Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

• DOI: 10.1194/jlr.m035592
• Publication Date: 2013-07-05T03:39:13Z
• Authors: Seongah Han; Taro E. Akiyama; Stephen F. Previs; Kithsiri Herath; Thomas P. Roddy; Kristian K. Jensen; Hong-Ping Guan; Beth A. Murphy; Lesley A. McNamara; Xun Shen; Walter Strapps; Brian K. Hubbard; Shirly Pinto; Cai Li; Jing Li
• Keywords: Blood Glucose; Male; 0301 basic medicine; 0303 health sciences; Lipogenesis; dyslipidemia; Gene Expression; Mice, Obese; QD415-436; Biochemistry; Lipoproteins, LDL; Mice; 03 medical and health sciences; Cholesterol; Diabetes Mellitus, Type 2; Liver; Gene Knockdown Techniques; diabetes mellitus; Receptors, Glucagon; Animals; RNA Interference; glucose; RNA, Small Interfering; Triglycerides

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon key regulator for homeostasis, antagonizing the receptor (GCGR) possible therapeutic strategy treatment diabetes mellitus. To study effect hepatic GCGR inhibition on regulation lipid metabolism, we generated siRNA-mediated knockdown (si-GCGR) in db/db mouse. The markedly reduced plasma levels; however, total cholesterol was increased. detailed analysis showed an increase LDL fraction, and no change VLDL HDL fractions. Further studies that result over-expression lipogenic genes elevated de novo synthesis. Inhibition signaling via had both metabolism mice. Glucagon counterregulatory hormone insulin has important physiological role regulating preventing hypoglycemia animals humans. primary target organ liver. interaction increases production by activating glycogenolysis gluconeogenesis, abnormal associated with hyperglycemia patients (1D'Alessio D. dysregulated secretion diabetes.Diabetes Obes. Metab. 2011; 13: 126-132Crossref PubMed Scopus (177) Google Scholar). Several preclinical clinical have demonstrated utility inhibiting as one potential For example, pharmacologic or antisense oligonucleotide (ASO) interventions suppress been shown to reverse animal models (2Petersen K.F. Sullivan J.T. Effects novel antagonist (Bay 27–9955) glucagon-stimulated humans.Diabetologia. 2001; 44: 2018-2024Crossref (195) Scholar, 3Qureshi S.A. Rios C.M. Xie Yang X. Tota L.M. Ding V.D. Li Z. Bansal A. Miller C. Cohen S.M. et al.A inhibits glucagon-mediated biological effects.Diabetes. 2004; 53: 3267-3273Crossref (94) 4Winzell M.S. Brand C.L. Wierup N. Sidelmann U.G. Sundler F. Nishimura E. Ahren B. antagonism improves islet function mice resistance induced high-fat diet.Diabetologia. 2007; 50: 1453-1462Crossref (53) 5Sloop K.W. Cao J.X. Siesky A.M. Zhang H.Y. Bodenmiller D.M. Cox A.L. Jacobs S.J. Moyers J.S. Owens R.A. 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Mu Woods Zycband Ronan Liu Roy R.S. Zhu al.Glucagon are resistant diet-induced obesity streptozotocin-mediated beta loss hyperglycaemia.Diabetologia. 142-150Crossref (166) More importantly, intervention antagonists revealed impressive circulating glucose-lowering humans (11Kelly R.P. Garhyan Abu-Raddad E.J. Fu Lim Prince M.J. Pinaire J.A. Loh M. Deeg M.A. Short-term LY2409021 effectively (FBG) HbA1c mellitus (T2DM) [abstract].Diabetes. 60: A84Google 12Engel S.S. Xu Andryuk P.J. Davies Amatruda Kaufman Goldstein B.J. Efficacy tolerability MK-0893, (GRA), : A85Google However, concerns over association between dyslipidemia, specifically, LDL-C from data antagonist, raised Although most robust lowering, effects lipids mechanisms linking action not elucidated. dyslipidemia typically involves triglycerides (TGs), HDL-C levels, cardiovascular risk (13Krauss R.M. Lipids lipoproteins Care. 27: 1496-1504Crossref (491) Cardiovascular further when complicated levels. Thus, it evaluate impact especially synthesis, TG secretion, HDL-C. objective present explore ablation mouse model. Male, 8 week-old were purchased Jackson Laboratory (Bar Harbor, ME) regular chow diet (Diet 7012; Harlan Teklad) throughout study. Following week stabilizing period, 10 used small interfering RNA (siRNA) injection. siRNA packaged nanoparticles, previously described (14Tadin-Strapps Peterson L.B. Cumiskey Rosa R.L. Mendoza V.H. Castro-Perez Puig O. Strapps W.R. Yendluri al.siRNA-induced liver ApoB lowers serum LDL-cholesterol model human-like lipids.J. Lipid 52: 1084-1097Abstract Full Text PDF (56) Chemically modified siRNAs synthesized characterized, 5ʹ-UGGUCAAGUGUCUGUUUGA-3ʹand 5ʹ-GGACTTCTCTCAATTTTCT-3ʹ sequences nontargeting siRNA, respectively. encapsulated injected tail vein 3 mg/kg dose days 0 6. Animals euthanized at day 11, samples collected immediately following euthanasia. Animal procedures practiced conformity Public Health Service policy guidelines Institutional Care Use Committee Merck. Ambient (nonfasted) measured 9:00 AM bleeds glucometer (One Touch Ultra; LifeScan, Inc., Milpitas, CA) 4 11 after At fasted 5 h, collected. Plasma separated centrifugation stored -80°C until analysis. Total commercial kits (Wako Chemicals; Richmond, VA) according manufacturers' protocol. ALPCO ELISA kit (ALPCO Diagnostics; Salem, NH) Infinity Triglycerides (Thermo Fisher Scientific; Waltham, MA), respectively, instructions. (Linco Research Immunoassay; St. Charles, MO). Nonesterified fatty acid (NEFA) commercially available enzyme-coupled spectrophotometric assays Chemicals, Inc.), β hydroxybutylate (bHB) Liquid Enzymatic Reagent Kit (Stanbio Laboratory; Boerne, TX). apoB apoA-I LC/MS assay, protein convertase subtilisin/kexin 9 (PCSK9) murine-specific ELISA, 15Chen Wang S.P. Krsmanovic M.L. Gagen V. R. Shah He T. Stout al.Small molecule activation lecithin acyltransferase modulates lipoprotein hamsters.Metabolism. 2012; 61: 470-481Abstract (37) All performed recommended instrument operation, calibration, quality control, assay guidelines. content (16Han Liang C.P. Westerterp Senokuchi Welch Q. Matsumoto Accili Tall A.R. regulates atherogenesis mice.J. 2009; 119: 1029-1041PubMed examine proteins, lysates prepared Western blot carried out earlier Scholar) anti-Ldlr antibody (Abcam, PLC; MA) anti-α tubulin PLC). (VLDL-C; d < 1.006 g/ml), (d = 1.006–1.063 1.063–1.21 g/ml) isolated sequential density ultracentrifugation TLA-100 rotor (Beckman Coulter, Inc.; Brea, CA), Sodium bromide (NaBr) (Sigma-Aldrich, Louis, MO) prepare solution. ApoB-containing run SDS-PAGE gels visualized 0.05% Coomassie blue staining (Sigma-Aldrich). P407 (Pluronic F-127; Invitrogen, Grand Island, NY) measure rate plasma. Mice overnight (10 ml/kg body weight). Blood 0, 1, 2, 4, 6 h injection, quantified, above. Lipoproteins fractionated fast liquid chromatography (FPLC) gel filtration Superose-6 size exclusion column (GE Healthcare LifeSciences, Piscataway, NJ) attached Dionex HPLC system Scientific, Inc.). effluent mixed enzymatic reagent Chemicals), continuously photodiode array detector 600 nm absorbance. first, second, third peaks attributed VLDL, LDL-C, HDL-C, area under each peak calculated Chromeleon software, (17Han Flattery McLaren Raubertas Lee S.H. Geoghagen J.M. Roddy T.P. al.Comparison separation methodologies human cynomolgus monkey samples.J. Cardiovasc. Transl. 5: 75-83Crossref (13) Liver homogenized, Easy (Qiagen, Valencia, manufacturer's Two micrograms sample transcribed cDNA (Life Technologies Corp., Carlsbad, mRNA interest RT-PCR, SYBR Green PCR core reagents primers (18Jensen K.K. Previs S.F. Herath Bhat Hu P.L. D.G. Shin M.K. al.Demonstration decoupling synthesis combining gene 2H2O quantification.Am. Physiol. Endocrinol. 302: E209-E217Crossref (16) relative amounts specific amplicons primer set estimated cycle threshold (Ct) value normalized copy number housekeeping P values determined two-tailed equal variance Student's t-test, comparing ΔCt si-Control si-GCGR groups. (Sigma-Aldrich) 20 weight into h. tracer incorporation, (35 µl) spiked known amount heptadecanoic (C17:0, internal standard) then saponified 1 N potassium hydroxide (KOH) 80% ethanol (heated 65°C 60 minutes). Samples acidified (25 µl hydrochloric (HCl), extracted chlor09 oform. evaporation solvent, residue first reacted trimethylsilyldiazomethane form FA methyl esters, evaporated dryness, pyridine-acetic anhydride (1:1, v/v) 30 min generate acetate. dryness. 2H labeling derivatized Agilent 5973N-MSD equipped 6890 GC (Agilent Technologies; Santa Clara, DB5-MS capillary (30 m x 0.25 mm µm). mass spectrometer operated electron mode, selective ion monitoring m/z 270 271 palmitate, 284 285 C17:0 (internal standard), 368 369 cholesterol; selected dwell time ms per ion. quantify contribution precursor product ratio assumed general equation used: %newlymadematerial=productlabeling/(precursor labeling×n)×100Eq. which palmitate cholesterol, water, n= exchangeable hydrogens (assumed 22 25 respectively) (19Lee W.N. Bassilian Ajie H.O. Schoeller D.A. Edmond Bergner E.A. Byerley L.O. In vivo measurement acids D2O isotopomer analysis.Am. 1994; 266: E699-E708Crossref 20Diraison Pachiaudi Beylot deuterated water: determination average deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract (76) Values expressed means ± SEM. statistical significance differences <0.05 considered significant. Eleven reduction (61%) observed (Fig. 1A). There difference cumulative group compared 1B). Compared mice, ambient si-GCGR-treated sustained 1C, D). si-GCGRsi-GCGR-treated similar two groups 1E, F). Lipoprotein 2-fold those group, significant 2A). addition, there expression, PCSK9 lipase activity these 2B, C; see supplementary Fig. IA). use isolate fractions slight apoB-100 fractions, tendency toward 2D, E). FPLC confirmed fraction 2F). TGs 3A). This may potentially resulted genes. differential sterol-regulatory element-binding isoform-1c (Srebf1) SREBP-2 (Srebf2) controls 3B). profound SREBP pathways, such synthase (Fasn), acetyl-CoA carboxylase A B (Acaca, Acacb), stearoyl-CoA desaturase (Scd1), elongation very long-chain (Elovl5, Elovl6), long-chain-fatty acid-CoA ligase (Acsl3, Acsl5), Acyl-CoA synthetase short-chain family member (Acss2), ATP citrate lyase (Acly), acetoacetyl-CoA (Aacs), mevalonate decarboxylase (Mvd), farnesyl diphosphate (Fdps) 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), whereas MLX interactor (Chrebp) unchanged Microsomal triglyceride transfer (Mttp) moderate other candidate molecules involved apoB-containing including apoB, Lpl, PCSK9, Ldlr As expected signaling, glucokinase (Gck) pyruvate kinase (Pklr) increased, phosphoenolpyruvate carboxykinase (Pepck) decreased Upregulation glycogen biosynthetic gluconeogenic knockdown. These findings suggest produce upregulation genes, eventually lead contents. Stable isotope determine whether translated lipogenesis, tracer, quantified GC/MS. (>2-fold) (>5-fold) 4A, B). Presumably, changes reflect because source newly made lipids. altered inhibitor administered injection blocks clearance circulation; thus, accumulation reflects results well basal 4C, groups, NEFA, bHB, alanine transaminase, aspartate aminotransferase 4E, F; IB). Targeting treat great interest. strong lowering Previous (9Parker 11Kelly 21Mu Brady Dallas-Yang Wright Lu al.Chronic moderately raises peptide without severe hypertrophy obese mice.Diabetologia. 54: 2381-2391Crossref (58) Consistent observations, indicate blocking can improve therapies consistently improvement different even trials, questions regarding raised. Interestingly, several illustrated hypolipidemic actions glucagon, respect TG, contents, oxidation (22Eaton Hypolipemic experimental lipemia rat.J. 1973; 14: 312-318Abstract 23Longuet Sinclair E.M. Maida Baggio L.L. Maziarz Charron Drucker D.J. required adaptive metabolic response fasting.Cell 2008; 8: 359-371Abstract 24Xiao Pavlic Szeto Patterson B.W. Lewis G.F. acute hyperglucagonemia intestinal healthy humans.Diabetes. 383-390Crossref (44) 25Aubry Marcel Y.L. Davignon types hyperlipoproteinemia.Metabolism. 1974; 23: 225-238Abstract (27) underscore importance understanding implications downregulating metabolism. Previously published examining somewhat inconsistent effects, insofar all Gcgr-/- ASO-treated exhibited dyslipidemic phenotypes (5Sloop 8Gelling some studies, but (or reduced) another study, 16 (23Longuet contents current si-RNA Si-GCGR production. aggravated characteristics lipogenesis. We did observe any NEFA concentrations flux liver, nor β-hydroxybutyrate and/or shown). LDLR unchanged; therefore, increasing probably less due fractional catabolism. On basis our results, propose lipogenesis driving force knock down fructose-1,6-bisphosphatase converts fructose 1,6 biphosphate 6-phosphate upregulated (see II; tested impairment leptin CETP transgenic LDLR+/- mouse, targeting Gcgr. ~20% ~24% induction CETP-transgenic III). gluconeogenesis glycolysis could build-up acetyl-CoA, be carbon substrate either secreted VLDL. Certainly, Another explanation knocking sensitized insulin-mediated It documented upregulates biosynthesis One inhibitory pathway mediated (26Xiong Collins Q.F. An Lupo Jr, Robidoux W. p38 mitogen-activated plays lipogenesis.J. Biol. Chem. 282: 4975-4982Abstract (79) recent global (27Yang MacDougall McDowell M.T. Xi Wei Zavadoski W.J. Molloy M.P. Baker Kuhn Cabrera al.Polyomic profiling reveals alterations glucagon-receptor mice: anti-glucagon diabetes.BMC Genomics. 12: 281Crossref Importantly, monoclonal antibody-treated 28Gu Yan Winters K.A. Komorowski Vonderfecht Atangan Sivits Hill Bi al.Long-term causes reversible alpha-cell hyperglucagonemia.J. Pharmacol. Exp. Ther. 331: 871-881Crossref (85) Scholar), might caused Indeed, stable (2H cholesterol) significantly interesting note expressions report multiple discrepancy genetic background, dysmetabolic status degree deficiency. glucagon/GCGR only protects against excessive Given atherosclerosis cause morbidity mortality (29Nigro Osman Dart Little Insulin atherosclerosis.Endocr. Rev. 242-259Crossref (260) carefully antagonized. directly applying useful Whether decoupled level will field. Understanding provide insights guide design next generation authors thank Christine McCrary Sisk Kathleen Newcomb Merck Sharp & Dohme their editorial assistance preparation this manuscript Xun Shen Corp. generating set. Download .zip (.17 MB) Help zip files nonesterified