ß­ecdysterone protects against apoptosis by promoting autophagy in nucleus pulposus cells and ameliorates disc degeneration.

Increasing cell apoptosis is one of the major causes of intervertebral disc degeneration (IDD). ß-ecdysterone has been demonstrated to protect PC12 cells against neurotoxicity. A previous study revealed that ß­ecdysterone may be involved in the regulation of autophagy in osteoblasts. Therefore, we hypothesized that ß­ecdysterone may possess therapeutic effects on IDD via autophagy stimulation. The effect of ß­ecdysterone on IDD was explored by in vitro experiments. The results demonstrated that ß­ecdysterone attenuated the apoptosis induced by tert­butyl hydroperoxide via promoting autophagy in nucleus pulposus cells. Beclin­1, an indispensable protein for the stimulation of autophagy, is upregulated and stabilized by ß­ecdysterone in a dose­ and time­dependent manner in nucleus pulposus cells. Inhibition of autophagy with 3­methyladenine partially abrogated the protective function of ß­ecdysterone against apoptosis of nucleus pulposus cells, indicating that autophagy participated in the protective effect of ß­ecdysterone on IDD. Additionally, ß­ecdysterone promoted the expression of anabolic genes while inhibiting the expression of catabolic genes in nucleus pulposus cells. Collectively, the present study demonstrated that ß­ecdysterone may protect nucleus pulposus cells against apoptosis by autophagy stimulation and ameliorate disc degeneration, which indicates that ß­ecdysterone may be a potential therapeutic agent for IDD.

The cell biology of systemic insulin function.

Insulin is the paramount anabolic hormone, promoting carbon energy deposition in the body. Its synthesis, quality control, delivery, and action are exquisitely regulated by highly orchestrated intracellular mechanisms in different organs or "stations" of its bodily journey. In this Beyond the Cell review, we focus on these five stages of the journey of insulin through the body and the captivating cell biology that underlies the interaction of insulin with each organ. We first analyze insulin's biosynthesis in and export from the ß-cells of the pancreas. Next, we focus on its first pass and partial clearance in the liver with its temporality and periodicity linked to secretion. Continuing the journey, we briefly describe insulin's action on the blood vasculature and its still-debated mechanisms of exit from the capillary beds. Once in the parenchymal interstitium of muscle and adipose tissue, insulin promotes glucose uptake into myofibers and adipocytes, and we elaborate on the intricate signaling and vesicle traffic mechanisms that underlie this fundamental function. Finally, we touch upon the renal degradation of insulin to end its action. Cellular discernment of insulin's availability and action should prove critical to understanding its pivotal physiological functions and how their failure leads to diabetes.

Biotransformation of anabolic compound methasterone with Macrophomina phaseolina, Cunninghamella blakesleeana, and Fusarium lini, and TNF-α inhibitory effect of transformed products.

Microbial transformation of methasterone (1) was investigated with Macrophomina phaseolina, Cunninghamella blakesleeana, and Fusarium lini. Biotransformation of 1 with M. phaseolina yielded metabolite 2, while metabolites 3-7 were obtained from the incubation of 1 with C. blakesleeana. Metabolites 8-13 were obtained through biotransformation with F. lini. All metabolites, except 13, were found to be new. Methasterone (1) and its metabolites 2-6, 9, 10, and 13 were then evaluated for their immunomodulatory effects against TNF-α, NO, and ROS production. Among all tested compounds, metabolite 6 showed a potent inhibition of proinflammatory cytokine TNF-α (IC50=8.1±0.9µg/mL), as compared to pentoxifylline used as a standard (IC50=94.8±2.1µg/mL). All metabolites were also evaluated for the inhibition of NO production at concentration of 25µg/mL. Metabolites 6 (86.7±2.3%) and 13 (62.5±1.5%) were found to be the most potent inhibitors of NO as compared to the standard NG-monomethyl-l-arginine acetate (65.6±1.1%). All metabolites were found to be non-toxic against PC3, HeLa, and 3T3 cell lines. Observed inhibitory potential of metabolites 6 and 13 against pro-inflammatory cytokine TNF-α, as well as NO production makes them interesting leads for further studies.

Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat.

The actual levels of steroid hormones in organs are vital for endocrine, reproductive and neuronal health and disorders. We developed an accurate method to determine the levels of steroid hormones and steroid conjugates in various organs by an efficient preparation using a solid-phase-extraction cartridge. Each steroid was identified by the precursor ion spectra using liquid chromatography-electrospray ionization time-of-flight mass spectrometry, and the respective steroids were quantitatively analysed in the selected reaction monitoring mode by liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS). The data showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver. The glucuronide conjugates of steroid hormones and the precursors were detected in all organs except liver, but sulfate conjugates of these steroids were observed only in the target organs of the hormones and kidney. Interestingly, these steroids and the conjugates were not observed in the liver except pregnenolone. In conclusion, an accurate determination of tissue steroids was developed using LC-MS analysis. Biosynthesis of steroid hormones from the precursors was estimated even in the target organs, and the delivery of these steroid conjugates was also suggested via the circulation without any significant hepatic participation.

Sex and season influence gonadal steroid biosynthetic pathways, end-product production and steroid conjugation in blotched blue-tongued lizards (Tiliqua nigrolutea).

We examined differences in gonadal steroid production and biosynthetic pathway activity with changing reproductive condition and between sexes in the scincid lizard, Tiliqua nigrolutea. We observed clear seasonal and sexual variation in the production of androgens and steroid conjugates, but detected no 17beta-estradiol or 5alpha-dihydrotestosterone produced by the gonads. An alternative steroid, more polar than estradiol, was detected: an investigation of this steroid is reported separately [Gen. Comp. Endocrinol. 129 (2002) 114]. There were seasonal and sex-related differences in steroid biosynthetic pathway activity. The Delta5 pathway metabolite, dehydroepiandrosterone, was detected only in males, and only from incubations using regressed testicular tissue. There was also a seasonal difference between the sexes in rates of progesterone accumulation, although the absence of corresponding elevated plasma concentrations suggests that the role of progesterone switches from a directly acting hormone to a precursor for others during the reproductive cycle in females. These results suggest that within the traditional view that vertebrate biosynthetic pathway activity and end-products are phylogenetically conserved, there is likely to be considerably species- and/or genus-specific variation.