Bernard I. Weinstein

Altered expression of 3α-hydroxysteroid dehydrogenases in human glaucomatous optic nerve head astrocytes

3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms (AKR1C1-AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that in glaucomatous human optic nerve heads, increased expression of 3alpha-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3alpha-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3alpha-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3alpha-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma.

Glucocorticoid localization by radioautography in the rabbit eye following systemic administration of 3H-dexamethasone.

Dexamethasone was localized radioautographically in the nuclei of target cells of the rabbit eye, following i.v. administration of the labeled steroid. Nuclear receptors in stromal and endothelial cells of the outflow pathway region suggest that glucocorticoids may alter the outflow facility by specific responses of target cells.

Metformin and carbohydrate-modified diet: a novel obesity treatment protocol: preliminary findings from a case series of nondiabetic women with midlife weight gain and hyperinsulinemia.

The authors conducted a retrospective analysis of a new obesity treatment protocol, metformin and hypocaloric, carbohydrate-modified diet, in high-risk, nondiabetic hyperinsulinemic women with progressive midlife weight gain (refractory to diet and exercise). Thirty consecutive nondiabetic women with glucose-mediated area-under-the-curve (AUC) insulin elevations (>or=100 microU/mL) in two body mass index (BMI) categories (group I: 25 to 32.9 kg/m(2) and group II: 33 to 41.7 kg/m(2)) participated in a 1-year treatment program of metformin (mean daily doses of 1,500 mg/day [group I] and 2,000 mg/day [group II]) and carbohydrate-modified dietary regimens. Follow-up body weight (at 3, 6, and 12 months), percentage of patients meeting goal weight attainment (10% reduction in body weight or BMI normalization), and fasting insulin levels (as available) are reported in 26 women (18/18 in group I and 8/12 in group II) who returned for one or more follow-up visits. Significant weight loss was observed at 3, 6, and 12 months in both group I (3.47 [SE 0.68], 6.41 [0.72], and 8.06 [0.96] kg, P < 0.0001) and group II (4.4 [0.8], 9.7 [2.3], 15.1 [3.3], P = 0.001, 0.004, 0.011). Twenty-five of 26 (96%) patients lost >or=5% of their body weight at 6 months and 21/26 (81%) patients lost >or=10% of their body weight at 12 months. Posttreatment fasting insulin decrement (-35.5 [8.2]%) was the most significant predictor of 1-year weight loss (R(2)=0.656, regression coefficient = 0.810, P = 0.005). Following completion of the 1-year intervention study, weight stabilization (within 1 kg) was observed at a 6-month surveillance in 8/9 patients who attained goal weight and continued metformin without additional nutritional counseling, in contrast to weight gain (>or=4 kg or 50% of lost weight) in 5/6 patients who discontinued metformin. The authors concluded that metformin and carbohydrate-modified hypocaloric diet could be an effective novel treatment for long-term weight management in nondiabetic, hyperinsulinemic women.

Prostaglandin synthesis and release from cultured human trabecular-meshwork cells and scleral fibroblasts

Human trabecular-meshwork (HTM) cells in culture convert arachidonic acid to two products: PGE2 and 6-keto PGF1 alpha. Prostaglandin PGE2 was the major product of arachidonic-acid metabolism. The synthesis and release of PGE2 and 6-keto PGF1 alpha was inhibited by a 15-min pre-treatment with indomethacin (5 X 10(-6) M) or a 4-24-hr incubation with 10(-7) M dexamethasone. The effects of dexamethasone could be prevented by cycloheximide (0.5 micrograms ml-1), or actinomycin D (2 micrograms ml-1). Prostaglandin E2 synthesis and release by these HTM cells from two different individuals could be stimulated by bradykinin, arachidonic acid and the calcium ionophore, A23187.

Human hepatic cortisol reductase activities : enzymatic properties and substrate specificities of cytosolic cortisol Δ4-5β-reductase and dihydrocortisol-3α-oxidoreductase(s)

The metabolism of cortisol by human liver homogenates has been studied. Cortisol delta 4-reductase and dihydrocortisol-3-oxidoreductase activities were distributed in all subcellular fractions. The products of the soluble enzymes were identified. Cortisol and 5 beta-dihydrocortisol were reduced to 3 alpha,5 beta-tetrahydrocortisol, and 5 alpha-dihydrocortisol was reduced to 3 alpha,5 alpha-tetrahydrocortisol. The soluble enzymes showed a wide range of substrate specificity. The 21 substituted cortisol derivatives were not metabolized. The apparent Km values of cortisol delta 4-5 beta-reductase and dihydrocortisol-3 alpha-oxidoreductase for their substrates (cortisol, 5 alpha-dihydrocortisol, and 5 beta-dihydrocortisol) all ranged from 18 to 27 microM. Dexamethasone inhibited the reduction of all of these substrates and the inhibition was abolished by 21 substitution of the dexamethasone. Testosterone was a competitive inhibitor of the reduction of cortisol, 5 alpha-dihydrocortisol, and 5 beta-dihydrocortisol with a Ki ranging from 11 to 32 microM. NADPH was the preferred cofactor for the cortisol delta 4-5 beta-reductase and dihydrocortisol-3 alpha-oxidoreductase. No end product inhibition was observed.

Normal and conditionally transformed bovine lens epithelial cell lines containing alpha- and gamma-crystallin☆

Abstract Bovine lens cell lines were developed which maintain their ability to synthesize both α- and γ-crystallin even after 120 generations in culture. Although these cells have the morphologic appearance of epithelial cells with no evidence of elongation or aggregation characteristic of fiber cells or lentoid bodies, biochemically they contain γ-crystallin and thus appear to be expressing some differentiated function. In addition, these cells have been transformed with a temperature sensitive mutant of SV 40 which resulted in an increased growth potential while still maintaining its capacity to synthesize both crystallins. A radioimmunoassay is described for the quantitation of γ-crystallin in cell homogenates. These cell lines will be useful for studying the effect of hormones and various growth factors on the differentiation of the mammalian lens.

Purification and properties of human hepatic 3α-hydroxysteroid dehydrogenase

Abstract 3α-Hydroxysteroid dehydrogenase (3α-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5β-[ 3 H]dihydrocortisol (5β-DHF) as substrate. Electrophoretically homogeneous enzyme was obtained using a procedure that involved ammonium sulfate precipitation and three successive column chromatography steps: DEAE-cellulose, hydroxylapatite and Blue-Sepharose. The enzyme is a monomer since the native molecular weight was found to be 37,000, using a calibrated Sephadex G-75 column, and the denatured subunit molecular weight was determined to be 38,500, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme had a pI of 5.6–5.9. The 3-ketosteroids: cortisol, testosterone, progesterone and androstenedione, were not substrates for 3α-HSD indicating that a saturated 4,5 double bond was required for substrate activity. The conformation at the 5 position, however, did not influence substrate activity since 5α- and 5β-DHF and 5α-dihydrotesterone were all reduced at similar rates. The purified enzyme preferred NADPH to NADH as a cofactor and showed a broad peak of activity in the pH range of 6.8–7.4. The apparent K m for 5β-DHF was 18 μM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4°C. Freezing and thawing of the enzyme resulted in a large loss of activity during early stages of the purification. This is the first report of the purification to homogeneity of 3α-HSD from human tissue.

Human hepatic 3α-hydroxysteroid dehydrogenase: Possible identity with human hepatic chlordecone reductase

3 alpha-Hydroxysteroid dehydrogenase is a cytosolic, monomeric, NADPH-dependent oxidoreductase which reduces 3-keto-5-dihydrosteroids to their tetrahydro products. We present here the first partial amino acid sequence data for the human liver enzyme and show these sequences to be identical to the deduced amino acid sequence for human hepatic chlordecone reductase. In addition, these two enzymes exhibit similar substrate and cofactor specificities and immunological reactivity. The results suggest that the natural substrates for chlordecone reductase are 3-keto-5-dihydrosteroids and that these two proteins may be identical.

Human Trabecular Meshwork Cells

In man, most of the aqueous humor drainage and the major resistance to outflow occur across the trabecular meshwork and inner wall of Schlemm’s canal. Pros-taglandins are known to alter intraocular presure (IOP) and have been implicated in both physiological and pathophysiological aspects of IOP regulation. In the present study, we have characterized pathways of arachidonic acid metabolism in human trabecular meshwork (HTM) cells and measured basal and hormone-stimulated PGE2 release. The principal product of [1-14C]PGH2 metabolism and [1 − 14C]arachidonic acid metabolism was PGE2. Immunoreactive PGE2 was released in excess of immunoreactive 6-keto-PGF1α during 15-min incubations in phosphate-buffered saline. Incubation wth indomethacin (5 μM) for 20 min substantially reduced (80–85% inhibition) PGE2 formation from AA. Overnight incubation with dexamethasone (ID50 = 3.3 × 10−9M) inhibited basal and A23187-stimulated PGE2 release. The effects of dexamethasone on PGE2 synthesis and release could be prevented by pretreatment of the HTM cells with 2 μg/ml cyclo-heximide. A23187 (10−6-10−5 M) elicited a significant P <0.05 compared to control) increase in PGE2 release (RIA determinations).