Positive Regulation by GABABR1 Subunit of Leptin Expression through Gene Transactivation in Adipocytes

Background: The view that c-aminobutyric acid (GABA) plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes. Methodology/Principal Findings: GABAB receptor 1 (GABABR1) subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABA BR ligands. However, no prominent expression was seen with mRNA for GABA BR2 subunit required for heteromeric orchestration of the functional GABABR by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABA BR1-null mice than in wild-type mice. Knockdown by siRNA of GABA BR1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells. Conclusions/Significance: Our results indicate that GABABR1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner o

Chronic Fluid Flow Is an Environmental Modifier of Renal Epithelial Function

Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is been known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca++, which can then result in a variety of activated signaling pathways. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5th decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because ADPKD is a slowly progressing disease, I asked how fluid flow may act, via the primary cilium, to alter epithelial physiology during the course of cell turnover. I performed an experiment to determine under what conditions fluid flow can result in a change of function of renal epithelial tissue. A wildtype epithelial cell line derived the cortical collecting duct of a heterozygous offspring of the Immortomouse (Charles River Laboratory) was selected as our model system. Gentle orbital shaking was used to induce physiologically relevant fluid flow, and periodic measurements of the transepithelial Sodium current were performed. At the conclusion of the experiment, mechanosensitive proteins of interest were visualized by immunostaining. I found that fluid flow, in itself, modifies the transepithelial sodium current, cell proliferation, and the actin cytoskeleton. These results significantly impact the understanding of both the mechanosensation function of primary cilia as well as the understanding of ADPKD disease progression

Excitotoxicity Triggered by Neurobasal Culture Medium

Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12–15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca2+ influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium

Acute Insulin Stimulation Induces Phosphorylation of the Na-Cl Cotransporter in Cultured Distal mpkDCT Cells and Mouse Kidney

The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin

SLO-2 Is Cytoprotective and Contributes to Mitochondrial Potassium Transport

Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance “big” K+ channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K+ transport and APC. The canonical Ca2+-activated BK (or “maxi-K”) channel SLO1 was dispensable for both mitochondrial K+ transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K+ channel SLO2 was required, and that SLO2-dependent mitochondrial K+ transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K+

Role of Glomerular Proteoglycans in IgA Nephropathy

Mesangial matrix expansion is a prominent feature of the most common form of glomerulonephritis, IgA nephropathy (IgAN). To find molecular markers and improve the understanding of the disease, the gene and protein expression of proteoglycans were investigated in biopsies from IgAN patients and correlated to clinical and morphological data. We collected and microdissected renal biopsies from IgAN patients (n = 19) and from healthy kidney donors (n = 14). Patients were followed for an average time of 4 years and blood pressure was according to target guidelines. Distinct patterns of gene expression were seen in glomerular and tubulo-interstitial cells. Three of the proteoglycans investigated were found to be of special interest and upregulated in glomeruli: perlecan, decorin and biglycan. Perlecan gene expression negatively correlated to albumin excretion and progress of the disease. Abundant decorin protein expression was found in sclerotic glomeruli, but not in unaffected glomeruli from IgAN patients or in controls. Transforming growth factor beta (TGF-β), known to interact with perlecan, decorin and biglycan, were upregulated both on gene and protein level in the glomeruli. This study provides further insight into the molecular mechanisms involved in mesangial matrix expansion in IgAN. We conclude that perlecan is a possible prognostic marker for patients with IgAN. In addition, the up-regulation of biglycan and decorin, as well as TGF-β itself, indicate that regulation of TGF-β, and other profibrotic markers plays a role in IgAN pathology

Alteration of Forkhead Box O (Foxo4) Acetylation Mediates Apoptosis of Podocytes in Diabetes Mellitus

The number of kidney podocytes is reduced in diabetic nephropathy. Advanced glycation end products (AGEs) accumulate in patients with diabetes and promote the apoptosis of podocyte by activating the forkhead box O4 (Foxo4) transcription factor to increase the expression of a pro-apoptosis gene, Bcl2l11. Using chromatin immunoprecipitation we demonstrate that AGE-modified bovine serum albumin (AGE-BSA) enhances Foxo4 binding to a forkhead binding element in the promoter of Bcl2lll. AGE-BSA also increases the acetylation of Foxo4. Lysine acetylation of Foxo4 is required for Foxo4 binding and transcription of Bcl2l11 in podocytes treated with AGE-BSA. The expression of a protein deacetylase that targets Foxo4 for deacetylation, sirtuin (Sirt1), is down regulated in cultured podocytes by AGE-BSA treatment and in glomeruli of diabetic patients. SIRT1 over expression in cultured murine podocytes prevents AGE-induced apoptosis. Glomeruli isolated from diabetic db/db mice have increased acetylation of Foxo4, suppressed expression of Sirt1, and increased expression of Bcl2l11 compared to non-diabetic littermates. Together, our data provide evidence that alteration of Foxo4 acetylation and down regulation of Sirt1 expression in diabetes promote podocyte apoptosis. Strategies to preserve Sirt1 expression or reduce Foxo4 acetylation could be used to prevent podocyte loss in diabetes

Expression and Function of Serotonin 2A and 2B Receptors in the Mammalian Respiratory Network

Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT2A, 5-HT2B, and 5-HT2C receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT2ARs, expression and function of 5-HT2BRs within the respiratory network are still unclear. 5-HT2BR utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT2ARs and 5-HT2BRs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT2BR. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation

Proliferation of Acid-Secretory Cells in the Kidney during Adaptive Remodelling of the Collecting Duct

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H+-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H+-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH4Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH4Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions

The Organophosphate Chlorpyrifos Interferes with the Responses to 17β-Estradiol in the Digestive Gland of the Marine Mussel Mytilus galloprovincialis

BACKGROUND: Many pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists are low compared with those of natural ligands, their ability to act via multiple mechanisms might enhance the biological effect. The organophosphate Chlorpyrifos (CHP) has been shown to be weakly estrogenic and cause adverse neurodevelopmental effects in mammals. However, no information is available on the endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue of both estrogens and pesticides, the possible effects of CHP on the responses to the natural estrogen 17β-estradiol (E(2)) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Mussels were exposed to CHP (4.5 mg/l, 72 hrs) and subsequently injected with E(2) (6.75 ng/g dw). Responses were evaluated in CHP, E(2) and CHP/E(2) treatment groups at 24 h p.i. by a biomarker/transcriptomic approach. CHP and E(2) induced additive, synergistic, and antagonistic effects on lysosomal biomarkers (lysosomal membrane stability, lysosome/cytoplasm volume ratio, lipofuscin and neutral lipid accumulation). Additive and synergistic effects were also observed on the expression of estrogen-responsive genes (GSTπ, catalase, 5-HTR) evaluated by RT-Q-PCR. The use of a 1.7K cDNA Mytilus microarray showed that CHP, E(2) and CHP/E(2), induced 81, 44, and 65 Differentially Expressed Genes (DEGs), respectively. 24 genes were exclusively shared between CHP and CHP/E(2), only 2 genes between E(2) and CHP/E(2). Moreover, 36 genes were uniquely modulated by CHP/E(2). Gene ontology annotation was used to elucidate the putative mechanisms involved in the responses elicited by different treatments. CONCLUSIONS: The results show complex interactions between CHP and E(2) in the digestive gland, indicating that the combination of certain pesticides and hormones may give rise to unexpected effects at the molecular/cellular level. Overall, these data demonstrate that CHP can interfere with the mussel responses to natural estrogens