Insulin-dependent suppression in glutamyl hydrolase activity and elevated cellular methotrexate polyglutamates

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Folates and antifolates are converted to polyglutamates for enhanced cellular retention and protein binding.
• The precise regulation of polyglutamate formation and breakdown remains incompletely understood.
• Insulin was previously observed to increase methotrexate (MTX) polyglutamate accumulation in rat hepatoma cells.

Purpose of the Study:

• To investigate the mechanism by which insulin influences the accumulation of methotrexate (MTX) polyglutamates.
• To determine the role of gamma-glutamyl hydrolase (GH) activity in insulin-mediated polyglutamate synthesis.
• To examine insulin's effects on both folate and antifolate polyglutamate metabolism.

Main Methods:

• Measuring gamma-glutamyl hydrolase (GH) activity in response to insulin treatment.
• Quantifying the synthesis and accumulation of MTX and folate polyglutamates in cultured cells.
• Comparing insulin's effects in parental H35 cells versus a GH-overexpressing H35D cell line.

Main Results:

• Insulin significantly decreased gamma-glutamyl hydrolase (GH) activity, inversely correlating with increased MTX polyglutamate synthesis.
• Insulin's effects on GH activity were dose-dependent, time-dependent, and influenced by cAMP signaling.
• Insulin treatment also led to increased folate polyglutamate levels and was effective in H35D cells with elevated GH.
• H35D cells showed higher GH activity and reduced MTX polyglutamylation compared to insulin-treated H35 cells.

Conclusions:

• Insulin-induced reduction in gamma-glutamyl hydrolase (GH) activity is the primary mechanism driving increased cellular polyglutamate accumulation.
• This regulatory pathway impacts the cellular retention of both folate and antifolate compounds.
• Understanding insulin's modulation of GH offers insights into controlling intracellular drug levels and efficacy.