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Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer

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Abstract

Deficiency of IQGAP2, a scaffolding protein expressed primarily in liver leads to rearrangements of hepatic protein compartmentalization and altered regulation of enzyme functions predisposing development of hepatocellular carcinoma and diabetes. Employing a systems approach with proteomics, metabolomics and fluxes characterizations, we examined the effects of IQGAP2 deficient proteomic changes on cellular metabolism and the overall metabolic phenotype. Iqgap2 −/−mice demonstrated metabolic inflexibility, fasting hyperglycemia and obesity. Such phenotypic characteristics were associated with aberrant hepatic regulations of glycolysis/gluconeogenesis, glycogenolysis, lipid homeostasis and futile cycling corroborated with corresponding proteomic changes in cytosolic and mitochondrial compartments. IQGAP2 deficiency also led to truncated TCA-cycle, increased anaplerosis, increased supply of acetyl-CoA for de novo lipogenesis, and increased mitochondrial methyl-donor metabolism necessary for nucleotides synthesis. Our results suggest that changes in metabolic networks in IQGAP2 deficiency create a hepatic environment of a ‘pre-diabetic’ phenotype and a predisposition to non-alcoholic fatty liver disease which has been linked to the development of hepatocellular carcinoma.

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Acknowledgements

Authors would sincerely like to thank Dr. Schmidt VA (SUNY, Stony Brook) for the kind gift of Iqgap2 −/− mice for the study. Proteome research efforts (J.E.B, A.N, C.Z) were supported by grants 5R01GM086688, 1U19AI10777, 5R01AI101307, and 5R01HL110879. I.J.K., was supported by DK58132-01A2 and Diabetes Research and Training Center (DRTC), NIH grant P60DK020541, I.J.K and C.G were supported by NIAID grant U19AI091175-01. L.W.N.P was supported by the biomedical mass spectrometry laboratory at Harbor-UCLA, partly supported by the Clinical and Translational Science Institute at UCLA (UL1 TR000124) and the Center of Excellence for Pancreatic Diseases (PO1 AT00396).

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Authors and Affiliations

  1. Department of Medicine, Diabetes Center, Stable Isotope and Metabolomics Core Facility, Albert Einstein College of Medicine, Bronx, NY, 10461, USA

    B. Vaitheesvaran, K. Hartil, P. Ó Broin & I. J. Kurland

  2. Department of Genome Sciences, University of Washington, Seattle, WA, 98109, USA

    A. Navare, C. Zheng & J. E. Bruce

  3. Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA

    C. Guha

  4. Department of Genetics., Division of Computational Genetics, Albert Einstein College of Medicine, Bronx, NY, 10461, USA

    P. Ó Broin & A. Golden

  5. Department of Pediatrics, Division of Endocrinology and Metabolism, University of California, Los Angeles, CA, 90502, USA

    W. N. Lee

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  1. B. Vaitheesvaran
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Supplemental Figure S1

Indirect Calorimetry: Oxygen Consumption (VO2) and activity for Iqgap2 -/- and control were determined during the diurnal cycle and fasted to fed transitions. Day (light cycle) and night (dark cycle) 12 hours, (over)night fast – 15hrs, day re-fed - 5hrs in duration. n=8, (a) represents VO2 calculated normalizing measurements to total body mass, (b) represents VO2 calculated using lean body mass. (c) represents ambulatory activity determined simultaneously using an Opto-Varimetrix-3 sensor system. Consecutive adjacent infrared beam breaks in either the x- or y-axes were scored as an activity (total z counts). Activity was graphed by normalizing measurements to total body mass. Data are mean ±SEM. Error bars are represented only in one direction for clarity. Supplementary material 1 (JPEG 765 kb)

Supplemental Figure S2

Bar graphs illustrating 18hr Fast and refed plasma levels of important amino acids for Iqgap2 -/- and control mice. Data are mean ± SEM. * represents p<0.05 using Students T test comparisons for 18hr fast Iqgap2 -/- and control mice. The refed aminoacid levels were comparable between the groups. F-phenylalanine, S – serine, I- Isoleucine, L- leucine, T- threonine, V-valine, M-methionine, E-glutamic acid, A-alanine, G-glycine. Supplementary material 2 (JPEG 128 kb)

Supplemental Figure S3

Bar graphs illustrating stable isotope [U-13C6]-glucose (M+6) infusion studies a) represents hepatic glucose production calculated using isotope infusion rate and final M+6 glucose enrichments and is expressed as mg/kg/min. b) represents ‘F’ - sum of fraction mass isotopomers (M1/∑m+M2/∑m+M3/∑m) that recycled from the infused isotope [U-13C6]-glucose and contributed to Cori cycling. Data are mean ± SEM. ** represents p<0.001 using Students T test comparisons for 18hr fast Iqgap2 -/- and control mice. Supplementary material 3 (JPEG 478 kb)

Supplemental Figure S4a

Immunoblot analysis illustrating 18hr Fast and refed expressions of key metabolic proteins involved in hepatic anaplerotic (PC) and cataplerotic reactions (PEPCK, M2-PK is a unique isoform of pyruvate kinase expressed during hepatocarcinogenesis) for Iqgap2 -/- and control mice. PC-pyruvate carboxylase, PEPCK-phosphoenol pyruvatec carboxy kinase, M2-PK- M2 isoform of pyruvate kinase.Supplementary material 4 (JPEG 199 kb)

Supplemental Figure S4b

Critical proteins ( PC, PK and PEPCK) and loading control PCNA were quantifed using ImageJ software and are shown as bar graphs. Values were normalized to Control and are data±SEM . * represents p<0.05 for Iqgap2 -/-against Control mice using Students’s Ttest. Supplementary material 5 (JPEG 305 kb)

Supplemental Figure S5

Immunoblot analysis illustrating 18hr Fast and refed expressions of key metabolic proteins involved in lipid synthesis for Iqgap2 -/- and control. FAS-fatty acid synthase, ACL- ATP citrate lyase, ACC – acetyl coA carboxylase, DGAT1 – diacylglycaerol acyl transferase 1, RAPTOR- regulatory-associated protein of mTOR.Supplementary material 6 (JPEG 239 kb)

Supplemental Figure S6

Thin layer chromatography (TLC) analysis for lipids illustrating 18hr Fast and refed triglycerides levels in Iqgap2 -/- and control mice. Supplementary material 7 (JPEG 163 kb)

Supplemental Figure S7

Bar graphs illustrating 18hr Fast and refed hepatic levels for important antioxidant metabolites for Iqgap2 -/- and control mice. Data are mean ± SEM. ** represents p<0.01 using Students T test. Supplementary material 8 (JPEG 337 kb)

Supplemental Figure S8

Peptide-level MS1 integrated area calculation was performed on at least 5 peptides specific to pyruvate kinase R/L isoform. The stacked chromatograms of biological replicates of Fast Iqgap2 -/- and control are shown with a chromatographic peak eluting around 61-63 minute, corresponding to peptide GSQVLVTVDPK. Integrated area under individual peak is highlighted. The inset mass spectrum corresponding to the chromatographic peak shows isotopic distribution of the singly charged protonated precursor ion of GSQVLVTVDPK at m/z 1142.64. The ratio of average peak area of fast Iqgap2 -/- to the average peak area of fast control showed 2.6-fold increase for the peptide. Supplementary material 9 (JPEG 228 kb)

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Vaitheesvaran, B., Hartil, K., Navare, A. et al. Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer. Metabolomics 10, 920–937 (2014). https://doi.org/10.1007/s11306-014-0639-9

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