: SALAYE LIPIKA , BYCHKOVA IELIZAVETA , LORENZO FELIPE , SINK SANDY T. , MCCLAIN DON 
Published: June 2018

Iron is a risk factor for type 2 diabetes, but its relationship to other aspects of metabolic syndrome is less clear. To investigate iron’s relation to nonalcoholic steatohepatitis (NASH), mice were fed a “fast food” (FF)-diet (40% energy as fat, 12% SFA, 0.2% cholesterol, and 18.9 g/L glucose and 23.1 g/L fructose in their drinking water) for 10 or 24 weeks. The diets contained either 4 (low iron, LI), 35 (normal iron, NI) or 2000mg iron/kg chow (high iron, HI). HOMA-IR values were 2.2-fold higher (p&lt;0.001) in the FF-groups at 10 weeks, but did not increase further with HI (1.48-fold, p=0.5). At 10 weeks all groups had similar glucose excursions during glucose and pyruvate tolerance testing. There was no significant difference in IP-GTT or IP-PTT area under the glucose curve. Liver triglycerides (TG) were significantly increased by FF (4.2-fold, p&lt;0.01) but not iron at 10 weeks. The effect of FF was greater at 24 weeks (22.4 fold p&lt;0.001), and at 24 weeks iron further increased liver TG (3.3 fold HIFF vs. LIFF, p&lt;0.001). A marker of liver injury (ALT) increased 10.3-fold (p&lt;0.001) on the HIFF diet compared to LI normal chow, but only 3.2-fold (p&lt;0.05) on LIFF. The fibrotic gene collagen1alpha increased 14.3-fold (p&lt;0.001) in HIFF but not at all (0.8-fold) on LIFF compared to LI normal chow. Similar protection was afforded by the LI diet to increases in expression of the inflammatory genes TGF-beta and TNF-alpha seen in HIFF. RNA sequencing data revealed transcriptional regulation of a large family of fat metabolic genes by FF, whereas only the TGF-beta signaling pathway was significantly altered by iron content. We conclude that dietary iron restriction can protect from NASH induced by a high-fat and -carbohydrate diet. A likely candidate mechanism is through a known mediator of hepatic fibrosis, TGF-beta.

Disclosure
L. Salaye: None. I. Bychkova: None. F. Lorenzo: None. S.T. Sink: None. D. McClain: None.
Pyruvate Carboxylase Is Required for Hepatic Gluconeogenesis and TCA Cycle Function
Authors: CAPPEL DAVID , DEJA STANISLAW , FU XIAORONG , BURGESS SHAWN C. 
Published: June 2018

Production of glucose by the liver is crucial for maintaining whole-body gluconeogenesis and is dependent on carbon substrates provided by anaplerotic pathways into the TCA cycle. In the liver, the large majority of anaplerotic flux is through the conversion of pyruvate to oxaloacetate by the action of pyruvate carboxylase (PC). To investigate the role of PC in hepatic gluconeogenesis, we produced a liver-specific PC knockout mouse (LPKCO). LPCKO mice have impaired ability to convert pyruvate into glucose. Surprisingly, they display no difference in blood glucose under fed and overnight fasted conditions. LPCKO mice, however, have elevated concentrations of ketone bodies in plasma under both fed and fasted conditions, suggesting a switch from gluconeogenesis to ketogenesis. Additionally, we observed changes in kidney gene expression that suggest a compensatory upregulation of renal gluconeogenesis. Using stable isotope tracers, we analyzed anaplerotic and TCA cycle fluxes in LPCKO livers and found that LPCKO livers are deficient in anaplerotic flux and have a large reduction but not total ablation of hepatic gluconeogenesis. The LPCKO livers also show a significant reduction in TCA cycle turnover and reduced concentrations of several TCA cycle metabolites. On a high-fat diet (HFD), LPCKO mice gain slightly less weight than their non-transgenic littermates. After 12 weeks of HFD the LPCKO mice have significantly lower blood glucose and plasma insulin compared to non-transgenic littermates. When subjected to a glucose tolerance test, HFD-fed LPCKO mice show significantly better glucose tolerance than non-transgenic littermates. Overall, we demonstrate that flux through hepatic PC is required for anaplerosis and TCA cycle function in the liver and that restricting hepatic gluconeogenesis and TCA cycle turnover may help to protect against the deleterious metabolic effects of high fat diet.

Disclosure
D. Cappel: None. S. Deja: None. X. Fu: None. S.C. Burgess: None.
Foxo1-Ser253 Phosphorylation Regulates Glucose Homeostasis in Mice
Authors: YAN HUI , WU YUXIN , PAN QUAN , SHEN ZHENG , ZHENG HONGTING , WHITE MORRIS F. , SUN YUXIANG , GUO SHAODONG 
Published: June 2018

The forkhead transcription factor Foxo1 is a key mediator in insulin signaling pathway that controls hepatic glucose production (HGP) and pancreatic beta-cell function. Upon activation of the protein kinase Akt, insulin promotes glycogen synthesis and inhibits gluconeogenesis in the liver, reducing blood glucose. We previously demonstrated human Foxo1-Ser256, an equivalent to mouse Foxo1-Ser253, is a key phosphorylation site for insulin and Akt suppression, promoting Foxo1 nuclear export and suppressing the expression of the target gene for liver gluconeogenesis. Here we investigated the role of Foxo1-Ser253 phosphorylation in control of glucose hemostasis in vivo, by generating Foxo1-S253A/A knock-in (KI) mice, in which Foxo1-Ser253 replaced by alanine (A mutation) that blocks phosphorylation. Foxo1-S253A/A mice displayed mild increases in feeding blood glucose and insulin level, but reductions in fasting blood glucose and glucagon concentration, as well as a decrease in the ratio of the number of pancreatic α-cells/β-cells per islet. Foxo1-S253A/A mice exhibited slight increases in insulin sensitivity but barely changed glucose uptake among tissues, and an enhanced energy expenditure. Further analyses indicate that Foxo1-S253A/A enhanced Foxo1 ability and promoted the effect of glucagon on HGP. This is the first report demonstrating that Foxo1-Ser253 phosphorylation status itself is sufficient to affect HGP and glucose homeostasis, as well as regulate the synthesis of insulin and glucagon in in vivo.

Disclosure
H. Yan: None. Y. Wu: None. Q. Pan: None. Z. Shen: None. H. Zheng: None. M.F. White: Advisory Panel; Self; Housey Pharmaceutical Research Laboratories. Y. Sun: None. S. Guo: None.
Meal Intake Is Suppressive of Ketogenesis in Patients with Type 1 Diabetes
Authors: GHANIM HUSAM , GREEN KELLY , HEJNA JEANNE M. , KUHADIYA NITESH D. , BATRA MANAV , CHAUDHURI AJAY , DANDONA PARESH 
Published: June 2018

Aim: To investigate whether the intake of a meal suppresses ketogenesis in patients with type 1 diabetes.
Methods: 30 patients with type 1 diabetes with no C-peptide, fasted overnight, were divided into 3 groups of 10 patients each. The first (group 1) continued the fast for 5 hours after arrival in our clinical research unit while the second (group 2) was administered a high fat high calorie (HFHC) meal without preprandial insulin and the third (group 3) was administered the meal with preprandial insulin. Blood samples were obtained prior to and after the meal at hourly intervals for 5 hours.
Results: In group 1, glucose and glucagon concentrations remained unchanged while FFA concentrations increased (by 74±16%) as did acetoacetate (AcAc) and beta-hydroxybutyrate (BHB) concentrations (by 52±13% and 64±16%, respectively). In group 2, the intake of the meal without preprandial insulin induced marked increase in glucose, glucagon and FFAs (by 235±32%, 81±18% and 75±17%, respectively) concentrations. However, the meal induced significantly lower increase in AcAc or BHB concentrations (increased by 29±9% and 35±11%, respectively) compared to group 1. In group 3, given preprandial insulin before the meal, there was a significant reduction in glucose and glucagon concentrations as compared to group 2 and a fall in FFA concentrations (by 41±12%) below baseline levels. There was no significant increase in AcAc and BHB.
Conclusions: While a prolonged fast was associated with maintenance of glucose and increases in FFA, AcAc and BHB concentrations, the intake of the meal prevented increases in AcAc and BHB in spite of increases in glucose, glucagon and FFA concentrations. These actions are consistent with an anti-ketogenic effect of the meal intake even in type 1 diabetes with an absence of β cell function.

Disclosure
H. Ghanim: None. K. Green: None. J.M. Hejna: None. N.D. Kuhadiya: Speaker's Bureau; Self; AstraZeneca, Novo Nordisk A/S, Janssen Scientific Affairs, LLC.. Advisory Panel; Self; AstraZeneca. Consultant; Self; Dexcom, Inc. M. Batra: Speaker's Bureau; Self; Eli Lilly and Company. A. Chaudhuri: Speaker's Bureau; Self; AstraZeneca, Eli Lilly and Company, Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk Inc. P. Dandona: Advisory Panel; Self; AstraZeneca. Consultant; Self; AstraZeneca. Research Support; Self; AstraZeneca.
Mfge8 Binding of the avß5 Integrin Promotes Insulin Resistance in Mice
Authors: DATTA RITWIK , KHALIFEH-SOLTANI AMIN , HA ARNOLD , ATABAI KAMRAN 
Published: June 2018

Milk fat globule EGF factor 8 (Mfge8) promotes obesity by inducing fatty acid uptake through ligation of the αvβ5 integrin. Global deletion of Mfge8 in mice increases insulin sensitivity through effects on lean body mass. The primary objective of the present study was to assess whether acute disruption of the Mfge8 signaling pathway in wild type mice modulates insulin sensitivity independent of body composition. We therefore evaluated insulin sensitivity in age and sex-matched wild type mice with insulin (ITT) and glucose tolerance tests (GTT) in the presence of a blocking antibody (ALULA) targeting the β5 integrin. Intraperitoneal administration of 5mg/kg ALULA 1h prior to ITT resulted in significantly lower blood glucose levels compared to control antibody treated mice. ALULA treated mice had a significantly lower blood glucose levels in GTT. Serum insulin level measured during GTT in ALULA treated mice was lower compared to control group, suggesting enhanced sensitivity to insulin. We validated our findings in vitro using differentiated 3T3L1 adipocytes and hepG2 liver cells. Both cell types had significantly higher uptake of the glucose analog 2NBDG after insulin treatment in presence of ALULA as compared with insulin treatment with control antibody. Insulin is known to induce glucose uptake in adipocytes through a PI3K/Akt signaling pathway. β5 integrin blockade followed by insulin administration augmented insulin mediated phosphorylation of Akt and As160 in white adipose tissue (WAT) and skeletal muscle (SM) in mice indicating that the 㬥 integrin normally dampens insulin signaling. In preliminary data we have found that after insulin treatment, the β5 integrin associates with the mature insulin receptor 㬥 (IR㬥) in WAT and SM as shown in coimmunoprecipitation studies. Further mechanistic insight to this novel regulation of IR㬥 by 㬥5 integrin will direct us towards testing the therapeutic potential of blocking 㬥5 integrin producing insulin resistance in diabetes.

Disclosure
R. Datta: None. A. Khalifeh-Soltani: None. A. Ha: None. K. Atabai: None.
Diabetes Prevention Using a Simulation Model That Explains Individual Variability in Response to Diet Change
Authors: CHEN JULIA H. , FUKASAWA MOMOKO , CHEN QIAN , BURNS SAMUEL P. , KUMAR KEI , NIRENGI SHINSUKE , TAKAHASHI KAORU , SUGANUMA AKIKO , KUZUYA HIDESHI , SAKANE NAOKI , DWIVEDI GAURAV 
Published: June 2018

Objectives: Limited understanding of the causes of variability in individual response to similar lifestyle changes makes it challenging to design individualized diet plans for diabetes prevention. We previously reported a simulation model of lifestyle disease and tested it using data from DPP and a low carb-low fat diet crossover study. Here we used the model to analyze data from J-DOIT1 to understand the causes of variability and to optimize diet therapy.
Methods: J-DOIT1 is a large-scale intervention study of 2,607 subjects with a 5.5 year follow-up. We selected 60 baseline-matched individuals from placebo and intervention arms. Model was calibrated to each subject’s body weight, FPG, HbA1c time courses to estimate physiology and lifestyle parameters (e.g., RMR, diet intake). Simulation analysis was used to optimize individual diet.
Results: Individuals with the most and least improved biomarkers showed no significant difference in model-estimated energy balance or in self-reported lifestyle at baseline. Neither caloric deficit nor linear combinations of model-estimated parameters were predictive of outcomes, showing the value of our complex physiological model for prediction. The model suggests that unique sets of optimal diets exist for achieving individual health goals (Fig. 1).
Fig. 1. Optimized changes in fat and carbohydrate intake for two individuals to reduce body weight by 5-7%. Fig. 1. Optimized changes in fat and carbohydrate intake for two individuals to reduce body weight by 5-7%.

Disclosure
J.H. Chen: None. M. Fukasawa: None. Q. Chen: None. S.P. Burns: None. K. Kumar: None. S. Nirengi: None. K. Takahashi: None. A. Suganuma: None. H. Kuzuya: None. N. Sakane: Research Support; Spouse/Partner; Pwc. G. Dwivedi: None.
Comparison of Dietary Fat Intake Using Food Records vs. RBC Fatty Acid Biomarkers in a Multiethnic Population—The Federal Women Study
Authors: COURVILLE AMBER B. , BERNSTEIN SHANNA , GALVAN-DE LA CRUZ MIRELLA , ONUZURUIKE ANTHONY , MATTHAN NIRUPA R. , LICHTENSTEIN ALICE H. , SUMNER ANNE E. , CHUNG STEPHANIE T. 
Published: June 2018

Erythrocyte membrane fatty acids (RBC FA) may be a useful biomarker of dietary fat type to assess its impact on health and disease. Our objective was to examine the association of dietary fat type and RBC FA composition in 2 groups of women of African descent (blacks) compared to whites. Dietary fat type was assessed with food records (5±2 days [mean±SD]) and RBC FA composition (mol%) by gas chromatography in 1federally employed women (53% African American, 14% African immigrants, 33% white; age 44±9y, range 24-62y, BMI 30.5±5.9 kg/m2, range 20.3-45.2 kg/m2). Dietary fat type (SFA, MUFA, n-3 PUFA, n-6 PUFA, and trans FA) was calculated as a proportion of total dietary fat. Blacks reported consuming higher SFA and n-6 PUFA (all P&lt;0.05). Dietary report of MUFA, trans FA or n-3 PUFA intake were similar by race (all P≥0.20). In contrast, when RBC FA composition was measured, blacks had higher MUFA and n-3PUFA (both P&lt;0.05). RBC composition of SFA, n-6 PUFA and trans FA were similar by race (all P&gt;0.05). Spearman correlations were calculated to determine the relationship between dietary FA and RBC FA. There was a positive correlation between dietary FA and RBC n-6 PUFA (r=0.3, P&lt;0.02).
In summary, RBC FA was a good marker of n-6 PUFA intake, however, food records appeared to provide a more informative assessment of overall dietary fat type exposure.

Disclosure
A.B. Courville: None. S. Bernstein: None. M. Galvan-De La Cruz: None. A. Onuzuruike: None. N.R. Matthan: None. A.H. Lichtenstein: None. A.E. Sumner: None. S.T. Chung: None.
White- and Brown-Rice Supplementation Improves Insulin Tolerance, Brown Fat Activation, Energy Expenditure, and Changes the Gut Microflora in Diet-Induced Obesity
Authors: YOKOMIZO HISASHI , ISHIKADO ATSUSHI , SHINJO TAKANORI , PARK KYOUNGMIN , MAEDA YASUTAKA , WU I-HSIEN , POBER DAVID M. , DEVKOTA SUZANNE , MATSUMOTO MOTONOBU , KOSTIC ALEKSANDAR , KING GEORGE L. 
Published: June 2018

Increased consumption of white rice (WR) with parallel decreased use of brown rice (BR) have been associated with increased prevalence of type 2 diabetes (T2DM), suggesting WR may elevate the risk of T2DM. To test whether WR or BR have differential effects on glucose metabolism in diet-induced obesity, C57BL/6 mice were fed 6 different diets: control diet (CD, 10%), high fat diet (HFD, 46%) and CD and HFD with either WR or BR. Diets with WR or BR were controlled with comparable carbohydrate and protein contents. After 18 weeks, the median weights of HFD mice were greater (36%) than those on CD. Surprisingly, adding WR or BR to HFD decreased weight gains equally by 53% with parallel reduction of abdominal, visceral, subcutaneous, and total fat vs. HFD. Activity and food intake did not change among mice on HFD. However, HFD decreased heat, VO2, and VCO2 production vs. CD which were normalized by adding WR or BR, per indirect calorimetry with metabolic cages. Intraperitoneal glucose tolerance test on HFD were improved by either WR or BR, although mice on BR+HFD exhibited greater improvements than mice on WR+HFD (P&lt;0.05). Adding WR or BR to HFD decreased HFD-induced elevations of plasma leptin and MCP-1 levels. HFD decreased brown fat tissue mass and markers, UCP-1 and PGC1α vs. CD mice which improved when WR or BR was added to HFD. Gut microbiota composition, assessed by 16S rRNA sequencing and metagenomic analysis, showed clear differences between HFD vs. CD or vs. the addition of BR or WR. The ratio of Firmicutes to Bacteroidetes was increased significantly in HFD vs. CD diet, which has been associated with obesity and metabolic syndrome, but decreased by the addition of WR (63%) or BR (81%). Thus, BR and, surprisingly, WR may activate BAT, increase energy expenditure to reduce body weight and improve insulin sensitivity while on HFD, possibly by altering gut microbiome, indicating that common substances may exist in BR and WR to lower the risks for T2DM.

Disclosure
H. Yokomizo: None. A. Ishikado: Employee; Self; Sunstar Inc.. Employee; Spouse/Partner; Sunstar Inc.. T. Shinjo: None. K. Park: None. Y. Maeda: None. I. Wu: None. D.M. Pober: None. S. Devkota: None. M. Matsumoto: Employee; Self; Sunstar Inc.. A. Kostic: None. G.L. King: Research Support; Self; Sanofi-Aventis.
Angiotensin-Converting Enzyme 2 Deficiency Increases Translocation of Gut Bacteria by Depletion of Bone Marrow-Derived Circulating Angiogenic Cells
Authors: DUAN YAQIAN , FENG DONGNI , BELI ELENI , CALZI SERGIO LI , LONGHINI ANA LEDA F. , LAMENDELLA REGINA , WRIGHT JUSTIN R. , MARKEL TROY A. , OBUKHOV ALEXANDER G. , GRANT MARIA B. 
Published: June 2018

A healthy microbiome is central to good nutrition and efficient metabolism both of which can be altered in diabetes. Disruption of gut-vascular integrity may lead to translocation of microbial antigens and bacteria into the circulation. Bone marrow-derived circulating angiogenic cells (CACs) are essential to maintain vascular integrity. We tested the hypothesis that angiotensin-converting enzyme 2 (ACE2), the key enzyme in the vasoprotective axis of renin-angiotensin system (RAS), acts to regulate the infiltration of bone marrow-derived CACs in the gut and to maintain gut-vascular integrity in diabetes and could influence the systemic translocation of pathogenic antigens and bacteria from the gut.
Diabetic ACE2 knockout (ACE2-/y)/C57BL/6-Ins2WT/C96Y (Akita) mice were examined at 9 months after the onset of diabetes and compared to age-matched controls. Beta diversity analysis of the gut microbiome revealed a distinct bacterial profile among the different genotypes (n=9-17 per group, p=0.002). The fecal samples from ACE2-/y-Akita group compared to Akita group were enriched with a great diversity of bacterial taxa that have been reported to contribute to the pathogenesis of diabetes and showed activation of peptidoglycan biosynthesis pathways by metatranscriptome analysis (n=4 per group, p=0.005). Flow cytometry analysis showed that loss of ACE2 led to reduced infiltration of bone marrow-derived CACs (CD45+Flk-1+CD31+) into the gut (n=8-14 per group, p&lt;0.0001), without modifying levels of proinflammatory or immune cell phenotypes. The CAC depletion mediated by ACE2 deficiency was associated with increased levels of gut bacteria in the blood as assessed by SourceTracker analysis (n=9 per group). These data suggested a loss of the protective arm of RAS contributes to the depletion of CACs and disruption of gut-vascular barrier that may promote bacterial translocation in diabetes.

Disclosure
Y. Duan: None. D. Feng: None. E. Beli: None. S. Li Calzi: None. A.F. Longhini: None. R. Lamendella: None. J.R. Wright: None. T.A. Markel: None. A.G. Obukhov: None. M.B. Grant: None.