Discoidal HDL and apoA-I-derived peptides improve glucose uptake in skeletal muscle
2013; Elsevier BV; Volume: 54; Issue: 5 Linguagem: Inglês
10.1194/jlr.m032904
ISSN1539-7262
AutoresJonathan Dalla‐Riva, Karin G. Stenkula, Jitka Petrlová, Jens O. Lagerstedt,
Tópico(s)Diabetes Treatment and Management
ResumoLipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined whether nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and whether a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain-specific peptides of apoA-I showed that the lipid-free C-terminal 190–243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190–243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. Discoidal HDL and the 190–243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle, and the C-terminal α-helical content of apoA-I may be an important determinant of this effect. Lipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined whether nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and whether a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain-specific peptides of apoA-I showed that the lipid-free C-terminal 190–243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190–243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. Discoidal HDL and the 190–243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle, and the C-terminal α-helical content of apoA-I may be an important determinant of this effect. Apolipoprotein A-I (apoA-I) is the primary protein component of high-density lipoprotein (HDL) and as such is important for reverse cholesterol transport (1Zannis V.I. Chroni A. Krieger M. Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL.J. Mol. Med. (Berl.). 2006; 84: 276-294Crossref PubMed Scopus (307) Google Scholar). The apoA-I protein exists in a variety of structural organizations in the different forms of HDL and in the lipid-free state (2Rye K.A. Bursill C.A. Lambert G. Tabet F. Barter P.J. The metabolism and anti-atherogenic properties of HDL.J. Lipid Res. 2009; 50 (Suppl.): S195-S200Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The lipid-bound forms include both discoidal planar HDL particles of different diameters and mature spherical HDL particles of varying sizes and lipid compositions (Fig. 1). Key features of apoA-I that determine its function include high structural plasticity resulting in major changes in secondary, tertiary, and quaternary structures between apo- and lipid-bound states, along with an amphipathic character of the helices formed by lipid association. It is known that reduced plasma HDL is an independent risk factor for cardiovascular disease (3Despres J.P. Lemieux I. Dagenais G.R. Cantin B. Lamarche B. HDL-cholesterol as a marker of coronary heart disease risk: the Quebec cardiovascular study.Atherosclerosis. 2000; 153: 263-272Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) and type 2 diabetes (4Gatti A. Maranghi M. Bacci S. Carallo C. Gnasso A. Mandosi E. Fallarino M. Morano S. Trischitta V. Filetti S. Poor glycemic control is an independent risk factor for low HDL cholesterol in patients with type 2 diabetes.Diabetes Care. 2009; 32: 1550-1552Crossref PubMed Scopus (40) Google Scholar), with diabetic patients having an increased risk for cardiovascular complications (5Laakso M. Hyperglycemia and cardiovascular disease in type 2 diabetes.Diabetes. 1999; 48: 937-942Crossref PubMed Scopus (642) Google Scholar). Such outcomes are typically regarded as secondary to diabetes; however, recent data show that apoA-I in HDL directly contributes to peripheral glucose metabolism (6Han R. Lai R. Ding Q. Wang Z. Luo X. Zhang Y. Cui G. He J. Liu W. Chen Y. Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism.Diabetologia. 2007; 50: 1960-1968Crossref PubMed Scopus (120) Google Scholar, 7Drew B.G. Duffy S.J. Formosa M.F. Natoli A.K. Henstridge D.C. Penfold S.A. Thomas W.G. Mukhamedova N. de Courten B. Forbes J.M. et al.High-density lipoprotein modulates glucose metabolism in patients with type 2 diabetes mellitus.Circulation. 2009; 119: 2103-2111Crossref PubMed Scopus (310) Google Scholar, 8Zhang Q. Zhang Y. Feng H. Guo R. Jin L. Wan R. Wang L. Chen C. Li S. High density lipoprotein (HDL) promotes glucose uptake in adipocytes and glycogen synthesis in muscle cells.PLoS ONE. 2011; 6: e23556Crossref PubMed Scopus (44) Google Scholar, 9Drew B.G. Rye K.A. Duffy S.J. Barter P. Kingwell B.A. The emerging role of HDL in glucose metabolism.Nat. Rev. Endocrinol. 2012; 8: 237-245Crossref PubMed Scopus (177) Google Scholar). How the various conformations of apoA-I contribute to this effect is yet to be fully clarified. The rate of glucose uptake in skeletal muscle, the principal site for plasma glucose clearance, is determined by cell surface levels of GLUT4, which is controlled by both the insulin signaling pathway and the AMP kinase (AMPK) contraction-induced pathway (10Tremblay F. Dubois M.J. Marette A. Regulation of GLUT4 traffic and function by insulin and contraction in skeletal muscle.Front. Biosci. 2003; 8: d1072-d1084Crossref PubMed Scopus (33) Google Scholar). As these are largely independent signaling routes, AMPK represents a therapeutic target for the maintenance of plasma glucose despite insulin resistance (11Towler M.C. Hardie D.G. AMP-activated protein kinase in metabolic control and insulin signaling.Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1056) Google Scholar). This is exemplified by impaired contraction-induced glucose uptake in skeletal muscle of mice expressing dominant negative AMPK (12Mu J. Brozinick Jr, J.T. Valladares O. Bucan M. Birnbaum M.J. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle.Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar) and improved glucose control in diabetic subjects during an acute bout of exercise (13Musi N. Fujii N. Hirshman M.F. Ekberg I. Froberg S. Ljungqvist O. Thorell A. Goodyear L.J. AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise.Diabetes. 2001; 50: 921-927Crossref PubMed Scopus (308) Google Scholar). It has been shown that lipid-free apoA-I and spherical HDL can induce glucose uptake in C2C12 myotubes (6Han R. Lai R. Ding Q. Wang Z. Luo X. Zhang Y. Cui G. He J. Liu W. Chen Y. Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism.Diabetologia. 2007; 50: 1960-1968Crossref PubMed Scopus (120) Google Scholar) and in human myotubes differentiated from muscle satellite cells from diabetic donors (7Drew B.G. Duffy S.J. Formosa M.F. Natoli A.K. Henstridge D.C. Penfold S.A. Thomas W.G. Mukhamedova N. de Courten B. Forbes J.M. et al.High-density lipoprotein modulates glucose metabolism in patients with type 2 diabetes mellitus.Circulation. 2009; 119: 2103-2111Crossref PubMed Scopus (310) Google Scholar) via the activation of AMPK, which provides promise for apoA-I/HDL as a novel diabetic treatment. Despite these findings, it is not clear whether discoidal HDL is also capable of specifically regulating muscle glucose uptake and whether this occurs via AMPK. Given that HDL subspecies interact differently with cellular receptors at the vascular wall for cholesterol efflux and that discoidal HDL is a potent structure for this interaction (14Favari E. Calabresi L. Adorni M.P. Jessup W. Simonelli S. Franceschini G. Bernini F. Small discoidal pre-beta1 HDL particles are efficient acceptors of cell cholesterol via ABCA1 and ABCG1.Biochemistry. 2009; 48: 11067-11074Crossref PubMed Scopus (112) Google Scholar), we hypothesized that discoidal HDL would be highly effective in the stimulation of glucose uptake in muscle. Herein, we investigated the effects of synthetic discoidal HDL (rHDL) and apoA-I-derived peptides on glucose uptake, intracellular signaling, and GLUT4 translocation to the plasma membrane using L6 myotubes and flexor digitorum brevis (FDB) fibers. We show that rHDL produces insulin-like effects in these models, and we identify a novel peptide candidate that induces responses comparable to those of rHDL. L6 myoblasts (ATCC #CRL-1458) were grown in α-MEM (Invitrogen) supplemented with 10% FBS (Sigma) and 1% antibiotic/antimycotic (penicillin, streptomycin, amphotericin B; Invitrogen). Differentiation to myotubes was achieved by switching from growth media to 2% FBS α-MEM for 6–12 days. Cells were maintained at 37°C and 5% CO2. Human apoA-I variants (full-length and truncated variants produced by site-directed mutagenesis, corresponding to amino acids 1–243 and to amino acids 1–189, 44–189, 44–243, and 190–243, respectively) were expressed in Escherichia coli strain BL21 Star (DE3)pLysS cells (Invitrogen) from the human apoA-I gene containing a hexa-His affinity tag at the N-terminus (15Lagerstedt J.O. Budamagunta M.S. Oda M.N. Voss J.C. Electron paramagnetic resonance spectroscopy of site-directed spin labels reveals the structural heterogeneity in the N-terminal domain of apoA-I in solution.J. Biol. Chem. 2007; 282: 9143-9149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, the gene (full-length or truncated variants of the gene) was cloned into the pEXP-5 plasmid (Novagen Inc.), transferred into the bacteria, and cultivated at 37°C in LB medium with 50 µg/ml of ampicillin and 34 µg/ml of chloramphenicol. Protein expression was induced for 3–4 h following the addition of 0.5 mmol/l isopropyl-β-thiogalactopyranoside (Sigma). Following cell disruption, apoA-I was purified from the soluble fraction of the cells using a His-Trap-Nickel-chelating column (GE Healthcare) and a mobile phase of phosphate-buffered saline (PBS), pH 7.4, with 3 mol/l guanidine. The protein was then washed in PBS (pH 7.4) containing 100 mmol/l imidazole, and then eluted with PBS containing 500 mmol/l imidazole. Imidazole was removed from the protein sample by using desalting columns (GE Healthcare) equilibrated with PBS, pH 7.4. Protein purity was analyzed by SDS-PAGE, and concentration was determined by the BCA method (Pierce) or using a nanodrop 2000c spectrophotometer (Thermo Scientific). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (Avanti Polar Lipids) was dissolved in chloroform:methanol (3:1), which was evaporated under a stream of nitrogen gas, and the resulting lipid film was resuspended in PBS. For POPC rHDL, deoxycholate was added to the POPC emulsion at a 2:1 molar ratio (deoxycholate:POPC) and incubated with apoA-I at a 156:1 molar ratio (phospholipid:protein) for 1 h at 22°C (16Lagerstedt J.O. Cavigiolio G. Budamagunta M.S. Pagani I. Voss J.C. Oda M.N. Structure of apolipoprotein A-I N terminus on nascent high density lipoproteins.J. Biol. Chem. 2011; 286: 2966-2975Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Deoxycholate was removed from the POPC rHDL preparation by extensive dialysis against PBS. DMPC rHDL was prepared according to Ref. 17Petrlova J. Duong T. Cochran M.C. Axelsson A. Morgelin M. Roberts L.M. Lagerstedt J.O. The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils.J. Lipid Res. 2012; 53: 390-398Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar. Briefly, the DMPC emulsion was passed through a polycarbonate membrane with 100 nm pore size using the LiposoFast system (Avestin) a minimum of 20 times. The resulting vesicles were incubated with apoA-I at a 156:1 molar ratio (phospholipid:protein) for 4 days at 22°C. ApoA-I dimers, indicative of rHDL formation, was confirmed by blue native PAGE (Invitrogen). POPC vesicles were prepared by first passing the POPC emulsion through a polycarbonate membrane with 400 nm pore size followed by passage through a 100 nm pore size membrane using the LiposoFast system (Avestin) a minimum of 20 times. Treatments were performed with POPC rHDL unless otherwise indicated. Circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Jasco CDF-426S Peltier set to 25°C. ApoA-I (full-length and 190–243 fragment) was diluted to 0.1 mg/ml in PBS (final concentration was 25 mmol/l phosphate, 25 mmol/l NaCl, pH 7.4), placed in a 0.1 mm quartz cuvette, and after extensive purging with nitrogen, scanned in the region 200–260 nm (scan speed was 20 nm/min). Averages of five scans were baseline-subtracted (PBS buffer; 25 mmol/l phosphate, 25 mmol/l NaCl), and the α-helical content was calculated from the molar ellipticity at 222 nm as previously described (18Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain.Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (264) Google Scholar). Prior to stimulation, cultured cells were serum starved for 4 h in serum-free α-MEM, and all subsequent treatments, including insulin or phenformin as positive controls (Sigma), were performed in serum-free α-MEM. After treatments, cells were washed with ice-cold PBS and lysed on ice using a nondenaturing lysis buffer (1% Triton X-100, 50 mmol/l Tris, 150 mmol/l NaCl, pH 8.0) containing protease and phosphatase inhibitors (Roche). Lysates were centrifuged at 16,000 g, 20 min at 4°C, and then BCA protein assay (Pierce) was performed on supernatants. Equal protein amounts were separated by SDS-PAGE and transferred to nitrocellulose membranes. pAMPK, AMPK, pACC, pAkt, Akt (Cell Signaling), and tubulin (Sigma) were used for immunodetection with IRDye 800CW and 680RD secondary antibodies (LI-COR). Blots were imaged using the Odyssey Fc system and quantified using Image studio v2.0 software. Prior to stimulation cells were starved for 2 h in serum-free α-MEM, and all subsequent treatments, which were performed in triplicate and included cytochalasin B (Sigma) as a measure of cell-associated nonspecific radioactivity, were performed in uptake buffer (140 mmol/l NaCl, 20 mmol/l HEPES, 5 mmol/l KCl, 2.5 mmol/l MgSO4, 1 mmol/l CaCl2, pH 7.4). After stimulation, treatments were replaced with 10 µmol/l 2-deoxy-D-glucose (Sigma) and 1 µCi/ml 2-[3H]deoxy-D-glucose (Perkin Elmer) in uptake buffer for 15 min at room temperature. Cells were then washed twice with ice-cold PBS and lysed with 1 mol/l NaOH on ice. Lysates were collected and radioactivity was quantified by scintillation counting. Two to five transgenic mice (C57/Bl6; 10–14 weeks old) with muscle-specific HA-GLUT4-GFP expression (gift from S. Cushman, Lund University Diabetes Centre, Sweden) (19Fazakerley D.J. Lawrence S.P. Lizunov V.A. Cushman S.W. Holman G.D. A common trafficking route for GLUT4 in cardiomyocytes in response to insulin, contraction and energy-status signalling.J. Cell Sci. 2009; 122: 727-734Crossref PubMed Scopus (35) Google Scholar, 20Lizunov V.A. Stenkula K.G. Lisinski I. Gavrilova O. Yver D.R. Chadt A. Al-Hasani H. Zimmerberg J. Cushman S.W. Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E950-E960Crossref PubMed Scopus (35) Google Scholar), were used for each condition. The animals were euthanized, and FDB muscles dissected out and incubated with oxygenated Krebs-Hensleit carbonate Hepes (KRBH) buffer (6 mmol/l KCl, 1 mmol/l Na2HPO4, 0.2 mmol/l NaHPO4, 1.4 mmol/l MgSO4, 1 mmol/l CaCl2, 128 mmol/l NaCl, 10 mmol/l HEPES, pH 7.4) with 0.5% (w/v) BSA. After dissection, muscles were continually oxygenated with 95% O2 / 5% CO2 and incubated at 37°C for 2 h in a water bath with slow shaking. After incubation, muscles were washed three times with oxygenated KRBH and were then either treated with insulin (100 nmol/l), apoA-I (full-length, lipid-free), rHDL, or apoA-I fragment 190–243 or kept basal for 1 h. After stimulation, basal (nonstimulated) and stimulated muscles were fixed for 10 min with 4% paraformaldehyde in PBS, washed three times with PBS containing 1% BSA, and incubated for 30–60 min with anti-HA (Covance), followed by 30 min with fluorescently labeled secondary antibodies ALEXA-647 (Invitrogen). Fixed cells were imaged using a confocal LSM 510 microscope (Zeiss) using a 40× objective, NA 1.3, using BP 505-530 and LP 650. Images were collected with the LSM software. All data are displayed as mean ± SEM unless indicated otherwise. Where appropriate, analysis was performed by two-tailed Student t-test or one-way ANOVA with Bonferroni舗s post hoc test using Microsoft Excel and Graph Pad Prism software. P ≤ 0.05 was considered significant. To investigate the effect of discodial HDL (rHDL) on GLUT4 translocation and glucose uptake, we produced recombinant human apoA-I and reconstituted HDL needed for cell incubations. L6 myotubes were incubated with 2 µmol/l (60 µg/ml) discoidal rHDL (expressed as total protein concentration of apo A-I; given two apoA-I molecules per particle, this corresponds to 1 µmol/l discodial rHDL) for 1 h. The rHDL treatment induced a glucose uptake that was 2.3 ± 0.39-fold (P ≤ 0.05) over basal, which was similar to insulin stimulation (2.4 ± 1.0-fold) (Fig. 2A). To test for the contribution of the constituent phospholipid to rHDL-induced glucose uptake, rHDL made with POPC was compared with 100 nm POPC vesicles containing no apoA-I protein. Incubation with empty POPC vesicles (0.10 mmol/l) did not induce glucose uptake (Fig. 2A). DMPC as the lipid constituent in protein-free lipid vesicles and as the phospholipid constituent of rHLD was also tested. Whereas rHDL particles synthesized from apoA-I (30 µg/ml) and DMPC (0.16 mmol/l) induced glucose uptake to a level similar to insulin-stimulated cells, DMPC vesicles alone did not stimulate glucose uptake (results not shown). Blue native PAGE was performed on all rHDL preparations to confirm the formation of 10 nm diameter discoidal apoA-I dimers. Fig. 2B is a representative Coomassie-stained gel that shows monomeric lipid-free apoA-I (∼28 kDa) and the size of POPC rHDL (∼10 nm diameter). These data clearly show that rHDL exerts a potent effect on glucose uptake in muscle and that the rHDL-mediated glucose uptake is apoA-I protein dependent. To support the glucose uptake observations, the ability of rHDL to increase the amount of GLUT4 in the plasma membrane was assessed by immunofluorescence microscopy of intact FDB muscle fibers, isolated from a transgenic mouse model with muscle-specific HA-GLUT4-GFP expression (20Lizunov V.A. Stenkula K.G. Lisinski I. Gavrilova O. Yver D.R. Chadt A. Al-Hasani H. Zimmerberg J. Cushman S.W. Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E950-E960Crossref PubMed Scopus (35) Google Scholar). The HA epitope present on the first exofacial loop of the HA-GLUT4-GFP construct allows detection of GLUT4 inserted into the plasma membrane. Intact FDB fibers were incubated ex vivo with rHDL, followed by fixation and HA antibody labeling of nonpermeabilized cells. Both insulin and rHDL treatment induced translocation of GLUT4 into the sarcolemma plasma membrane as detected by HA signal (Fig. 2C, upper panel). The lower panel in Fig. 2C displays total GLUT4 detected by GFP signal merged with the HA signal in nonstimulated and stimulated muscle fibers. Due to steric hindrance, labeling of the transverse tubules was limited, and therefore, the plasma membrane GLUT4 translocation was assessed only at the sarcolemma. The effect of apoA-I on muscle has previously been suggested to occur through a noninsulin-dependent signal pathway as described in studies using lipid-free apoA-I (6Han R. Lai R. Ding Q. Wang Z. Luo X. Zhang Y. Cui G. He J. Liu W. Chen Y. Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism.Diabetologia. 2007; 50: 1960-1968Crossref PubMed Scopus (120) Google Scholar) and apoA-I in mature plasma HDL (7Drew B.G. Duffy S.J. Formosa M.F. Natoli A.K. Henstridge D.C. Penfold S.A. Thomas W.G. Mukhamedova N. de Courten B. Forbes J.M. et al.High-density lipoprotein modulates glucose metabolism in patients with type 2 diabetes mellitus.Circulation. 2009; 119: 2103-2111Crossref PubMed Scopus (310) Google Scholar). To dissect the effect of discoidal rHDL on signaling pathways, we conducted western blotting using lysates from L6 myotubes incubated with rHDL. After 60 min of treatment (14 µmol/l apoA-I in rHDL), L6 myotube lysates showed increased (1.36 ± 0.071-fold; P ≤ 0.01) levels of phosphorylated AMPK (Fig. 3A, B) and its downstream target ACC (1.64 ± 0.26-fold; P ≤ 0.05) (Fig. 3C, D). Phenformin was used as a positive control, inducing a 1.98 ± 0.33-fold (P ≤ 0.05) and 2.7 ± 0.74-fold (P ≤ 0.05) elevation of phosphorylated AMPK and ACC, respectively, at a concentration of 0.4 mmol/l. In contrast, Akt phosphorylation was unaffected by rHDL (1.12 ± 0.27-fold), while insulin (100 nmol/l) had a 51 ± 12.5-fold (P ≤ 0.01) effect (Fig. 3E, F). Representative immunoblots of those quantified in Fig. 3A, C, and E are given in Fig. 3B, D, and F, respectively. The relative effect of specific regions of apoA-I to increase glucose uptake was investigated using full-length and truncated protein fragments corresponding to residues 1–243 (full-length), 1–189 (N-terminal/central domain), 44–189 (central domain), 44–243 (central/C-terminal domain), and 190–243 (C-terminal domain) of full-length apoA-I (Fig. 4A). As can be seen in Fig. 4B, all five peptides induced glucose uptake, with peptide fragment 190–243 displaying the largest and most consistent influence on L6 myotube glucose uptake (1.77 ± 0.23-fold change versus control; P ≤ 0.05). To verify this observation, confocal immunofluorescence imaging of FDB fibers labeled with HA-antibody was performed after ex vivo incubation with the 190–243 peptide. Relative to basal conditions, these images show greater membrane levels of GLUT4 protein in response to the 190–243 peptide (Fig. 4C), thus supporting the findings in Fig. 4B. We next used western blotting to examine the signaling pathway activated by the 190–243 peptide. Incubation of L6 myotubes with increasing concentrations (2, 10, and 20 µmol/l) of 190–243 peptide resulted in phosphorylation of AMPK (Fig. 4D). In contrast, no Akt phosphorylation was observed. Phenformin at 1 mmol/l and insulin at 100 nmol/l were used as positive controls for phosphorylation of AMPK and Akt, respectively. Finally, our initial analyses on lipid-free apoA-I-induced signaling in L6 myotubes treated with liver X receptor (LXR) agonist to induce overexpression of ABCA1 suggest a non-ABCA1-dependent signaling pathway (data not shown). We hypothesized that binding of cellular lipids to the 190–243 fragment upon incubation with L6 myotubes and FDB fibers may be necessary for its glucose uptake-inducing effect. It is known that the 190–243 fragment can promote cholesterol efflux from cultured cells and form discoidal particles by solubilization of lipid in solution (21Vedhachalam C. Chetty P.S. Nickel M. Dhanasekaran P. Lund-Katz S. Rothblat G.H. Phillips M.C. Influence of apolipoprotein (Apo) A-I structure on nascent high density lipoprotein (HDL) particle size distribution.J. Biol. Chem. 2010; 285: 31965-31973Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Tanaka M. Koyama M. Dhanasekaran P. Nguyen D. Nickel M. Lund-Katz S. Saito H. Phillips M.C. Influence of tertiary structure domain properties on the functionality of apolipoprotein A-I.Biochemistry. 2008; 47: 2172-2180Crossref PubMed Scopus (42) Google Scholar), which can be visualized as oligomers on native PAGE. To assess oligomer formation indicative of lipid binding, purified 190–243 peptide and conditioned media from cells treated with the 190–243 fragment for 1 h were run on a blue native PAGE and Coomassie stained (Fig. 5A). Under both conditions, 190–243 appeared as a single band at approximately 40 kDa corresponding to a tetramer. Lipid-free apoA-I and rHDL were included on the gel as a full-length protein monomer and dimer reference. Although the presence of minute amounts of lipids in the 190–243 tetramers cannot be excluded, the migration distance is clearly different from the rHDL particles formed by the 190–243 peptide in interaction with cultured baby hamster kidney (BHK) cells expressing human ABCA1 (∼10 nm rHDL formed; approximately corresponding to the 242 kDa marker protein) and from interaction with phospholipid multilamellar vesicles (∼17 nm rHDL) (21Vedhachalam C. Chetty P.S. Nickel M. Dhanasekaran P. Lund-Katz S. Rothblat G.H. Phillips M.C. Influence of apolipoprotein (Apo) A-I structure on nascent high density lipoprotein (HDL) particle size distribution.J. Biol. Chem. 2010; 285: 31965-31973Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Moreover, our findings on the oligomeric state of the 190–243 peptide is in agreement with those on the 198–243 peptide that self-associates as tetramers in solution (23Zhu H.L. Atkinson D. Conformation and lipid binding of a C-terminal (198–243) peptide of human apolipoprotein A-I.Biochemistry. 2007; 46: 1624-1634Crossref PubMed Scopus (32) Google Scholar). As depicted in Fig. 1, the structure of apoA-I in the apo-state is significantly different from the structural organization of the same protein in rHDL particles. This structural transition of the lipid-binding process involves a major increase in α-helical secondary structure (from about 44–55% in the apo-state to 78% α-helical secondary structure in discoidal HDL) (17Petrlova J. Duong T. Cochran M.C. Axelsson A. Morgelin M. Roberts L.M. Lagerstedt J.O. The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils.J. Lipid Res. 2012; 53: 390-398Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 24Saito H. Dhanasekaran P. Nguyen D. Deridder E. Holvoet P. Lund-Katz S. Phillips M.C. Alpha-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids.J. Biol. Chem. 2004; 279: 20974-20981Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). As rHDL is potent in stimulation of glucose uptake in myotubes, we speculated that lipid-free 190–243 fragment may adopt an amphiphatic α-helix in solution. To investigate this, CD spectroscopy spectra were obtained at a protein concentration of 0.1 mg/ml for the 190–243 fragment and full-length protein for comparison (Fig. 5). The helical content was estimated from their molar ellipticities at 222 nm to be 17% (or 21% at 0.2 mg/ml; data not shown) and 56% for 190–243 fragment and full-length apoA-I, respectively, suggesting an increase in helical structure of the fragment (see Discussion). This study analyzed the capability of discoidal HDL and the potency of subdomains of apoA-I to promote translocation of the GLUT4 glucose transporter to the plasma membrane and thereby induce glucose uptake. From our work, it is clear that discoidal HDL promotes glucose uptake in cultured skeletal muscle, eliciting an effect comparable to insulin. Furthermore, we have made the discovery that the 190–243 peptide, corresponding to the C-terminal domain of apoA-I, is itself an efficient agonist for glucose uptake. Currently only two studies have shown that both lipid-free apoA-I and mature spherical HDL can increase glucose uptake in skeletal muscle via the AMPK signaling pathway (6Han R. Lai R. Ding Q. Wang Z. Luo X. Zhang Y. Cui G. He J. Liu W. Chen Y. Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism.Diabetologia. 2007; 50: 1960-1968Crossref PubMed Scopus (120) Google Scholar, 7Drew B.G. Duffy S.J. Formosa M.F. Natoli A.K. Henstridge D.C. Penfold S.A. Thomas W.G. Mukhamedova N. de Courten B. Forbes J.M. et al.High-density lipoprotein modulates glucose metabolism in patients with type 2 diabetes mellitus.Circulation. 2009; 119: 2103-2111Crossref PubMed Scopus (310) Google Scholar). However, what had not been clearly addressed was the efficacy of discoidal HDL, an important consideration given the marked alterations in structure that apoA-I undergoes during HDL maturation (depicted in Fig. 1). While the study by Drew et al. (7Drew B.G. Duffy S.J. Formosa M.F. Natoli A.K. Henstridge D.C. Penfold S.A. Thomas W.G. Mukhamedova N. de Courten B. Forbes J.M. et al.High-density lipoprotein modulates glucose metabolism in patients with type 2
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