Effects of 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8) HCL and verapamil on the metabolism of free fatty acid by hepatocytes

Joseph O. Olubadewo, George Cook, Murray Heimberg

Research output: Contribution to journalArticle

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Abstract

The influence of calcium antagonists on hepatic lipid metabolism was investigated in freshly dispersed rat hepatocytes incubated with [1-14C]oleate and verapamil or 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8). Synthesis of triglyceride was calculated from the specific radioactivity of [1-14C]oleate in extracted total lipid, after separation of each lipid class by thin-layer chromatography. Ketogenesis was measured enzymatically or as the amount of radioactivity incorporated into neutralized acid-soluble extracts. Neither verapamil nor TMB-8 affected triglyceride synthesis. In contrast, TMB-8 and verapamil exerted a concentration-dependent inhibition of ketogenesis, with TMB-8 being more potent than verapamil (inhibition by 50 μM TMB-8 was 73 ± 9% versus 38 ± 2% inhibition by 50 μM verapamil). Increasing the concentrations of calcium (0 to 4.2 mM) or oleate (0 to 2.0 mM) increased the rate of ketogenesis but did not alter the antiketogenic potency of TMB-8 or verapamil, indicating that inhibition of ketogenesis by these drugs was not calcium dependent. Since the calcium antagonists did not affect ketogenesis from octanoic acid, and since carnitine stimulated ketogenesis from [1-14C]oleate by 25% and reversed the antiketogenic effects of TMB-8 and verapamil, it appeared that the two calcium antagonists inhibited ketogenesis by interfering with the activity of the outer mitochondrial carnitine palmitoyltransferase. In assays of the outer carnitine palmitoyltransferase in isolated mitochondria, both TMB-8 and verapamil were inhibitory. TMB-8 was the more potent inhibitor of this enzyme, and carnitine was able to overcome inhibition by each of the inhibitors. These results suggest that verapamil and TMB-8 may inhibit ketogenesis by mechanisms independent of their well known effects on cellular calcium concentrations, and that inhibition may be competitive with respect to carnitine concentration. However, direct inhibition of carnitine palmitoyltransferase may not explain completely the inhibition of ketogenesis by these drugs, since concentrations required for enzyme inhibition were greater than those required for inhibition of ketogenesis in isolated hepatocytes.

Original languageEnglish (US)
Pages (from-to)1463-1471
Number of pages9
JournalBiochemical Pharmacology
Volume37
Issue number8
DOIs
StatePublished - Apr 15 1988

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Verapamil
Nonesterified Fatty Acids
Metabolism
Hepatocytes
Oleic Acid
Calcium
Carnitine O-Palmitoyltransferase
Carnitine
Enzyme inhibition
Radioactivity
Triglycerides
8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate
Lipids
Thin layer chromatography
Mitochondria
Enzyme Inhibitors
Thin Layer Chromatography
Lipid Metabolism
Pharmaceutical Preparations
Rats

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Pharmacology

Cite this

Effects of 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8) HCL and verapamil on the metabolism of free fatty acid by hepatocytes. / Olubadewo, Joseph O.; Cook, George; Heimberg, Murray.

In: Biochemical Pharmacology, Vol. 37, No. 8, 15.04.1988, p. 1463-1471.

Research output: Contribution to journalArticle

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abstract = "The influence of calcium antagonists on hepatic lipid metabolism was investigated in freshly dispersed rat hepatocytes incubated with [1-14C]oleate and verapamil or 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8). Synthesis of triglyceride was calculated from the specific radioactivity of [1-14C]oleate in extracted total lipid, after separation of each lipid class by thin-layer chromatography. Ketogenesis was measured enzymatically or as the amount of radioactivity incorporated into neutralized acid-soluble extracts. Neither verapamil nor TMB-8 affected triglyceride synthesis. In contrast, TMB-8 and verapamil exerted a concentration-dependent inhibition of ketogenesis, with TMB-8 being more potent than verapamil (inhibition by 50 μM TMB-8 was 73 ± 9{\%} versus 38 ± 2{\%} inhibition by 50 μM verapamil). Increasing the concentrations of calcium (0 to 4.2 mM) or oleate (0 to 2.0 mM) increased the rate of ketogenesis but did not alter the antiketogenic potency of TMB-8 or verapamil, indicating that inhibition of ketogenesis by these drugs was not calcium dependent. Since the calcium antagonists did not affect ketogenesis from octanoic acid, and since carnitine stimulated ketogenesis from [1-14C]oleate by 25{\%} and reversed the antiketogenic effects of TMB-8 and verapamil, it appeared that the two calcium antagonists inhibited ketogenesis by interfering with the activity of the outer mitochondrial carnitine palmitoyltransferase. In assays of the outer carnitine palmitoyltransferase in isolated mitochondria, both TMB-8 and verapamil were inhibitory. TMB-8 was the more potent inhibitor of this enzyme, and carnitine was able to overcome inhibition by each of the inhibitors. These results suggest that verapamil and TMB-8 may inhibit ketogenesis by mechanisms independent of their well known effects on cellular calcium concentrations, and that inhibition may be competitive with respect to carnitine concentration. However, direct inhibition of carnitine palmitoyltransferase may not explain completely the inhibition of ketogenesis by these drugs, since concentrations required for enzyme inhibition were greater than those required for inhibition of ketogenesis in isolated hepatocytes.",
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N2 - The influence of calcium antagonists on hepatic lipid metabolism was investigated in freshly dispersed rat hepatocytes incubated with [1-14C]oleate and verapamil or 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8). Synthesis of triglyceride was calculated from the specific radioactivity of [1-14C]oleate in extracted total lipid, after separation of each lipid class by thin-layer chromatography. Ketogenesis was measured enzymatically or as the amount of radioactivity incorporated into neutralized acid-soluble extracts. Neither verapamil nor TMB-8 affected triglyceride synthesis. In contrast, TMB-8 and verapamil exerted a concentration-dependent inhibition of ketogenesis, with TMB-8 being more potent than verapamil (inhibition by 50 μM TMB-8 was 73 ± 9% versus 38 ± 2% inhibition by 50 μM verapamil). Increasing the concentrations of calcium (0 to 4.2 mM) or oleate (0 to 2.0 mM) increased the rate of ketogenesis but did not alter the antiketogenic potency of TMB-8 or verapamil, indicating that inhibition of ketogenesis by these drugs was not calcium dependent. Since the calcium antagonists did not affect ketogenesis from octanoic acid, and since carnitine stimulated ketogenesis from [1-14C]oleate by 25% and reversed the antiketogenic effects of TMB-8 and verapamil, it appeared that the two calcium antagonists inhibited ketogenesis by interfering with the activity of the outer mitochondrial carnitine palmitoyltransferase. In assays of the outer carnitine palmitoyltransferase in isolated mitochondria, both TMB-8 and verapamil were inhibitory. TMB-8 was the more potent inhibitor of this enzyme, and carnitine was able to overcome inhibition by each of the inhibitors. These results suggest that verapamil and TMB-8 may inhibit ketogenesis by mechanisms independent of their well known effects on cellular calcium concentrations, and that inhibition may be competitive with respect to carnitine concentration. However, direct inhibition of carnitine palmitoyltransferase may not explain completely the inhibition of ketogenesis by these drugs, since concentrations required for enzyme inhibition were greater than those required for inhibition of ketogenesis in isolated hepatocytes.

AB - The influence of calcium antagonists on hepatic lipid metabolism was investigated in freshly dispersed rat hepatocytes incubated with [1-14C]oleate and verapamil or 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8). Synthesis of triglyceride was calculated from the specific radioactivity of [1-14C]oleate in extracted total lipid, after separation of each lipid class by thin-layer chromatography. Ketogenesis was measured enzymatically or as the amount of radioactivity incorporated into neutralized acid-soluble extracts. Neither verapamil nor TMB-8 affected triglyceride synthesis. In contrast, TMB-8 and verapamil exerted a concentration-dependent inhibition of ketogenesis, with TMB-8 being more potent than verapamil (inhibition by 50 μM TMB-8 was 73 ± 9% versus 38 ± 2% inhibition by 50 μM verapamil). Increasing the concentrations of calcium (0 to 4.2 mM) or oleate (0 to 2.0 mM) increased the rate of ketogenesis but did not alter the antiketogenic potency of TMB-8 or verapamil, indicating that inhibition of ketogenesis by these drugs was not calcium dependent. Since the calcium antagonists did not affect ketogenesis from octanoic acid, and since carnitine stimulated ketogenesis from [1-14C]oleate by 25% and reversed the antiketogenic effects of TMB-8 and verapamil, it appeared that the two calcium antagonists inhibited ketogenesis by interfering with the activity of the outer mitochondrial carnitine palmitoyltransferase. In assays of the outer carnitine palmitoyltransferase in isolated mitochondria, both TMB-8 and verapamil were inhibitory. TMB-8 was the more potent inhibitor of this enzyme, and carnitine was able to overcome inhibition by each of the inhibitors. These results suggest that verapamil and TMB-8 may inhibit ketogenesis by mechanisms independent of their well known effects on cellular calcium concentrations, and that inhibition may be competitive with respect to carnitine concentration. However, direct inhibition of carnitine palmitoyltransferase may not explain completely the inhibition of ketogenesis by these drugs, since concentrations required for enzyme inhibition were greater than those required for inhibition of ketogenesis in isolated hepatocytes.

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