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Background: The mechanisms underlying the impact of pathophysiological elevations in insulin or glucose on hepatic cellular homocysteine kinetics is not fully understood., Objective: The objective of the study was to investigate the impact of elevated insulin/glucose on hepatic homocysteine kinetics at the cellular level., Design and Methods: Effects of insulin and glucose on homocysteine remethylation and transsulfuration metabolic fluxes were investigated in a cell model using stable isotopic tracers and gas chromatography/mass spectrometry. The methylation status was assessed by S-adenosylmethionine (adoMet), the adoMet to S-adenosylhomocysteine ratio, DNA methyltransferase activity, and methylated cytidine content of DNA. The expression profile of homocysteine remethylation, transmethylation, and transsulfuration-associated genes was determined., Results: Insulin increased cellular homocysteine production primarily by its inhibition of transsulfuration. When cells were exposed to elevated insulin and glucose, homocysteine remethylation was enhanced, which consequently increased intracellular adoMet concentrations by inducing adoMet synthase activity. Elevated glucose further enhanced DNA methyltransferase activity that subsequently led to increased global DNA methylation., Conclusions: We demonstrated the novel finding of a direct promoting effect of high cellular insulin or glucose exposure on homocysteine remethylation, adoMet synthase activity, and adoMet synthesis. We also provided new evidence indicating that when hepatic tissue is exposed to elevated insulin or glucose, the cellular methylation balance can be altered, which may have potential epigenetic impacts gene regulation in diabetic individuals. These findings in a cell line may or may not reflect what happens in humans. In vivo studies on the homocysteine transmethylation fluxes and DNA methylation in diabetic state are underway.

The effect of changing endocrine status on the uterine endometrium of the female Fischer rat was observed in vivo after neonatal androgenization, after aging, after estrogen (E) implant in ovariectomized (OVX) rats, and in vitro after progesterone (P) addition to monolayer cultures of uterine tumor cells. Neonatal androgenization produced cystic ovarian follicles and persistent vaginal cornification for 10-14 mo, indicative of an anovulatory, persistent E status. The constant estrous (CE) state, induced by androgenization, uniformly produced focal glandular hyperplasia of the endometrium at 9 mo of age. Focal glandular hyperplasia, which was absent in the cycling 6-mo-old estrous endometrium, was also apparent in the 12-mo-old rats which had ceased cycling and entered natural CE. Subcutaneous estradiol-17 beta (E2) implants in 12-mo-old OVX rats induced early cystic and adenomatous changes in the foci of hyperplasia. Acyclic control rats, 21 mo old, in persistent diestrus (PD) exhibited a 3-fold elevation of serum P associated with glandular atrophy of the endometrium. Ten nM P also inhibited proliferation of endometrial cells in cultures prepared from tumors of 21-mo-old rats. On the other hand, adenomatous hyperplasia was observed in 29-mo-old PD rats, despite the presence of low serum E and elevated serum P. These results indicate that either induced or natural constant E status leads to focal glandular hyperplasia in the Fischer rat. E2 implants in the 12-mo-old OVX Fischer rat induced early cystic and adenomatous changes in the focal hyperplasia. Inhibition of these focal hyperplasias was associated with elevated serum P at 21 mo. The development of adenomatous hyperplasia in the aged endometrium, on the other hand, occurred despite elevated serum P at 29 mo.

The studies in this report were designed to investigate whether the loss of pituitary luteinizing hormone (LH) responses to N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) in aged noncycling rats was the result of age, endocrine status associated with diestrus, or noncyclic low estrogen status. Pituitary monolayer cultures were prepared from female Fischer 344 rats. Aged (18-month-old) persistent diestrous (PD) rats, young diestrous (D) rats, or noncycling neonatally androgenized-constant estrous (AN-CE) rats were used. Enzymatically dispersed cells were maintained in the same batch of medium supplemented with dextran-coated charcoal adsorbed serums. Total LH contents were 1.75 +/- 0.04, 1.15 +/- 0.03, and 1.71 +/- 0.02 micrograms LH/dish in Day 5 cultures prepared from aged PD, young D, and AN-CE rats, respectively. Incubations with 5 mM DBcAMP for 4 h significantly (P less than 0.05) stimulated LH release in cultures prepared from young D and AN-CE rats but inhibited LH release in cultures prepared from aged PD rats even though a 4-h incubation with 10 nM LH releasing hormone (LHRH) stimulated LH release similarly in cultures of all three types of cells. The loss of DBcAMP-induced LH release in cultures prepared from aged PD rats was reversed by 17 beta-estradiol (E2). This treatment also reduced the basal LH release and increased the cellular LH content. These results indicate that the loss of DBcAMP-induced LH response in the aged rat is not an irreversible aging phenomenon but appears to be associated with the chronically low E2 status of aged PD rats but not young cycling D or noncycling AN-CE rats.