mTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model.

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo.

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

Niacin-induced hyperglycemia is partially mediated via niacin receptor GPR109a in pancreatic islets.

The widely used lipid-lowering drug niacin is reported to induce hyperglycemia during chronic and high-dose treatments, but the mechanism is poorly understood. Recently, the niacin receptor [G-protein-coupled receptor, (GPR) 109a], has been localized to islet cells while its potential role therein remains unclear. We, therefore, aimed at investigating how GPR109a regulates islet beta-cell function and its downstream signaling using high-fat diet-induced obese mice and INS-1E beta cells. Eight-week niacin treatment elevated blood glucose concentration in obese mice with increased areas under the curve at oral glucose and intraperitoneal insulin tolerance tests. Additionally, niacin treatment significantly decreased glucose-stimulated insulin secretion (GSIS) but induced peroxisome proliferator-activated receptor gamma (Pparg) and GPR109a expression in isolated pancreatic islets; concomitantly, reactive oxygen species (ROS) were transiently increased, with decreases in GSIS, intracellular cyclic adenosine monophosphate (cAMP) accumulation and mitochondrial membrane potential (ΔΨm), but with increased expression of uncoupling protein 2 (Ucp2), Pparg and Gpr109a in INS-1E cells. Corroborating these findings, the decreases in GSIS, ΔΨm and cAMP production and increases in ROS, Pparg and GPR109a expression were abolished in INS-1E cells by GPR109a knockdown. Our data indicate that niacin-induced pancreatic islet dysfunction is probably modulated through activation of the islet beta-cell GPR109a-induced ROS-PPARγ-UCP2 pathways.

p-FAK-Tyr(397) regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization.

During spermatogenesis, the molecular mechanism that confers spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES), a testis-specific F-actin-rich adherens junction, in the rat testis remains elusive. Herein, the activated form of focal adhesion kinase (FAK), p-FAK-Tyr(397), a component of the apical ES that was expressed predominantly and stage specifically in stage VII-early stage VIII tubules, was found to be a crucial apical ES regulator. Using an FAK-Y397E phosphomimetic mutant cloned in a mammalian expression vector for its transfection vs. FAK and vector alone in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation were manifested by entrapment of spermatids in the seminiferous epithelium in late stage VIII-X tubules and were mediated by a disruption on the spatiotemporal expression and/or mislocalization of actin regulatory protein actin-related protein 3, which induces branched actin polymerization, epidermal growth factor receptor pathway substrate 8 (an actin barbed end capping and bundling protein), and palladin (an actin cross-linking and bundling protein). This thus perturbed changes of F-actin organization at the apical ES to facilitate spermiation, which also led to a concomitant alteration in the distribution and upregulation of adhesion proteins nectin-2 and nectin-3 at the apical ES. As such, nectin-2 and -3 remained at the apical ES to anchor step 19 spermatids on to the epithelium, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr(397) that regulates spermatid adhesion at the apical ES in vivo.