The WFS1 gene, encoding a transmembrane glycoprotein of the endoplasmic reticulum called wolframin, is mutated in Wolfram syndrome, an autosomal recessive disorder defined by the association of diabetes mellitus, optic atrophy, and further organ abnormalities. Disruption of the WFS1 gene in mice causes progressive ?-cell loss in the pancreas and impaired stimulus-secretion coupling in insulin secretion. However, little is known about the physiological functions of this protein. We investigated the immunohistochemical expression of wolframin in human placenta throughout pregnancy in normal women and diabetic pregnant women. In normal placenta, there was a modulation of wolframin throughout pregnancy with a strong level of expression during the first trimester and a moderate level in the third trimester of gestation. In diabetic women, wolframin expression was strongly reduced in the third trimester of gestation. The pattern of expression of wolframin in normal placenta suggests that this protein may be required to sustain normal rates of cytotrophoblast cell proliferation during the first trimester of gestation. The decrease in wolframin expression in diabetic placenta suggests that this protein may participate in maintaining the physiologic glucose homeostasis in this organ.
Neural precursor cell-expressed, developmentally down-regulated-8 (NEDD8) is a 76-amino-acid ubiquitin-like polypeptide. NEDD8 affects the signaling of various molecules but the major cellular target proteins are cullins. The neddylation process is correlated closely with apoptosis, cell-cycle regulation, embryogenesis and development.
The renal handling of salt and protons and bicarbonate are intricately linked through shared transport mechanisms for sodium, chloride, protons, and bicarbonate. In the collecting duct, the regulated fine-tuning of salt and acid-base homeostasis is achieved by a series of transport proteins located in different cell types, intercalated and principal cells. Intercalated cells are considered to be of less importance for salt handling but recent evidence has suggested that the anion exchanger pendrin may participate in salt reabsorption and blood pressure regulation. Here, we examined the regulated expression of two functionally related but differentially expressed anion exchangers, AE1 and pendrin, by dietary electrolyte intake and aldosterone. Cortical expression of pendrin was regulated on mRNA and protein level. The combination of NaHCO? and DOCA enhanced pendrin mRNA and protein levels, whereas DOCA or NaHCO? alone had no effect. NaCl or KHCO? increased pendrin mRNA, KCl decreased its mRNA abundance. On protein level, NH?Cl, NaCl, and KCl reduced pendrin expression, the other treatments were without effect. In contrast, AE1 mRNA or protein expression in kidney cortex was regulated by none of these treatments. In kidney medulla, NaHCO?/DOCA or NaHCO? alone enhanced AE1 mRNA levels. AE1 protein abundance was increased by NH?Cl, NaHCO?/DOCA, and NaCl. Immunolocalization showed that during NH?Cl treatment the relative number of AE1 positive cells was increased and pendrin expressing cells reduced. Thus, pendrin and AE1 are differentially regulated with distinct mechanisms that separately affect mRNA and protein levels. Pendrin is regulated by acidosis and chloride intake, whereas AE1 is enhanced by acidosis, NaCl, and the combination of DOCA and NaHCO?.
The monocarboxylate transporter family (MCT) comprises 14 members with distinct transport properties and tissue distribution. The kidney expresses several members of the MCT family, but only little is known about their exact distribution and function. Here, we investigated selected members of the MCT family in the mouse kidney. MCT1, MCT2, MCT7, and MCT8 localized to basolateral membranes of the epithelial cells lining the nephron. MCT1 and MCT8 were detected in proximal tubule cells whereas MCT7 and MCT2 were located in the thick ascending limb and the distal tubule. CD147, a beta-subunit of MCT1 and MCT4, showed partially overlapping expression with MCT1 and MCT2. However, CD147 was also found in intercalated cells. We also detected SMCT1 and SMCT2, two Na(+)-dependent monocarboxylate cotransporters, on the luminal membrane of type A intercalated cells. Moreover, mice were given an acid load for 2 and 7 days. Acidotic animals showed a marked but transient increase in urinary lactate excretion. During acidosis, a downregulation of MCT1, MCT8, and SMCT2 was observed at the mRNA level, whereas MCT7 and SMCT1 showed increased mRNA abundance. Only MCT7 showed lower protein abundance whereas all other transporters remained unchanged. In summary, we describe for the first time the localization of various MCT transporters in mammalian kidney and demonstrate that metabolic acidosis induces a transient increase in urinary lactate excretion paralleled by lower MCT7 protein expression.
The pathogenetic bases of HAART-associated lipodystrophy are still poorly known, even if it is clear that adipose tissue and its metabolism are sensitive to antiretroviral therapy alone and/or in combination with HIV infection. The NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression, morphogenesis and tumorigenesis. We investigated the possible involvement of NEED8 in adipogenesis and, consequently, in HIV-related lipodystrophy. One hundred HIV-1-infected patients were included in the study. Using an in vitro model of adipogenesis we evaluated the effects on adipogenesis of the forced expression of NEDD8 together with efavirenz, stavudine, saquinavir, amprenavir and indinavir, belonging to the three main classes of anti-HIV medications. We showed that NEDD8 expression level is higher in the peripheral blood of HIV patients developing lipodystrophy. Coherently, forced expression of NEDD8 in an in vitro model of adipogenesis was able to perturb expression of some key proteins involved in adipogenesis, such as C/EBPalpha and PPARgamma, possibly acting throughout the NEDD8/p27/beta-catenin pathway. Moreover, three out of five evaluated drugs were able to affect adipocyte differentiation: efavirenz, stavudine and saquinavir. Finally, we have shown that NEDD8 was expressed in the fat tissue of lipodystrophic patients, being significantly higher in the lipodystrophic patients with respect to the controls, thus further confirming the altered NEDD8 expression in the fat tissue of HIV-infected patients affected by lipodystrophy. Taken together, our data support the hypothesis of an implication of NEDD8 through p27 and beta-catenin pathways in the disruption of adipogenesis and consequent lipodystrophy in patients affected by HIV infection under HAART therapy with qualitative and quantitative differences according to diverse antiretroviral treatments. These evidences indicate the NEDD8/beta-catenin/p27 pathway as a possible molecular target for prevention of lipodystrophy development in patients under HAART therapy.
The highly active antiretroviral therapy (HAART) can cause a metabolic syndrome consisting of lipodystropy/lipoatrophy, dyslipidemia, and type 2 diabetes mellitus with an increased cardiovascular risk. The pathogenetic bases of HAART-associated lipodystrophy are poorly known. A genetic screen was used to evaluate proteins that are modulated in HIV-1-infected patients with or without lipodystrophy syndrome, that are routinely treated with HAART regimens. The most significant modulation was represented by FAP48 expression. Stable over-expression of FAP48 was able to alter, in vitro, adipogenesis, acting both on calcineurin and glucocorticoid pathways. Finally, we demonstrated that FAP48 over-expression was able to influence the capacity of some HIV drugs, Saquinavir and Efavirenz, but not Stavudine, Amprenavir, and Indinavir to inhibit adipocyte formation. In conclusion, this molecule could be a potential target for novel therapeutic approaches to the HAART related lipodystrophy in HIV patients.
Highly active antiretroviral therapy (HAART therapy) for HIV-1 infection has significantly increased the survival and quality of life of patients with this disease. However, in several epidemiological studies the onset of metabolic syndrome is a phenomenon reported to be extremely frequent. In the present study, genes involved in the molecular cascade responsible for the alteration of fat tissue and of lipid and glucose metabolism in patients with HIV-1 infection treated with antiretroviral therapy were identified. Towards this goal, hybridization using Atlas cDNA Expression Arrays allowed simultaneous monitoring of the expression levels of approximately 250 genes and identification of a panel of changes in relation to different therapeutic groups and in the presence of metabolic syndrome, with some genes being up-regulated, while others are down-regulated in the different subgroups of patients. The results of this analysis have shown a panel of transcriptional changes associated with oxidative stress mechanisms that provide a basis for further studies on understanding of mechanisms that, in vivo, are the foundation the metabolic disorders in patients with HIV infection.
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