Differential effects of high fat diet-induced obesity on oocyte mitochondrial functions in inbred and outbred mice.

Maternal obesity can cause reduced oocyte quality and subfertility. Mitochondrial dysfunction plays a central role here, and most often inbred mouse models are used to study these pathways. We hypothesized that the mouse genetic background can influence the impact of high fat diet (HFD)-induced obesity on oocyte quality. We compared the inbred C57BL/6 (B6) and the outbred Swiss strains after feeding a HFD for 13w. HFD-mice had increased body weight gain, hypercholesterolemia, and increased oocyte lipid droplet (LD) accumulation in both strains. LD distribution was strain-dependent. In Swiss mouse oocytes, HFD significantly increased mitochondrial inner membrane potential (MMP), reactive oxygen species concentrations, mitochondrial ultrastructural abnormalities (by 46.4%), and endoplasmic reticulum (ER) swelling, and decreased mtDNA copy numbers compared with Swiss controls (P < 0.05). Surprisingly, B6-control oocytes exhibited signs of cellular stress compared to the Swiss controls (P < 0.05); upregulated gene expression of ER- and oxidative stress markers, high mitochondrial ultrastructural abnormalities (48.6%) and ER swelling. Consequently, the HFD impact on B6 oocyte quality was less obvious, with 9% higher mitochondrial abnormalities, and no additive effect on MMP and stress marks compared to B6 control (P > 0.1). Interestingly, mtDNA in B6-HFD oocytes was increased suggesting defective mitophagy. In conclusion, we show evidence that the genetic background or inbreeding can affect mitochondrial functions in oocytes and may influence the impact of HFD on oocyte quality. These results should create awareness when choosing and interpreting data obtained from different mouse models before extrapolating to human applications.

NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest.

BACKGROUND: NBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved. METHODS: Expression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21(CIP1/WAF1) were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool. RESULTS: We show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis. CONCLUSIONS: We demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins.

Macrophage reprogramming by mycolic acid promotes a tolerogenic response in experimental asthma.

RATIONALE: Mycolic acid (MA) constitutes a major and distinguishing cell wall biolipid from Mycobacterium tuberculosis. MA interferes with the lipid homeostasis of alveolar macrophages, inducing differentiation into foamy macrophages exhibiting increased proinflammatory function. OBJECTIVES: We verified the interference of this altered macrophage function with inhaled antigen-triggered allergic airway inflammation and underlying Th2 lymphocyte reactivity. METHODS: Using ovalbumin (OVA) as model allergen, C57BL/6 or BALB/C mice were sensitized by OVA-alum immunization. Experimental asthma, triggered subsequently by repetitive nebulized OVA inhalation, was assessed, using as readout parameters eosinophilia, peribronchial inflammation, and Th2 cytokine function. MEASUREMENTS AND MAIN RESULTS: A single intratracheal treatment of sensitized mice with MA, inserted into liposomes as carriers, prevented the onset of OVA-triggered allergic airway inflammation and promoted unresponsiveness to a secondary set of allergen exposures. The development of this tolerant condition required an 8-d lapse after MA instillation, coinciding with the appearance of foamy alveolar macrophages. MA-conditioned CD11b(+)F4/80(+) macrophages, transferred to the airways, mimicked the tolerogenic function of instilled MA; however, without the 8-d lapse requirement. Indicative of a macrophage-mediated tolerogenic antigen-presenting function, major histocompatibility complex (MHC)-mismatched donor macrophages failed to promote tolerance. Furthermore, Treg markers were strongly increased and established tolerance was lost after in situ depletion of CD25(+) Treg cells. Contrary to the interleukin-10 dependence of tolerogenic dendritic cells, IFN-gamma deficiency but not interleukin-10 deficiency abrogated the tolerogenic capacity of MA-conditioned macrophages. CONCLUSIONS: These results document an innate-driven Mycobacterium tuberculosis MA-triggered immune regulatory mechanism in control of pulmonary allergic responses by converting macrophages into IFN-gamma-dependent tolerogenic antigen-presenting cells.

Blocking tumor cell eicosanoid synthesis by GP x 4 impedes tumor growth and malignancy.

Using tumor cell-restricted overexpression of glutathione peroxidase 4 (GP x 4), we investigated the contribution of tumor cell eicosanoids to solid tumor growth and malignant progression in two tumor models differing in tumorigenic potential. By lowering cellular lipid hydroperoxide levels, GP x 4 inhibits cyclooxygenase (COX) and lipoxygenase (LOX) activities. GP x 4 overexpression drastically impeded solid tumor growth of weakly tumorigenic L929 fibrosarcoma cells, whereas B16BL6 melanoma solid tumor growth was unaffected. Yet, GP x 4 overexpression did markedly increase the sensitivity of B16BL6 tumors to angio-destructive TNF-alpha therapy and abolished the metastatic lung colonizing capacity of B16BL6 cells. Furthermore, the GP x 4-mediated suppression of tumor cell prostaglandin E(2) (PGE(2)) production impeded the induction of COX-2 expression by the tumor stress conditions hypoxia and inflammation. Thus, our results reflect a PGE(2)-driven positive feedback loop for COX-2 expression in tumor cells. This was further supported by the restoration of COX-2 induction capacity of GP x 4-overexpressing L929 tumor cells when cultured in the presence of exogenous PGE(2). Thus, although COX-2 expression and eicosanoid production may be enabled by PGE(2) from the tumor microenvironment, our results demonstrate the predominant tumor cell origin of protumoral eicosanoids, promoting solid tumor growth of weakly tumorigenic tumors and malignant progression of strongly tumorigenic tumors.