Defective Fatty Acid Uptake in the Spontaneously Hypertensive Rat Is a Primary Determinant of Altered Glucose Metabolism, Hyperinsulinemia, and Myocardial Hypertrophy
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m100942200
ISSN1083-351X
AutoresTahar Hajri, Azeddine Ibrahimi, Chris T. Coburn, F. F. Knapp, Ted Kurtz, Michal Pravenec, Nada A. Abumrad,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoGenetic linkage studies implicated deficiency of CD36, a membrane fatty acid (FA) transporter, in the hypertriglyceridemia and hyperinsulinemia of the spontaneously hypertensive rat (SHR). In this study we determined whether loss of CD36 function in FA uptake is a primary determinant of the SHR phenotype. In vivo, tissue distribution of iodinated, poorly oxidized β-methyliodophenyl pentadecanoic acid (BMIPP) was examined 2 h after its intravenous injection. Fatty acid transport was also measured in vitro over 20 to 120 s in isolated adipocytes and cardiomyocytes obtained from SHR and from a congenic line (SHRchr4) that incorporates a piece of chromosome 4 containing wild-type CD36. SHR heart and adipose tissue exhibited defects in FA uptake and in conversion of diglycerides to triglycerides that are similar to those observed in the CD36 null mouse. However, a key difference in SHR tissues is that fatty acid oxidation is much more severely impaired than fatty acid esterification, which may underlie the 4–5-fold accumulation of free BMIPP measured in SHR muscle. Studies with isolated adipocytes and cardiomyocytes directly confirmed both the defect in FA transport and the fact that it is underestimated by BMIPP. Heart, oxidative muscle, and adipose tissue in the SHR exhibited a large increase in glucose uptake measured in vivo using [18F]fluorodeoxyglucose. Supplementation of the diet with short-chain fatty acids, which do not require CD36-facilitated transport, eliminated the increase in glucose uptake, the hyperinsulinemia, and the heart hypertrophy in the SHR. This indicated that lack of metabolic energy consequent to deficient FA uptake is the primary defect responsible for these abnormalities. Hypertension was not alleviated by the supplemented diet suggesting it is unrelated to fuel supply and any contribution of CD36 deficiency to this trait may be more complex to determine. It may be worth exploring whether short-chain FA supplementation can reverse some of the deleterious effects of CD36 deficiency in humans, which may include hypertrophic cardiomyopathy. Genetic linkage studies implicated deficiency of CD36, a membrane fatty acid (FA) transporter, in the hypertriglyceridemia and hyperinsulinemia of the spontaneously hypertensive rat (SHR). In this study we determined whether loss of CD36 function in FA uptake is a primary determinant of the SHR phenotype. In vivo, tissue distribution of iodinated, poorly oxidized β-methyliodophenyl pentadecanoic acid (BMIPP) was examined 2 h after its intravenous injection. Fatty acid transport was also measured in vitro over 20 to 120 s in isolated adipocytes and cardiomyocytes obtained from SHR and from a congenic line (SHRchr4) that incorporates a piece of chromosome 4 containing wild-type CD36. SHR heart and adipose tissue exhibited defects in FA uptake and in conversion of diglycerides to triglycerides that are similar to those observed in the CD36 null mouse. However, a key difference in SHR tissues is that fatty acid oxidation is much more severely impaired than fatty acid esterification, which may underlie the 4–5-fold accumulation of free BMIPP measured in SHR muscle. Studies with isolated adipocytes and cardiomyocytes directly confirmed both the defect in FA transport and the fact that it is underestimated by BMIPP. Heart, oxidative muscle, and adipose tissue in the SHR exhibited a large increase in glucose uptake measured in vivo using [18F]fluorodeoxyglucose. Supplementation of the diet with short-chain fatty acids, which do not require CD36-facilitated transport, eliminated the increase in glucose uptake, the hyperinsulinemia, and the heart hypertrophy in the SHR. This indicated that lack of metabolic energy consequent to deficient FA uptake is the primary defect responsible for these abnormalities. Hypertension was not alleviated by the supplemented diet suggesting it is unrelated to fuel supply and any contribution of CD36 deficiency to this trait may be more complex to determine. It may be worth exploring whether short-chain FA supplementation can reverse some of the deleterious effects of CD36 deficiency in humans, which may include hypertrophic cardiomyopathy. spontaneously hypertensive rat fatty acid β-methyliodophenyl pentadecanoic acid 2-fluorodeoxyglucose diglycerides triglycerides The spontaneously hypertensive rat (SHR)1 is a widely studied rodent model of human metabolic Syndrome X, in which hypertension is associated with dyslipidemia and with insulin resistance of glucose metabolism (1Aitman T.J. Gotoda T. Evans A.L. Imrie H. Heath K.E. Trembling P.M. Truman H. Wallace C.A. Rahman A. Dore C. Flint J. Kren V. Zidek V. Kurtz T.W. Pravenec M. Scott J. Nat. Genet. 1997; 16: 197-201Crossref PubMed Scopus (138) Google Scholar). Two quantitative trait loci for defective insulin action in the SHR were identified on chromosomes 4 and 12. Quantitative trait loci for defects in glucose and fatty acid metabolism and for hypertension map to the same locus on chromosome 4 (1Aitman T.J. Gotoda T. Evans A.L. Imrie H. Heath K.E. Trembling P.M. Truman H. Wallace C.A. Rahman A. Dore C. Flint J. Kren V. Zidek V. Kurtz T.W. Pravenec M. Scott J. Nat. Genet. 1997; 16: 197-201Crossref PubMed Scopus (138) Google Scholar). Microarray screening and congenic mapping identified CD36 on rat chromosome 4 as a defective SHR gene at the peak of linkage to these quantitative trait loci (2Aitman T.J. Glazier A.M. Wallace C.A. Cooper L.D. Norsworthy P.J. Wahid F.N. Al-Majali K.M. Trembling P.M. Mann C.J. Shoulders C.C. Graf D. St. Lezin E. Kurtz T.W. Kren V. Pravenec M. Ibrahimi A. Abumrad N.A. Stanton L.W. Scott J. Nat. Genet. 1999; 21: 76-83Crossref PubMed Scopus (641) Google Scholar). The SHR CD36 cDNA contains multiple sequence variants and the protein product is undetectable in SHR adipocyte plasma membrane (2Aitman T.J. Glazier A.M. Wallace C.A. Cooper L.D. Norsworthy P.J. Wahid F.N. Al-Majali K.M. Trembling P.M. Mann C.J. Shoulders C.C. Graf D. St. Lezin E. Kurtz T.W. Kren V. Pravenec M. Ibrahimi A. Abumrad N.A. Stanton L.W. Scott J. Nat. Genet. 1999; 21: 76-83Crossref PubMed Scopus (641) Google Scholar). CD36 was identified on platelets (3Tandon N.N. Lipsky R.H. Burgess W.H. Jamieson G.A. J. Biol. Chem. 1989; 264: 755-7570Google Scholar) as a receptor for thrombospondin, collagen (4Greenwalt D.E. Lipsky R.H. Ockenhouse C.F. Ikeda H. Tandon N.N. Jamieson G.A. Blood. 1992; 80: 1105-1115Crossref PubMed Google Scholar), and oxidized lipoproteins (5Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar). CD36 is also known as FAT for fatty acid translocase (6Abumrad N.A. el-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. J. Biol. Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar) since its function in long-chain FA transport was identified from its binding the reactive FA derivative sulfo-N-succinimidyl oleic acid, an irreversible inhibitor of FA uptake by rat adipocytes (7Harmon C.M. Abumrad N.A. J. Membr. Biol. 1993; 133: 43-49Crossref PubMed Scopus (164) Google Scholar). Results of in vitrostudies provided strong support for a role of CD36 in FA transport (8Abumrad N. Coburn C. Ibrahimi A. Biochim. Biophys. Acta. 1999; 1441: 4-13Crossref PubMed Scopus (209) Google Scholar). Recent work with CD36 null (9Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F. Silverstein R.L. J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 10Coburn C.T. Knapp Jr., F.F. Febbraio M. Beets A.L. Silverstein R.L. Abumrad N.A. J. Biol. Chem. 2000; 275: 32523-32529Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar) and transgenic (11Ibrahimi A. Bonen A. Blinn W.D. Hajri T. Li X. Zhong K. Cameron R. Abumrad N.A. J. Biol. Chem. 1999; 274: 26761-26766Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) mice documented that CD36 facilitates a major fraction of FA uptake by heart, oxidative skeletal muscle, and adipose tissues. Based on the role of CD36 as a FA transporter, it was proposed that a primary genetic defect in the SHR is compromised tissue utilization of FA (2Aitman T.J. Glazier A.M. Wallace C.A. Cooper L.D. Norsworthy P.J. Wahid F.N. Al-Majali K.M. Trembling P.M. Mann C.J. Shoulders C.C. Graf D. St. Lezin E. Kurtz T.W. Kren V. Pravenec M. Ibrahimi A. Abumrad N.A. Stanton L.W. Scott J. Nat. Genet. 1999; 21: 76-83Crossref PubMed Scopus (641) Google Scholar), which would contribute to the pathogenesis of insulin resistance by producing secondary alterations in basal glucose metabolism (12Randle P.J. Diabetes Metab. Rev. 1998; 14: 263-283Crossref PubMed Scopus (686) Google Scholar). Support for this interpretation was provided by the findings that both the hyperinsulinemia and insulin resistance were improved when a piece of chromosome 4 containing wild-type CD36 was integrated into the SHR genome (SHRchr4 line) (13Pravenec M. Zidek V. Simakova M. Kren V. Krenova D. Horky K. Jachymova M. Mikova B. Kazdova L. Aitman T.J. Churchill P.C. Webb R.C. Hingarh N.H. Yang Y. Wang J.M. Lezin E.M. Kurtz T.W. J. Clin. Invest. 1999; 103: 1651-1657Crossref PubMed Scopus (103) Google Scholar) or with transgenic rescue (14Pravenec M. Landa V. Zidek V. Musilova A. Kren V. Kazdova L. Aitman T.J. Glazier A.M. Ibrahimi A. Abumrad N.A. Qi N. Wang J.M. St. Lezin E.M. Kurtz T.W. Nat. Genet. 2001; 27: 156-158Crossref PubMed Scopus (161) Google Scholar). However, arguing against this interpretation are important phenotypic differences between the SHR and CD36 null mouse. The CD36 null mouse, in contrast to the SHR, is hypoglycemic (9Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F. Silverstein R.L. J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar) and hypoinsulinemic 2T. Hajri, unpublished observations. 2T. Hajri, unpublished observations. while the mouse with CD36 overexpression exhibits the opposite changes (11Ibrahimi A. Bonen A. Blinn W.D. Hajri T. Li X. Zhong K. Cameron R. Abumrad N.A. J. Biol. Chem. 1999; 274: 26761-26766Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Also complicating the link between CD36 deficiency and the SHR phenotype are reports that some SHR lines, which apparently have normal CD36 levels, exhibit symptoms of insulin resistance (13Pravenec M. Zidek V. Simakova M. Kren V. Krenova D. Horky K. Jachymova M. Mikova B. Kazdova L. Aitman T.J. Churchill P.C. Webb R.C. Hingarh N.H. Yang Y. Wang J.M. Lezin E.M. Kurtz T.W. J. Clin. Invest. 1999; 103: 1651-1657Crossref PubMed Scopus (103) Google Scholar, 15Gotoda T. Lizuka Y. Kato N. Osuga J. Bihoreau M.T. Murakami T. Yamori Y. Shimano H. Ishibashi S. Yamada N. Nat. Genet. 1999; 22: 226-228Crossref PubMed Scopus (56) Google Scholar). There is no information on whether SHR tissues have loss of CD36 function. The CD36 protein deficiency in SHR was based on reaction of adipose tissue membranes with a polyclonal antibody against rat CD36, which may have failed to recognize a mutated protein that is partially or completely functional. In addition, it is not known if CD36 has a dominant role in FA transport in rat tissues and if its loss would have similar consequences to those observed in the mouse. We have compared uptake of FA and glucose by tissues of SHR, WKY, and the congenic SHRchr4 line (13Pravenec M. Zidek V. Simakova M. Kren V. Krenova D. Horky K. Jachymova M. Mikova B. Kazdova L. Aitman T.J. Churchill P.C. Webb R.C. Hingarh N.H. Yang Y. Wang J.M. Lezin E.M. Kurtz T.W. J. Clin. Invest. 1999; 103: 1651-1657Crossref PubMed Scopus (103) Google Scholar) and tested whether diet supplementation with short-chain FA, which do not require CD36 for uptake, can improve the hyperinsulinemia and other abnormalities of the SHR. Breeding pairs for SHR (NCrlBR) and WKY (NCrlBR) controls were purchased from the Charles River Company. The congenic SHR line (SHR.BN-Il6/Npy) harboring a segment of chromosome 4 containing CD36 (referred to here as SHRchr4) was generated by replacing the deletion variant of CD36 in the SHR with a wild-type variant from the normotensive Brown Norway rat as previously described in detail (13Pravenec M. Zidek V. Simakova M. Kren V. Krenova D. Horky K. Jachymova M. Mikova B. Kazdova L. Aitman T.J. Churchill P.C. Webb R.C. Hingarh N.H. Yang Y. Wang J.M. Lezin E.M. Kurtz T.W. J. Clin. Invest. 1999; 103: 1651-1657Crossref PubMed Scopus (103) Google Scholar). Rats were housed in a facility equipped with a 12-h light cycle, were given unlimited access to water, and were fed a standard chow diet ad libitum. The basal diet used was the standard chow Purina diet (number 5001) rich in carbohydrate (50% of total weight) and containing a low proportion of fat (4.5%) mostly as polyunsaturated fatty acids. The supplemented diet was prepared by adding 21.5 g of short- and medium-chain triacylglycerols (Captex 300, Arbitec Co.) to 100 g of basal diet followed by thorough mixed with a mechanical food blender. The composition of Captex 300 was 6% as caproic (C6), 65.6% as caprylic (C8), and 29.2% as capric (C10). The final diet contained 21% fat (38% of total energy) of which 79% was short- and medium-chain fatty triacylglycerols. Rats were started on the diet at 1 week after weaning. Systolic arterial blood pressure was measured in awake rats by the indirect tail cuff method using a Physiograph (Narco Biosystem, Houston, TX) equipped with transducers and preamplifiers. Rats were fasted overnight (16 h) and tail vein blood was collected into heparinized or EDTA (for FA determination) containing tubes. Plasma free fatty acids were measured using the Wako kit (Wako Chemicals, Richmond, VA). Triglycerides, cholesterol, and glucose were measured using enzymatic kits from Sigma Diagnostics. Plasma insulin was tested using a radioimmunoassay kit for rat insulin from Linco Research Inc. (St. Louis, MO). Rats, fasted overnight were given a 25% glucose solution (1.5 g/kg) orally using an intubation needle. Blood was collected for glucose determination from the tail prior to and at 10, 20, 30, 45, 90, and 120 min after the load. Glucose was measured using a Precision Q.I.D. monitoring system. BMIPP was radioiodinated by the thallation-iodide exchange method and purified over a Sep-Pak RP-18 Light cartridge (Waters Corp.), as previously described (16Knapp Jr., F.F. Goodman M.M. Callahan A.P. Kirsch G. J. Nucl. Med. 1986; 27: 521-531PubMed Google Scholar). Specific activity of [125I]BMIPP was in the range of 2–4 Ci/mmol and radiochemical purity, as determined by thin layer chromatography (TLC), was greater than 99%. BMIPP was dissolved in warm absolute ethanol (about 100 μl) and added dropwise to a stirred solution of 6% FA-free bovine serum albumin at 40 °C. The solution was sterile filtered (0.22 μm, Millipore) before injection. Each rat was injected in a lateral tail vein with 200 μl of the radioisotope solution (14–25 μCi). The animals were sacrificed after 2 h and the tissues were rapidly removed, rinsed with saline, and blotted dry. Tissues were weighed and counted in a NaI Auto-Gamma counter. A sample of the injected solution was counted to determine the total injected dose. 2-[18F]FDG in saline was injected via a lateral tail vein (about 15 μCi per rat) either alone or together with BMIPP. Tissues were harvested 2 h later as for BMIPP and counted for18F activity. When both BMIPP and 2-FDG were used in the same animal, the tissues were first counted for 2-FDG and then 24 h later for [125I]BMIPP, once the 18F radioactivity (half-life of 110 min) had decayed. Lipids were extracted from frozen tissue according to Folch et al. (17Folch J. Lees M. Sloane Stanley G. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Aliquots were chromatographed next to known standards on aluminum-backed silica plates (Analtech, Inc.). Petroleum ether:diethyl ether:glacial acetic acid (70:30:1, v/v/v) was used to resolve polar lipids, diglycerides, fatty acids, and triglycerides to determine the BMIPP distribution in each lipid class as a percentage of total counts on the plate. CD36 expression was measured by flow cytometry and Western blotting. Flow cytometry was performed on platelets prepared by centrifuging platelet-rich plasma at 1800 × g for 10 min. A 10-μl aliquot of washed platelets (about 3 × 107 cells) suspended in 0.02m phosphate-buffered saline with 9 mm EDTA and 0.1% bovine serum albumin (PEB) was incubated with a 1:100 dilution of anti-CD36 antibody and then with 1:150 fluorescein isothiocyanate-labeled secondary antibody. The cells were washed, resuspended in PEB, and assayed by flow cytometry (FACScan, Becton Dickinson). CD42 polyclonal antibody was used as a platelet marker. Data were analyzed as the percentage of gated cells expressing CD36. For Western blots, adipose tissue was homogenized in 1 ml of ice-cold TES buffer (20 mm Tris, 1 mm EDTA, 250 mm sucrose). The homogenate was centrifuged (16,000 ×g) to yield a pellet (P1), which was layered on 38% sucrose and centrifuged at 86,000 × g to pellet out mitochondria and nuclei (P2). The band at the interphase was harvested, diluted (1:7) in TES buffer, and centrifuged at 350,000 ×g to yield a total microsomal pellet (P3). Samples from both P1 and P3 (20–50 μg of protein) were subjected to electrophoresis according to Laemmli (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) followed by transfer to a nylon-supported nitrocellulose membrane. Briefly, the membrane was incubated with anti-CD36 antibody (2 h, 1:1000 dilution), washed, then incubated (1 h, 1:10,000) with secondary antibody labeled with horseradish peroxidase. Detection of immune complex was according to the directions of the ECL kit (Amersham Pharmacia Biotech). Adipocytes were isolated from the epididymal fat of male rats after tissue digestion with collagenase type I (1 mg/ml, Worthington Biochem). Suspensions were kept in a lipocrit of 30% and assayed as described earlier (19Harmon C.M. Luce P. Beth A.H. Abumrad N.A. J. Membr. Biol. 1991; 121: 261-268Crossref PubMed Scopus (165) Google Scholar) and in the legend of Fig. 4. Cardiomyocytes were isolated from perfused hearts as described by Luiken et al. (20Luiken J.J. van Nieuwenhoven F.A. America G. van der Vusse G.J. Glatz J.F. J. Lipid Res. 1997; 38: 745-758Abstract Full Text PDF PubMed Google Scholar). Suspensions were kept at about 25% cell density and were assayed within 2 h after isolation using the same assay conditions as for adipocytes. To evaluate whether the SHR phenotype involves loss of CD36 function in FA uptake, we compared biodistribution of the slowly oxidized FA analog [125I]BMIPP in WKY controls and SHR. As documented previously in studies on both humans and laboratory animals (10Coburn C.T. Knapp Jr., F.F. Febbraio M. Beets A.L. Silverstein R.L. Abumrad N.A. J. Biol. Chem. 2000; 275: 32523-32529Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 21Knapp Jr., F.F. Kropp J. Franken P.R. Visser F.C. Sloof G.W. Eisenhut M. Yamamichi Y. Shirakami Y. Kusuoka H. Nishimura T. Q. J. Nucl. Med. 1996; 40: 252-269PubMed Google Scholar), BMIPP, a 3-methyl branched (15C) FA analogue, is rapidly extracted from blood-like native FA (21Knapp Jr., F.F. Kropp J. Franken P.R. Visser F.C. Sloof G.W. Eisenhut M. Yamamichi Y. Shirakami Y. Kusuoka H. Nishimura T. Q. J. Nucl. Med. 1996; 40: 252-269PubMed Google Scholar, 22Torizuka K. Yonekura Y. Nishimura T. Tamaki N. Uehara T. Ikekubo K. Hino M. Kaku Igaku. 1991; 28: 681-690PubMed Google Scholar). It equilibrates within 2–3 min and is incorporated into cellular lipids at rates almost identical to those of native FA (22Torizuka K. Yonekura Y. Nishimura T. Tamaki N. Uehara T. Ikekubo K. Hino M. Kaku Igaku. 1991; 28: 681-690PubMed Google Scholar, 23Ambrose K.R. Owen B.A. Goodman M.M. Knapp Jr., F.F. Eur J Nucl Med. 1987; 12: 486-491Crossref PubMed Scopus (71) Google Scholar, 24Kropp J. Ambrose K.R. Knapp Jr., F.F. Nissen H.P. Biersack H.J. Int. J. Radiat. Appl. Instrum. Part B. 1992; 19: 8-283Google Scholar). The stable iodination of BMIPP coupled with its prolonged tissue retention allow sensitive comparisons of tissue capacities for FA uptake in vivo. The CD36 null mouse exhibits a 60–80% defect in BMIPP uptake by oxidative skeletal muscle, heart, and adipose tissue and the magnitude of the defect is similar when data from BMIPP are compared with those with the rapidly oxidized FA analog, 15-(p-iodophenyl)pentadecanoic acid, or with native palmitate (10Coburn C.T. Knapp Jr., F.F. Febbraio M. Beets A.L. Silverstein R.L. Abumrad N.A. J. Biol. Chem. 2000; 275: 32523-32529Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). The biodistribution data for BMIPP in SHR and WKY showed that of the several tissues examined, which included blood, liver, intestine, lung, hip muscle, heart, and adipose tissue, impaired BMIPP uptake in SHR was observed only in heart (25%) and fat tissue (60%) (Fig.1). In CD36 null mice, tissues where FA uptake was deficient exhibited a defect in converting labeled diglycerides (DG) to triglycerides (TG) with a severalfold increase in the ratio of DG to TG (10Coburn C.T. Knapp Jr., F.F. Febbraio M. Beets A.L. Silverstein R.L. Abumrad N.A. J. Biol. Chem. 2000; 275: 32523-32529Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). As shown in Fig. 2, such a block in DG to TG conversion was also observed in SHR heart. TableI, which documents BMIPP lipid distribution for different tissues, shows that the defect in intracellular processing of BMIPP is observed in oxidative (diaphragm) but not in glycolytic (hip) muscles. It is also measured in the kidney and in adipose tissue but not in the liver. Another point documented by Table I is that the defect in intracellular incorporation of BMIPP is observed early during growth since the rats used in these experiments were aged 9–10 weeks, as opposed to 14–16 weeks in the experiment shown in Fig. 2.Table IIncorporation of BMIPP into cellular lipidsDiglyceridesTriglyceridesPolar lipids% of total radioactivityWKY (n = 6)Liver48 ± 126.7 ± 0.623 ± 1Kidney49 ± 123 ± 225 ± 1Adipose41 ± 345 ± 311 ± 5Hip muscle61 ± 411 ± 122 ± 2Diaphragm51 ± 331 ± 217 ± 4Heart42 ± 245 ± 312.3 ± 0.9SHR (n = 6)Liver45 ± 228 ± 125 ± 1Kidney56.6 ± 0.61-ap < 0.005.18 ± 11-bp < 0.05.23.1 ± 0.7Adipose53 ± 31-bp < 0.05.36 ± 31-bp < 0.05.8 ± 3Hip muscle67 ± 213 ± 117 ± 2Diaphragm60.8 ± 0.71-bp < 0.05.20.5 ± 0.71-ap < 0.005.14.1 ± 0.2Heart48.7 ± 0.91-bp < 0.05.29 ± 11-ap < 0.005.16.7 ± 0.31-bp < 0.05.Tissue samples from an experiment similar to that shown in Fig. 3 were Folch-extracted and the lipids evaluated by TLC. Values reflect the percent of total counts recovered in each lipid fraction. Polar lipids include phospholipids and monoacylglycerol-3-phosphates. Means, shown ± S.E., are from one experiment typical of two others.1-a p < 0.005.1-b p < 0.05. Open table in a new tab Tissue samples from an experiment similar to that shown in Fig. 3 were Folch-extracted and the lipids evaluated by TLC. Values reflect the percent of total counts recovered in each lipid fraction. Polar lipids include phospholipids and monoacylglycerol-3-phosphates. Means, shown ± S.E., are from one experiment typical of two others. A significant finding in the oxidative tissues of the SHR is shown in Table II. Despite the defect in BMIPP uptake present in heart and diaphragm, there was a severalfold increase in accumulation of free BMIPP, a finding that was not observed in the CD36 null mouse.Table IIIncorporation of BMIPP into cellular free FA in myocardium from WKY and SHR as compared to that in myocardium from wild type and CD36 null miceWKYSHRWild typeCD36 −/−% of total BMIPP in lipids countsHeart1.1 ± 0.55.1 ± 0.72-ap < 0.005.3.1 ± 0.51.9 ± 0.3Diaphragm1.5 ± 0.34.6 ± 0.32-ap < 0.005.Rat data are from the experiments shown in Table II while the mouse data are from (10Coburn C.T. Knapp Jr., F.F. Febbraio M. Beets A.L. Silverstein R.L. Abumrad N.A. J. Biol. Chem. 2000; 275: 32523-32529Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Means are shown ± S.E. Rat data are from one experiment (n = 6) typical of two others. Mouse data are from one experiment (n = 3–5) typical of two more. Values for mouse diaphragm were not determined.2-a p < 0.005. Open table in a new tab Rat data are from the experiments shown in Table II while the mouse data are from (10Coburn C.T. Knapp Jr., F.F. Febbraio M. Beets A.L. Silverstein R.L. Abumrad N.A. J. Biol. Chem. 2000; 275: 32523-32529Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Means are shown ± S.E. Rat data are from one experiment (n = 6) typical of two others. Mouse data are from one experiment (n = 3–5) typical of two more. Values for mouse diaphragm were not determined. The defective FA utilization present in some SHR tissues was paralleled by a compensatory increase in glucose uptake, as shown in Fig.3. A large increase in uptake of the glucose tracer 2-[18F]FDG was observed in heart and diaphragm. Smaller but still significant increases were measured in intercostal and hip muscles and in adipose tissue. These data are in line with our findings with the CD36 null mouse. 3T. Hajri and N. Abumrad, unpublished observations. Since the ability to detect metabolic defects in the SHR may depend on the type of wild-type strain used for comparison, we examined BMIPP and 2-FDG uptake in tissues from SHR and a congenic line (SHRchr4) that incorporates a piece of chromosome 4 containing wild-type CD36. We also compared FA uptake by adipocytes isolated from the two lines.Panel A of Fig. 4 shows CD36 expression by adipose tissue from congenic SHRchr4, WKY, and SHR using a polyclonal anti-rat CD36 antibody. No detectable CD36 protein was observed in SHR adipose tissue (panel A) or platelets (not shown), while expression on both adipose tissue (panel A) and platelets (not shown) could be observed in the SHRchr4 line. Panel B shows that CD36 expression reversed the defects in uptake of FA and glucose in the SHR. Uptake of 2-FDG by SHRchr4 heart and adipose tissue was decreased by 400 and 46%, respectively, which supported an improvement in FA utilization. In line with this, initial rates of FA uptake by cardiomyocytes (panel C) and adipocytes (panel D) obtained from SHRchr4 were increased 2–3-fold above levels measured in cells from the parent SHR line, documenting reversal of the defect in FA transport with CD36 expression. We next tested whether the defect in FA uptake constitutes a primary cause of the hyperinsulinemia and insulin resistance observed in the SHR. We examined whether supplying SHR tissues with the FA they can utilize would reverse the defects caused by lack of CD36. The saturable component of FA uptake, presumably facilitated by CD36, does not recognize short-chain FA (25Abumrad N.A. Park J.H. Park C.R. J. Biol. Chem. 1984; 259: 8945-8953Abstract Full Text PDF PubMed Google Scholar). Also in line with this is the finding that pure CD36 does not bind short-chain FA. SHR and WKY rats were started on the diet supplemented with short- and medium-chain FA at about 5 weeks of age and maintained on it for a period of 3 months. As shown in TableIII, there were no significant differences in body weights between WKY and SHR at the end of the 3-month diet treatment. However, a significant decrease in SHR heart weight and in the heart to body weight ratio was observed on the FA-supplemented diet. The effect was small and not significant in WKY. The decrease in heart weight occurred without a change in blood pressure, which remained significantly higher in the SHR and was largely unaffected by the diet.Table IIIBody and heart weights and mean systolic blood pressure of rats fed chow without (basal) or with short-chain FA supplementationBasal+ Short-chain FASHRWKYSHRWKYBW (g)265 ± 34293 ± 34277 ± 30280 ± 34Heart W (g)2.04 ± 0.26a1.68 ± 0.221.65 ± 0.11a1.5 ± 0.02H/B (%)0.72 ± 0.02b0.57 ± 0.020.62 ± 0.03b0.55 ± 0.02SBP198 ± 7.5c128 ± 6c190 ± 4.1d116 ± 14.3dRats were maintained on the diet beginning at week 5 and for a period of 3 months. Values of the same row sharing the same superscript are significantly different with p < 0.01.n = 5 for the basal diet and 6 for the +FA diet. Open table in a new tab Rats were maintained on the diet beginning at week 5 and for a period of 3 months. Values of the same row sharing the same superscript are significantly different with p < 0.01.n = 5 for the basal diet and 6 for the +FA diet. Table IV shows that blood glucose levels were slightly increased by the FA diet in both rat groups but were not significantly different between the groups on either diet. In contrast, insulin levels, which were about 2.4 times higher in SHR were normalized by the FA-supplemented diet while no effect was observed on blood insulin in the WKY. The FA diet normalized the insulin/glucose ratio in SHR, which dropped from 1.4 to 0.4. Short-chain FA supplementation significantly decreased blood-free FA and slightly increased blood TG in both groups.Table IVPlasma parameters of rats fed chow without (basal) or with short-chain FA supplementationBasal+ Short-chain FASHRWKYSHRWKYBW (g)265 ± 34293 ± 34277 ± 30280 ± 34Glucose (mg/dl)63 ± 2a70 ± 4b98.3 ± 7a95 ± 7bInsulin (ng/dl)87.5 ± 10.9a,b36.9 ± 6.6a42.0 ± 9.2b32.3 ± 7.4Ins/Gluc1.39 ± 0.1a,b0.53 ± 0.09a0.43 ± 0.09b0.34 ± 0.09FFA (nmol/dl)190 ± 16a165 ± 11b115 ± 11a124 ± 8bTG (mg/dl)49 ± 736 ± 366 ± 2b54 ± 3bCholesterol (mg/dl)75 ± 6a123 ± 3a70 ± 9b118 ± 10bRats were kept on the diet for a period of 3 months. Blood samples were obtained from the tail ve
Referência(s)