Highly insulin-responsive isolated rat heart muscle cells yielded by a modified isolation method

Fischer, Yvan; Rose, Horst; Kammermeier, H.

doi:10.1016/0024-3205(91)90310-8

Cited by 65 publications

(50 citation statements)

References 27 publications

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“…Cardiac myocytes from lean and obese Zucker rats were isolated using a Langendorff perfusion system and a modified Krebs-Henseleit bicarbonate medium (12,26). After being isolated, myocytes were washed and treated with 0.5% DMSO or 0.4 mmol/l SSO in DMSO for 30 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Enhanced Sarcolemmal FAT/CD36 Content and Triacylglycerol Storage in Cardiac Myocytes From Obese Zucker Rats

et al. 2004

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In obesity, the development of cardiomyopathy is associated with the accumulation of myocardial triacylglycerols (TAGs), possibly stemming from elevation of myocardial long-chain fatty acid (LCFA) uptake. Because LCFA uptake is regulated by insulin and contractions, we examined in cardiac myocytes from lean and obese Zucker rats the effects of insulin and the contraction-mimetic agent oligomycin on the initial rate of LCFA uptake, subcellular distribution of FAT/CD36, and LCFA metabolism. In cardiac myocytes from obese Zucker rats, under basal conditions, FAT/CD36 was relocated to the sarcolemma at the expense of intracellular stores. In addition, the LCFA uptake rate, LCFA esterification rate into TAGs, and the intracellular unesterified LCFA concentration each were significantly increased. All these metabolic processes were normalized by the FAT/CD36 inhibitor sulfo-N-succinimidyloleate, indicating its antidiabetic potential. In cardiac myocytes isolated from lean rats, in vitro administration of insulin induced the translocation of FAT/ CD36 to the sarcolemma and stimulated initial rates of LCFA uptake and TAG esterification. In contrast, in myocytes from obese rats, insulin failed to alter the subcellular localization of FAT/CD36 and the rates of LCFA uptake and TAG esterification. In cardiac myocytes from lean and obese animals, oligomycin stimulated the initial rates of LCFA uptake and oxidation, although oligomycin only induced the translocation of FAT/CD36 to the sarcolemma in lean rats. The present results indicate that in cardiac myocytes from obese Zucker rats, a permanent relocation of FAT/CD36 to the sarcolemma is responsible for myocardial TAG accumulation. Furthermore, in vitro these cardiac myocytes, although sensitive to contraction-like stimulation, were completely insensitive to insulin, as the basal conditions in hyperinsulinemic, obese animals resemble the insulin-stimulated condition in lean littermates.

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Section: Methodsmentioning

confidence: 99%

Enhanced Sarcolemmal FAT/CD36 Content and Triacylglycerol Storage in Cardiac Myocytes From Obese Zucker Rats

et al. 2004

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“…Cardiac myocytes were isolated from male adult rats according to the procedure described by Fisher et al (11), as modified by Luiken et al (19). Briefly, rats were anesthetized using Somnotol (50 -60 mg/100 g ip) combined with heparin (300 IU/100 g ip).…”

Section: Methodsmentioning

confidence: 99%

Insulin stimulates fatty acid transport by regulating expression of FAT/CD36 but not FABPpm

Chabowski

Coort²,

Callés-Escandon³

et al. 2004

American Journal of Physiology-Endocrinology and Metabolism

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Because insulin has been shown to stimulate long-chain fatty acid (LCFA) esterification in skeletal muscle and cardiac myocytes, we investigated whether insulin increased the rate of LCFA transport by altering the expression and the subcellular distribution of the fatty acid transporters FAT/CD36 and FABPpm. In cardiac myocytes, insulin very rapidly increased the expression of FAT/CD36 protein in a time-and dose-dependent manner. During a 2-h period, insulin (10 nM) increased cardiac myocyte FAT/CD36 protein by 25% after 60 min and attained a maximum after 90 -120 min (ϩ40 -50%). There was a dose-dependent relationship between insulin (10 Ϫ12 to 10 Ϫ7 M) and FAT/CD36 expression. The half-maximal increase in FAT/CD36 protein occurred at 0.5 ϫ 10 Ϫ9 M insulin, and the maximal increase occurred at 10 Ϫ9 to 10 Ϫ8 M insulin (ϩ40 -50%). There were similar insulin-induced increments in FAT/CD36 protein in cardiac myocytes (ϩ43%) and in Langendorff-perfused hearts (ϩ32%). In contrast to FAT/CD36, insulin did not alter the expression of FABPpm protein in either cardiac myocytes or the perfused heart. By use of specific inhibitors of insulin-signaling pathways, it was shown that insulin-induced expression of FAT/CD36 occurred via the PI 3-kinase/Akt insulin-signaling pathway. Subcellular fractionation of cardiac myocytes revealed that insulin not only increased the expression of FAT/CD36, but this hormone also targeted some of the FAT/CD36 to the plasma membrane while concomitantly lowering the intracellular depot of FAT/ CD36. At the functional level, the insulin-induced increase in FAT/ CD36 protein resulted in an increased rate of palmitate transport into giant vesicles (ϩ34%), which paralleled the increase in plasmalemmal FAT/CD36 (ϩ29%). The present studies have shown that insulin regulates protein expression of FAT/CD36, but not FABPpm, via the PI 3-kinase/Akt insulin-signaling pathway. fatty acid translocase; plasma membrane-associated fatty acid-binding protein; cardiac myocytes; transport; plasma membrane; low-density microsomes; perfusion; heart LONG-CHAIN FATTY ACIDS (LCFAs) are taken up into cells by passive diffusion and by protein-mediated mechanisms involving a number of fatty acid-binding proteins (12,25). Overexpression of fatty acid transporters, including fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm), increase LCFA uptake (13,14). In recent years, it has been shown that rates of LCFA transport can be regulated acutely and chronically. In heart and skeletal muscle exposed briefly to insulin (15 min) and in contracting muscle (Ͻ30 min), rates of LCFA transport are increased due to the translocation of FAT/CD36 from an intracellular depot to the plasma membrane (4, 18, 19). Expression of FAT/CD36 mRNA is upregulated by LCFAs in neonatal cardiac myocytes (30), although the functional metabolic consequences of these changes on LCFA transport and metabolism have not been determined. In more detailed studies, FAT/CD36 mRNA and protein expression and plasma...

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“…Isolation of Cardiomyocytes and Glucose Transport Assays-Cardiomyocytes from adult female Sprague-Dawley rats (180 -220 g, fed ad libitum) were obtained as described previously (31). Treatment of cardiomyocytes for all experiments was performed in medium A containing 6 mM KCl, 1 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 , 1.4 mM MgSO 4 , 128 mM NaCl, 10 mM HEPES, 1 mM CaCl 2 , and 2% bovine serum albumin (fatty acid free, pH 7.4) at 37°C, equilibrated with oxygen.…”

Section: Methodsmentioning

confidence: 99%

“…Treatment of cardiomyocytes for all experiments was performed in medium A containing 6 mM KCl, 1 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 , 1.4 mM MgSO 4 , 128 mM NaCl, 10 mM HEPES, 1 mM CaCl 2 , and 2% bovine serum albumin (fatty acid free, pH 7.4) at 37°C, equilibrated with oxygen. The rate of 2-deoxy-D-glucose uptake was determined as described elsewhere (31).…”

Section: Methodsmentioning

confidence: 99%

Insulin-induced Recruitment of Glucose Transporter 4 (GLUT4) and GLUT1 in Isolated Rat Cardiac Myocytes

Fischer

Thomas

Sevilla

et al. 1997

Journal of Biological Chemistry

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155

111

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Using isolated rat cardiomyocytes we have examined: 1) the effect of insulin on the cellular distribution of glucose transporter 4 (GLUT4) and GLUT1, 2) the total amount of these transporters, and 3) the co-localization of GLUT4, GLUT1, and secretory carrier membrane proteins (SCAMPs) in intracellular membranes. Insulin induced 5.7-and 2.7-fold increases in GLUT4 and GLUT1 at the cell surface, respectively, as determined by the nonpermeant photoaffinity label [ 3 H]2-N-[4(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)propyl-2-amine. The total amount of GLUT1, as determined by quantitative Western blot analysis of cell homogenates, was found to represent a substantial fraction (ϳ30%) of the total glucose transporter content. Intracellular GLUT4-containing vesicles were immunoisolated from low density microsomes by using monoclonal anti-GLUT4 (1F8) or anti-SCAMP antibodies (3F8) coupled to either agarose or acrylamide. With these different immunoisolation conditions two GLUT4 membrane pools were found in nonstimulated cells: one pool with a high proportion of GLUT4 and a low content in GLUT1 and SCAMP 39 (pool 1) and a second GLUT4 pool with a high content of GLUT1 and SCAMP 39 (pool 2). The existence of pool 1 was confirmed by immunotitration of intracellular GLUT4 membranes with 1F8-acrylamide. Acute insulin treatment caused the depletion of GLUT4 in both pools and of GLUT1 and SCAMP 39 in pool 2. In conclusion: 1) GLUT4 is the major glucose transporter to be recruited to the surface of cardiomyocytes in response to insulin; 2) these cells express a high level of GLUT1; and 3) intracellular GLUT4-containing vesicles consist of at least two populations, which is compatible with recently proposed models of GLUT4 trafficking in adipocytes.

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Highly insulin-responsive isolated rat heart muscle cells yielded by a modified isolation method

Cited by 65 publications

References 27 publications

Enhanced Sarcolemmal FAT/CD36 Content and Triacylglycerol Storage in Cardiac Myocytes From Obese Zucker Rats

Enhanced Sarcolemmal FAT/CD36 Content and Triacylglycerol Storage in Cardiac Myocytes From Obese Zucker Rats

Insulin stimulates fatty acid transport by regulating expression of FAT/CD36 but not FABPpm

Insulin-induced Recruitment of Glucose Transporter 4 (GLUT4) and GLUT1 in Isolated Rat Cardiac Myocytes

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