RNAi-mediated germline knockdown of FABP4 increases body weight but does not improve the deranged nutrient metabolism of diet-induced obese mice.

OBJECTIVE: To investigate the impact of reduced adipocyte fatty acid-binding protein 4 (FABP4) in control of body weight, glucose and lipid homeostasis in diet-induced obese (DIO) mice. METHODS: We applied RNA interference (RNAi) technology to generate FABP4 germline knockdown mice to investigate their metabolic phenotype. RESULTS: RNAi-mediated knockdown reduced FABP4 mRNA expression and protein levels by almost 90% in adipocytes of standard chow-fed mice. In adipocytes of DIO mice, RNAi reduced FABP4 expression and protein levels by 70 and 80%, respectively. There was no increase in adipocyte FABP5 expression in FABP4 knockdown mice. The knockdown of FABP4 significantly increased body weight and fat mass in DIO mice. However, FABP4 knockdown did not affect plasma glucose and lipid homeostasis in DIO mice; nor did it improve their insulin sensitivity. CONCLUSION: Our data indicate that robust knockdown of FABP4 increases body weight and fat mass without improving glucose and lipid homeostasis in DIO mice.

Gamma oscillation underlies hyperthermia-induced epileptiform-like spikes in immature rat hippocampal slices.

BACKGROUND: Recently a hyperthermic rat hippocampal slice model system has been used to investigate febrile seizure pathophysiology. Our previous data indicates that heating immature rat hippocampal slices from 34 to 41 degrees C in an interface chamber induced epileptiform-like population spikes accompanied by a spreading depression (SD). This may serve as an in vitro model of febrile seizures. RESULTS: In this study, we further investigate cellular mechanisms of hyperthermia-induced initial population spike activity. We hypothesized that GABA(A) receptor-mediated 30-100 Hz gamma oscillations underlie some aspects of the hyperthermic population spike activity. In 24 rat hippocampal slices, the hyperthermic population spike activity occurred at an average frequency of 45.9 +/- 14.9 Hz (Mean +/- SE, range = 21-79 Hz, n = 24), which does not differ significantly from the frequency of post-tetanic gamma oscillations (47.1 +/- 14.9 Hz, n = 34) in the same system. High intensity tetanic stimulation induces hippocampal neuronal discharges followed by a slow SD that has the magnitude and time course of the SD, which resembles hyperthermic responses. Both post-tetanic gamma oscillations and hyperthermic population spike activity can be blocked completely by a specific GABA(A) receptor blocker, bicuculline (5-20 microM). Bath-apply kynurenic acid (7 mM) blocks synaptic transmission, but fails to prevent hyperthermic population spikes, while intracellular diffusion of QX-314 (30 mM) abolishes spikes and produces a smooth depolarization in intracellular recording. CONCLUSION: These results suggest that the GABA(A) receptor-governed gamma oscillations underlie the hyperthermic population spike activity in immature hippocampal slices.

Design and synthesis of novel antibacterial agents with inhibitory activity against DNA polymerase III.

4-Substituted 2-amino-6-(anilino)pyrimidines have been found to be selective inhibitors of DNA polymerase III, a replicative enzyme known to be essential in the DNA synthesis of Gram-positive bacteria. Among the analogues, 18 displayed an IC(50) of 10 microM against DNA polymerase III from Staphylococcus aureus.

Cloning, expression and characterization of rhesus macaque types 1 and 2 5alpha-reductase: evidence for mechanism-based inhibition by finasteride.

The rhesus macaque types 1 and 2 5alpha-reductase (5aR1 and 5aR2) were cloned and expressed in COS cells to facilitate comparison of rhesus and human 5aRs. The deduced protein sequences of the rhesus SaRs shared 94% and 96% identity with the human type 1 and 2 isozymes, respectively. Despite a four amino acid insertion at the N-terminal region of rhesus 5aR1, the biochemical properties of rhesus and human homologs are very similar with respect to pH optimum, Km values for testosterone and progesterone, and inhibition by a variety of inhibitors. As expected, the biochemical properties of the human and rhesus 5aR2 are also very similar. The mechanism of inhibition of the rhesus 5aR1 and 5aR2 by finasteride was investigated in more detail. Finasteride displays time dependent inhibition of the rhesus 5aR1 and 5aR2 with second order rate constants of 4 x 10(3) M(-1) s(-1) and 5.2 x 10(5) M(-1)s(-1). Inhibition of rhesus 5aR2 with 3H-finasteride resulted in 3H bound to the enzyme which is not released by dialysis. Heat denaturation of the [rhesus SaR2:inhibitor] complex releases dihydrofinasteride, a breakdown product presumably related to the NADP+-adduct previously identified with the human SaRs (Bull et al., Mechanism-based inhibition of human steroid 5alpha-reductase by finasteride: Enzyme catalyzed formation of NADP-dihydrofinasteride, a potent bisubstrate analog inhibitor. J. Amer. Chem. Soc., 1996, 118, 2359-2365). Taken together, these results provide good evidence that the rhesus macaque is a suitable model to evaluate the pharmacological properties of finasteride and other 5aR inhibitors.

Inhibition of rat alpha-reductases by finasteride: evidence for isozyme differences in the mechanism of inhibition.

The mechanism of inhibition of the rat types 1 and 2 5alpha-reductase by finasteride was investigated using recombinantly expressed enzymes. These studies revealed that finasteride is a potent, reversible inhibitor of the rat type 1 5alpha-reductase with Ki=10.2+/-1.3 nM. Finasteride is a potent inhibitor of the rat type 2; however, in this case the compound binds to the type 2 isozyme-NADPH complex to form a ternary complex with Ki=1.19+/-0.10 nM, which then rearranges to a high affinity complex (E:I) with a pseudo first order rate constant of 1.62+/-0.22 x 10(-3)/s. The second order rate constant is k3/Ki=1.37+/-0.31 x 10(6) M/s. Heat denaturation of the (type 2 enzyme:inhibitor) complex releases dihydrofinasteride and presumably the NADP+-adduct previously identified with the human 5alpha-reductases. The effects of finasteride were also studied in intact COS cells transiently expressing the rat types 1 and 2 5alpha-reductase. Results with whole cell assays confirm differences in mechanism of inhibition of rat types 1 and 2 5alpha-reductase by finasteride.

Effects of finasteride, a type 2 5-alpha reductase inhibitor, on fetal development in the rhesus monkey (Macaca mulatta).

In genetic male fetuses, dihydrotestosterone (DHT) plays an important role in normal prostatic and external genital differentiation. The enzyme steroid 5-alpha reductase (5 alpha R) catalyzes the conversion of testosterone (T) to DHT. The importance of 5 alpha R in sexual differentiation is evident from the study of human genetic males who congenitally lack this enzyme and consequently develop ambiguous genitalia. These individuals are specifically deficient in the type 2 isozyme, whereas the normal type 1 isozyme activity has been found. The purpose of this study was to determine 1) the suitability of the rhesus monkey for testing the safety of 5 alpha R inhibitors when administered during pregnancy and 2) the potential risk of administering a known type 2 5 alpha R inhibitor, finasteride, during the critical period of internal and external genital differentiation in rhesus monkeys. In vitro studies were also performed on selected rhesus monkey tissues to determine the distribution of the 5 alpha R isozymes. Gravid monkeys were treated once daily from gestational days (GD) 20 to 100. Sonographic monitoring was performed during the course of gestation to monitor viability, growth, and organ system development. Detailed fetal evaluations for developmental abnormalities were performed at term (GD 152 +/- 2). A group of 13 pregnant monkeys ("positive control") were given a high oral dose (2 mg/kg/day) of finasteride to demonstrate that inhibiting type 2 5 alpha R results in specific external genital abnormalities in male fetuses. Thirty-two pregnant monkeys were administered an intravenous (i.v.) formulation of finasteride at doses of 8, 80, or 800 ng/day. The highest i.v. dose selected was at least 60-750 times the semen levels of finasteride in man given orally 5 or 1 mg/day, respectively. Seventeen vehicle-control pregnant monkeys were also included. Administration of a high oral dose (2 mg/kg/day) of finasteride resulted in external genital abnormalities characterized by hypospadias, preputial adhesions to the glans, a small underdeveloped scrotum, a small penis, and a prominent midline raphe in male fetuses; however, no developmental abnormalities were seen in female fetuses. Similarly, no abnormalities were observed in either male or female fetuses of mothers given iv doses (8, 80, or 800 ng/day) of finasteride during pregnancy. The in utero sonographic findings in fetuses correlated with the gross findings at term. These studies have shown that external genital abnormalities can be produced in male monkey fetuses when exposed to a high oral dose (2 mg/kg/day) of finasteride, whereas no abnormalities were observed in fetuses exposed to the i.v. formulation of finasteride. Detailed in vitro studies demonstrated that the rhesus monkey also has two 5 alpha R isozymes (types 1 and 2) with a tissue distribution similar to that seen in man and, furthermore, that finasteride is a potent, mechanism-based inhibitor with selectivity for both human and rhesus type 2 5 alpha R. These studies have demonstrated that the monkey is a suitable model for assessing the safety of 5 alpha R inhibitors administered during pregnancy.

MK386: a potent, selective inhibitor of the human type 1 5alpha-reductase.

Steroid 5alpha-reductase is required for the conversion of testosterone to dihydrotestosterone. Localization of type 1 5alpha-reductase in the sebaceous gland of skin offers the possibility for selective inhibition of this isozyme as a treatment for acne. The goals of these studies are to demonstrate the mechanism of inhibition of MK386 and its selectivity for type 1 5alpha-reductase. The apparent potency of MK386 differed depending on the source of the enzyme (i.e. recombinant vs. native), yet selectivity for type 1 5alpha-reductase was unchanged. Our results indicate that the apparent potency of MK386 is modulated by the membrane concentration of the assay. These results suggest that MK386 has a high affinity for the lipid-rich membrane environment of 5alpha-reductase. MK386 was also found to be a slow binding inhibitor of type 1 5alpha-reductase. However, the cause of this time-dependent inhibition is unrelated to partitioning of the inhibitor into the membrane because similar studies with type 2 5alpha-reductase indicate that MK386 is a reversible, competitive inhibitor. A number of counterscreens were developed to demonstrate that MK386 is a poor inhibitor of other steroid metabolizing enzymes.

Expression of the type 1 and 2 steroid 5 alpha-reductases in human fetal tissues.

Androgens play a key role in human fetal development. Therefore, it is important to understand the distribution of the isozymes of 5 alpha-reductase, the enzyme that converts testosterone to the more potent androgen, dihydrotestosterone. The expression of the two isozymes of 5 alpha-reductase was studied in human fetal skin and fetal prostate by measuring the in vitro enzyme activity in crude preparations. Low levels of the type 1 5 alpha-reductase were found in fetal scalp and back skin. Studies with fetal prostate samples confirmed that the type 2 enzyme is expressed in levels similar to those found in adult tissues. These results provide the first evidence that both isozymes of 5 alpha-reductase are expressed during human fetal development.

Identification and selective inhibition of an isozyme of steroid 5 alpha-reductase in human scalp.

Steroid 5 alpha-reductase (EC 1.3.1.22) catalyzes the reduction of testosterone to dihydrotestosterone. The 5 alpha-reductase found in human scalp has been compared with the enzyme found in prostate. The scalp reductase has a broad pH optimum centered at pH 7.0. This is distinctly different from the pH optimum of 5.5 observed with the prostatic form of the enzyme. These enzymes also differ in the Km for testosterone, which is 25-fold higher for the scalp reductase. The most significant difference between the two enzymes is their affinity for inhibitors. Two 4-azasteroids and a 3-carboxyandrostadiene are potent inhibitors of the prostatic reductase but are weak inhibitors of the scalp reductase. In contrast, several N-4-methylazasteroids are good inhibitors of the scalp reductase. These findings support a proposal that different isozymes of 5 alpha-reductase may exist in scalp and prostate. The scalp reductase was also compared to 5 alpha-reductase 1, one of the two enzymes recently cloned from human prostate [Andersson, S. & Russell, D. W. (1990) Proc. Natl. Acad. Sci. USA 87, 3640-3644; and Andersson, S., Berman, D. M., Jenkins, E. P. & Russell, D. W. (1991) Nature (London) 354, 159-161]. The characteristics of the cloned reductase 1 are comparable to those of the scalp reductase.

Differences between calcium channel inhibitors in their effects on phencyclidine-induced behavioral stimulation in mice.

Sixteen calcium channel inhibitors (CCI's) were tested in a model utilizing phencyclidine (PCP)-induced behavioral stimulation in mice. There were marked differences in the effects of CCI's both within subclasses and between subclasses of CCI's. All of the dihydropyridines and possibly flunarizine were effective in blocking PCP-induced behavioral stimulation. Papaverine derivatives, including verapamil, and several other CCI's, were ineffective.