24, 25-dihydroxycholecalciferol but not 25-hydroxycholecalciferol suppresses apolipoprotein A-I gene expression

AimsLigands for the vitamin D receptor (VDR) regulate apolipoprotein A-I (apo A-I) gene expression in a tissue-specific manner. The vitamin D metabolite 24, 25-dihydroxycholecalciferol (24, 25-(OH)₂D₃) has been shown to possess unique biological effects. To determine if 24, 25-(OH)₂D₃ modulates apo A-I gene expression, HepG2 hepatocytes and Caco-2 intestinal cells were treated with 24, 25-(OH)₂D₃ or its precursor 25-OHD₃.Main methodsApo A-I protein levels and mRNA levels were measured by Western and Northern blotting, respectively. Changes in apo A-I promoter activity were measured using the chlorampenicol acetytransferase assay.Key findingsTreatment with 24, 25-(OH)₂D₃, but not 25-OHD₃, inhibited apo A-I secretion in HepG2 and Caco-2 cells and apo A-I mRNA levels and apo A-I promoter activity in HepG2 cells. To determine if 24, 25-(OH)₂D₃ represses apo A-I gene expression through site A, the nuclear receptor binding element that is essential for VDRs effects on apo A-I gene expression, HepG2 cells were transfected with plasmids containing or lacking site A. While the site A-containing plasmid was suppressed by 24, 25-(OH)₂D₃, the plasmid lacking site A was not. Likewise, treatment with 24, 25-(OH)₂D₃ suppressed reporter gene expression in cells transfected with a plasmid containing site A in front of a heterologous promoter. Finally, antisense-mediated VDR depletion failed to reverse the silencing effects of 24, 25-(OH)₂D₃ on apo A-I expression.SignificanceThese results suggest that the vitamin D metabolite 24, 25-(OH)₂D₃ is an endogenous regulator of apo A-I synthesis through a VDR-independent signaling mechanism.     

Biweekly intracavernous administration of papaverine for erectile dysfunction.

To determine the efficacy of the intracavernous administration of papaverine hydrochloride every 2 weeks to treat male impotence, we recruited 50 patients from a sexual dysfunction clinic. Of these, 8 did not complete the course of papaverine therapy. A total of 8 had complications, including priapism in 3 and ecchymoses or urethral bleeding in 5. In 30 patients who had substantial vasculogenic disease, there was a notable improvement in the penile blood pressure after papaverine treatment, although only 9 reported successful sexual intercourse. The penile blood pressure in the remaining 12 patients without substantial vascular disease increased modestly after therapy, and 5 persons reported satisfactory sexual activity. We conclude that administering papaverine intracavernosally every 2 weeks improves sexual potency in a subset of sexually impotent patients. Those with severe vasculogenic disease and a penile-brachial pressure index of less than 0.65 are not good candidates for this regimen.     

The effect of age on protein composition of rat cerebral microvessels

The effect of age on protein composition of cerebral microvessels was investigated by examining the content of glycosylation endproducts in cerebral microvessels isolated from young (3–6 month old), intermediate age (18 month) and aged (24–26 month old) Fischer 344 male rats and by quantitating various protein spots identified with two dimensional (2D) electrophoresis. The results indicate that aging in rats is not associated with significant increase in glycosylation of microvessel proteins. Of the 26 proteins in cerebral microvessels identified on the 2-D gel, ten showed significant age-related changes (p<0.0004) and in two of these the changes were significant as early as 18-months of age. A large acidic protein with a molecular weight of 144,000 and isoelectric point (pI) of 5.4 (Spot #1) was found only in aged rats. The results indicate that aging is associated with significant quantitative changes in protein composition of cerebral microvessels. It is possible that Spot #1 may be a novel biochemical marker of aging blood-brain barrier.     

Troglitazone stimulates acetyl-CoA carboxylase activity through a post-translational mechanism

Troglitazone, a thiazolidinedione, is known to act as an insulin sensitizer. The various effects of the drug include stimulation of glucose utilization and inhibition of gluconeogenesis and fatty acid oxidation. We studied the effect of troglitazone treatment on rat liver acetyl-CoA carboxylase (ACC), the key enzyme that catalyzes the formation of malonyl-CoA, the rate-limiting step in the synthesis of long chain fatty acids. Treatment of rats with troglitazone for 18 days resulted in more than 200% increase in the activity of hepatic acetyl-CoA carboxylase (1.01+/-0.14 and 2.33+/-0.28 mU/mg supernatant protein for control and troglitazone-treated rats, respectively) (p<0.001). The expression of acetyl-CoA carboxylase mRNA, as studied by RNAse protection assay, was not significantly different between the two groups of animals. The ACC from control and troglitazone-treated groups was purified by avidin-affinity chromatography. The purified enzyme migrated as a major protein band (Mr 262,000) on SDS-polyacrylamide gels. Troglitazone treatment was associated with increased citrate sensitivity of ACC. The specific activity of the purified preparation in troglitazone-treated rats was increased by 67% (2.5 vs. 1.5 U/mg). Quantitation of alkali-labile phosphate content of the purified preparation revealed 5.66+/-0.17 and 6.29+/-0.13 mol Pi/mol subunit of 262 Kda for control and troglitazone-treated rats, respectively (P<0.01). The subtle increase in phosphate content does not explain the observed activation of the enzyme. It is possible that additional mechanisms such as troglitazone related rearrangement of the occupancy of select phosphate binding sites or altered binding of the biotin cofactor may also contribute to the observed activation of ACC.     

Age-related changes in thyroid hormone effects on glucose transporter isoforms of rat heart.

To determine the age-related changes in thyroid hormone (TH) effects on cardiac glucose transporter one (GLUT-1) and four (GLUT-4) isoforms, male Fischer 344 rats at 4, 12, and 25 months of age were studied at euthyroid, hyperthyroid and hypothyroid conditions. Hyperthyroidism was induced with daily intraperitoneal injections of triiodothyronine (15 microg/100 gm) for 10 days. Hypothyroidism was achieved with 0.025% methimazole in the drinking water for 4 weeks. Immunoblot analysis indicated that at euthyroid basal conditions GLUT-1 protein was not significantly altered with age while GLUT-4 protein was significantly reduced in 25 month old rats (82.0 +/- 28.8% of a 4 month old rat p <0.01). In 4 months old rats, GLUT-1 was increased in both hypothyroidism (432.5 +/- 208.7% of age-matched euthyroid control) and to a lesser extent in hyperthyroidism (242.0 +/- 93.3% of control) p<0.01. In 25 month old rats, hyperthyroidism was also associated with increased GLUT-1 mass (190.8 +/- 117.6% of age-matched euthyroid control) p<0.01. Hypothyroidism in this age group was not associated with significant change in GLUT-1 protein. The cardiac GLUT-4 protein was increased during both hypothyroidism and hyperthyroidism. The changes of GLUT-4 in aged rats were similar to those found in young rats. It is concluded that TH effect on GLUT-1 expression in the heart is altered with age while TH effects on GLUT-4 are age independent.