Adiponectin Lowers Glucose Production by Increasing SOGA
2010; Elsevier BV; Volume: 177; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.100363
ISSN1525-2191
AutoresRachael B. Cowerd, Melissa M. Asmar, J. McKee Alderman, Elizabeth A. Alderman, Alaina L. Garland, Walker H. Busby, Wanda M. Bodnar, Ivan Rusyn, Benjamin D. Medoff, Roland Tisch, Elizabeth J. Mayer‐Davis, James A. Swenberg, Steven H. Zeisel, Terry P. Combs,
Tópico(s)Adipose Tissue and Metabolism
ResumoAdiponectin is a hormone that lowers glucose production by increasing liver insulin sensitivity. Insulin blocks the generation of biochemical intermediates for glucose production by inhibiting autophagy. However, autophagy is stimulated by an essential mediator of adiponectin action, AMPK. This deadlock led to our hypothesis that adiponectin inhibits autophagy through a novel mediator. Mass spectrometry revealed a novel protein that we call suppressor of glucose by autophagy (SOGA) in adiponectin-treated hepatoma cells. Adiponectin increased SOGA in hepatocytes, and siRNA knockdown of SOGA blocked adiponectin inhibition of glucose production. Furthermore, knockdown of SOGA increased late autophagosome and lysosome staining and the secretion of valine, an amino acid that cannot be synthesized or metabolized by liver cells, suggesting that SOGA inhibits autophagy. SOGA decreased in response to AICAR, an activator of AMPK, and LY294002, an inhibitor of the insulin signaling intermediate, PI3K. AICAR reduction of SOGA was blocked by adiponectin; however, adiponectin did not increase SOGA during PI3K inhibition, suggesting that adiponectin increases SOGA through the insulin signaling pathway. SOGA contains an internal signal peptide that enables the secretion of a circulating fragment of SOGA, providing a surrogate marker for intracellular SOGA levels. Circulating SOGA increased in parallel with adiponectin and insulin activity in both humans and mice. These results suggest that adiponectin-mediated increases in SOGA contribute to the inhibition of glucose production. Adiponectin is a hormone that lowers glucose production by increasing liver insulin sensitivity. Insulin blocks the generation of biochemical intermediates for glucose production by inhibiting autophagy. However, autophagy is stimulated by an essential mediator of adiponectin action, AMPK. This deadlock led to our hypothesis that adiponectin inhibits autophagy through a novel mediator. Mass spectrometry revealed a novel protein that we call suppressor of glucose by autophagy (SOGA) in adiponectin-treated hepatoma cells. Adiponectin increased SOGA in hepatocytes, and siRNA knockdown of SOGA blocked adiponectin inhibition of glucose production. Furthermore, knockdown of SOGA increased late autophagosome and lysosome staining and the secretion of valine, an amino acid that cannot be synthesized or metabolized by liver cells, suggesting that SOGA inhibits autophagy. SOGA decreased in response to AICAR, an activator of AMPK, and LY294002, an inhibitor of the insulin signaling intermediate, PI3K. AICAR reduction of SOGA was blocked by adiponectin; however, adiponectin did not increase SOGA during PI3K inhibition, suggesting that adiponectin increases SOGA through the insulin signaling pathway. SOGA contains an internal signal peptide that enables the secretion of a circulating fragment of SOGA, providing a surrogate marker for intracellular SOGA levels. Circulating SOGA increased in parallel with adiponectin and insulin activity in both humans and mice. These results suggest that adiponectin-mediated increases in SOGA contribute to the inhibition of glucose production. 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McArdle rat hepatoma cells were exposed to adipocyte conditioned media with or without adiponectin.16Brooks NL Trent CM Raetzsch CF Flurkey K Boysen G Perfetti MT Jeong YC Klebanov S Patel KB Khodush VR Kupper LL Carling D Swenberg JA Harrison DE Combs TP Low utilization of circulating glucose after food withdrawal in Snell dwarf mice.J Biol Chem. 2007; 282: 35069-35077Crossref PubMed Scopus (39) Google Scholar Cell lysates were digested with proteomics grade trypsin (Sigma, St. Louis, MO) and filtered through YM-10 molecular weight cutoff filters (Millipore, Bedford, MA). Tryptic digests were injected into an LCQ-Deca Ion Trap mass spectrometer coupled to a Surveyor HPLC system (Thermo Fisher Scientific, Waltham, MA). The solvent, 50% methanol and 0.1% formic acid, was delivered to the spectrometer at 200 μl/min. Peptide masses were acquired in positive mode using electrospray ionization under the following source conditions: spray voltage was 5 kV, sheath gas was 40 (arbitrary units), auxiliary gas was 20 (arbitrary units), and heated capillary temperature was 350 C. Total RNA was obtained from primary mouse hepatocytes using Triazol reagent (Invitrogen, Carlsbad, CA). mRNA was isolated using Oligotex mRNA Kit (Qiagen, Valencia, CA). Primers used to clone SOGA were designed using publicly available genomic and mRNA sequence data based on the open reading frame of SOGA peptides we detected by mass spectrometry. The 4.7-kb SOGA cDNA was isolated by annealing two PCR products using overlap extension. RNA ligase mediated RACE (Ambion, Austin, TX) was used to clone the 5′ SOGA mRNA sequence. Human- and murine-specific polyclonal antisera were produced in three New Zealand White rabbits (Franklin Rabbitry, NC) using a human-specific peptide antigen STQSLTSC*FARSSRSAIRHSPSKC and two partially overlapping murine-specific peptide antigens CSAQSLASCFIRPSRN and SAQSLASC*FIRPSRNPIRHSPSKC, where C* represents acemidomethyl cysteine. Synthetic peptides were purified by HPLC and analyzed on the LCQ-Deca Ion Trap mass spectrometer to confirm their molecular weight. Antigenic peptides (10 mg) were dissolved in 0.1 mol/L NaH2PO4 (pH 7.2)/0.05 mol/L NaCl and conjugated to keyhole limpet hemocyanin (KLH; 4 mg) before injection. KLH-conjugated peptides were dissolved in 3 ml of 0.03% trifluoroacetic acid and added to 3 ml complete Freund's adjuvant (Sigma). New Zealand White rabbits were injected intradermally using multiple injection sites. After 5 weeks, each animal was reinjected subcutaneously with KLH-conjugated antigen in 1 ml of 50% incomplete Freund's adjuvant (Sigma). Four weeks later, 20 ml blood were collected and rabbits were reimmunized. Injections and bleedings were performed at monthly intervals thereafter. The antibody production protocol was approved by UNC's Institutional Animal Care and Use Committee (IACUC). Rabbit antisera recognizing human and murine SOGA are available through Millipore (catalogue numbers ABS81 and ABS91). Mouse livers were perfused with a Krebs-Ringer-HEPES buffer containing collagenase IV (Sigma-Aldrich). Livers were isolated and cells were dispersed by gentle shaking and filtered through sterile 100 μm nylon gauze. Cells were washed twice with sterile phosphate-buffered saline and purified by centrifugation in 50% isotonic Percoll (Sigma-Aldrich). Cells were resuspended with Krebs-Ringer-HEPES + Ca2+ buffer to a total volume of 10 ml. Viability was validated via trypan blue exclusion and routinely exceeded 90%. Freshly isolated mouse hepatocytes were plated at 105 cells per well in 12-well culture plates coated with rat tail collagen I (BD Biosciences). Cells were maintained in William E culture medium (Invitrogen), 25 mmol/L glucose and 10% horse serum (HS). Adiponectin was provided from adipocyte-conditioned media as previously described.16Brooks NL Trent CM Raetzsch CF Flurkey K Boysen G Perfetti MT Jeong YC Klebanov S Patel KB Khodush VR Kupper LL Carling D Swenberg JA Harrison DE Combs TP Low utilization of circulating glucose after food withdrawal in Snell dwarf mice.J Biol Chem. 2007; 282: 35069-35077Crossref PubMed Scopus (39) Google Scholar SOGA siRNA, AICAR (500 μmol/L), or LY293004 (10 nmol/L) were introduced to the media 48 hours before the measurement of glucose production. siRNA sequences corresponding to bp 157–175 and 1812–1831 on the open reading frame of murine SOGA were selected using a rational design algorithm (Invitrogen). Transfection with a pool of 2 siRNAs targeting SOGA had a greater knockdown efficiency than transfecting with the individual siRNAs. Transfection was achieved by electroporation using the Mouse Hepatocyte Nucleofector Kit (LONZA) according to the manufacturer's protocol. In brief, freshly isolated mouse hepatocytes were diluted to 3 × 106 cells per tube in media without antibiotics and centrifuged at 2000 rpm for 2 minutes. The supernatant was removed and the cells were resuspended in 100 μl of nucleofector solution containing 100 nmol/L of siRNA. The cell suspension was transferred to an electroporation cuvette, which was placed in a Nucleofector I electroporation device and pulse charge was applied for 2 minutes using program T-28. Hepatocytes received 1.0 ml of media and were transferred to 12-well plates. SOGA expression and production of valine and glucose were assayed 48 hours after siRNA transfection. Media was replaced with glucose-free DMEM containing MG-132 (10 μmol/L), an inhibitor of the ubiquitin–proteasome pathway of protein degradation, for 6–8 hours to measure hepatocytes glucose production. Glucose was measured by colorimetric assay (Autokit Glucose CII). Valine in the medium was measured by a UPLC (Waters) coupled TSQ-Quantum ultra triple quad mass analyzer (ThermoFinigan) in the Biomarkers Facility Core at UNC. Valine was measured in selected reaction monitoring mode (SRM) using the MS/MS transition of 118→72. 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Cells were incubated for 30 minutes at 37°C, and the medium was replaced with GF/DMEM. Digital images were obtained at the Microscopy Services Laboratory of UNC with an Olympus IX81 Motorized Inverted Microscope, a ×40/1.30 Oil DIC lens, Camera pixel count: Hamamatsu C10600–10B 1344 × 1024 using the acquisition software Volocity 5.3.2 (Perkin-Elmer). Fluorescence Filter Cubes Specifications (Semrock, Inc.) were TXRED-4040B for rhodamine and Texas Red: Exciter 562 nm ± 20, Dichroic R 530–585/T 601–800, Emitter 642 ± 20. Lysosome and late autophagosome vacuole number was determined from digital images as isolated punctuate staining, greater than background staining intensity threshold, distinct from lipid droplets in clearly demarcated cells containing 2 nuclei. Spot recognition and enumeration according to the foregoing definition was determined by two individuals. Mice were housed in ventilated isolator cage systems in a pathogen-free barrier facility maintained at 23°C, 55% humidity on a 12-hour light/12-hour dark cycle. Mice received a standard chow diet consisting of 73% carbohydrate, 18% protein, 4% fat, and 5% ash (Purina). Young (3–6 months old) female C57Bl/6J calorie-restricted (CR) and ad libitum–fed (AL) mice were maintained as previously described.38Combs TP Berg AH Rajala MW Klebanov S Iyengar P Jimenez-Chillaron JC Patti ME Klein SL Weinstein RS Scherer PE Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin.Diabetes. 2003; 52: 268-276Crossref PubMed Scopus (474) Google Scholar Adjustments were made to ensure that CR mice received 70% of the ad libitum food intake. Blood samples were collected at 1:00 PM from the tail tip using heparinized capillary tubes (Fisher) and stored at –20°C. Male ob/ob mice (6–9 months old; FVB background strain) received a daily dose of pioglitazone at 6 mg/kg BW in 0.025% (wt/wt) carboxymethylcellulose by oral gavage for 4 days. Control mice received carboxymethylcellulose by oral gavage for 4 days. Blood was collected from the tail tip on day 5 and analyzed for glucose, adiponectin, and 25-kDa SOGA. Immediately after the collection of blood samples, ob/ob mice were sacrificed for tissue collection by cervical dislocation. Northern blot analysis for SOGA mRNA and 18S RNA was performed using 20 μg of liver RNA. NOD mice were bred and housed as previously described.39Wong CP Li L Frelinger JA Tisch R Early autoimmune destruction of islet grafts is associated with a restricted repertoire of IGRP-specific CD8+ T cells in diabetic nonobese diabetic mice.J Immunol. 2006; 176: 1637-1644PubMed Google Scholar Where indicated, diabetic NOD mice were injected with 5 units of insulin (NPH Human Insulin, Isophane Suspension; 100 U/ml Novolin; Novo Nordisk) 24 hours before blood collection. High fat fed adiponectin transgenic mice were produced as previously described.9Combs TP Pajvani UB Berg AH Lin Y Jelicks LA Laplante M Nawrocki AR Rajala MW Parlow AF Cheeseboro L Ding YY Russell RG Lindemann D Hartley A Baker GR Obici S Deshaies Y Ludgate M Rossetti L Scherer PE A transgenic mouse with a deletion in the collagenous domain of adiponectin displays elevated circulating adiponectin and improved insulin sensitivity.Endocrinology. 2004; 145: 367-383Crossref PubMed Scopus (446) Google Scholar Glucose was measured by colorimetric assay. Adiponectin and SOGA were measured by SDS-PAGE analysis using 1 μl plasma. The total concentration of protein in plasma, measured by BCA assay (Pierce), did not differ between groups. Experimental procedures were approved by IACUC. Thirteen healthy women between the ages of 20–63 years and body mass indexes between 20.2 and 31.9 kg/m2 were included for this study. Inclusion was contingent on a good age-typical health status, as ascertained by physical examination and standard clinical laboratory tests such as complete blood count, blood chemistries, fasting glucose, insulin, lipid and liver function tests, liver lipid content, and the presence of no known chronic disease including diabetes. Subjects were admitted to the Clinical and Translational Research Center of UNC and placed on a balanced weight maintenance diet for 10 days.40Fischer LM daCosta KA Kwock L Stewart PW Lu TS Stabler SP Allen RH Zeisel SH Sex and menopausal status influence human dietary requirements for the nutrient choline.Am J Clin Nutr. 2007; 85: 1275-1285Crossref PubMed Scopus (228) Google Scholar Circulating SOGA and adiponectin were measured from plasma samples collected from an intravenous catheter after an overnight fast. The race–ethnicity distribution of the participants was white (8), African American (3), Asian (1), and Native American (1), which reflected the local population characteristics of the Raleigh-Durham-Chapel Hill area. Plasma adiponectin and SOGA were determined by SDS-PAGE using polyclonal antisera against human adiponectin and human SOGA, horseradish peroxidase–linked secondary anti-rabbit IgG. Circulating adiponectin and SOGA levels were measured by enhanced chemiluminescence (ECL) signal intensity. Human studies were performed under an IRB approved protocol (CTRC-2645; Study: 07-1158). Student's t-test was used to identify significant differences when data within groups showed a normal distribution and Wilcoxon-Rank Sum test was used when data did not show a normal distribution. P values less than 0.05 were considered significant. Protein extracts from adiponectin-treated hepatoma cells were digested with trypsin and analyzed by mass spectrometry. Mass spectrometry revealed a peptide, KVLPSEEDDFLEVNSM, encoded by a gene located on chromosome 2 in mice (2qH1) and chromosom
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