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Abstract Beige fat activation involves a fuel switch to fatty acid oxidation following chronic cold adaptation. Mitochondrial acyl-CoA synthetase long-chain family member 1 (ACSL1) localizes in the mitochondria and plays a key role in fatty acid oxidation; however, the regulatory mechanism of the subcellular localization remains poorly understood. Here, we identify an endosomal trafficking component sortilin (encoded by Sort1) in adipose tissues that shows dynamic expression during beige fat activation and facilitates the translocation of ACSL1 from the mitochondria to the endolysosomal pathway for degradation. Depletion of sortilin in adipocytes results in an increase of mitochondrial ACSL1 and the activation of AMPK/PGC1α signaling, thereby activating beige fat and preventing high-fat diet (HFD)-induced obesity and insulin resistance. Collectively, our findings indicate that sortilin controls adipose tissue fatty acid oxidation by substrate fuel selection during beige fat activation and provides a potential targeted approach for the treatment of metabolic diseases.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2 x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by the Hoechst signal across the nucleus. We show a dose-dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

Brown and beige adipose tissues play a large role in non-shivering thermogenesis (NST) in mammals, and subsequently have been studied for decades as potential therapeutic targets to treat obesity and its related metabolic diseases. However, the mechanistic regulation of brown/beige adipose tissue induction and maintenance in humans is very limited due to the ethical reasons. In fact, metabolic signaling has primarily been investigated using rodent models. A better understanding of non-shivering thermogenesis in humans is thus vital and urgent in order to treat obesity by targeting human brown adipose tissue (BAT). In this review, we summarize the anatomical and physiological differences between rodent and human BAT, current useful and mostly non-invasive methods in studying human BAT, as well as recent advancements targeting thermogenic adipocytes as a means to combat metabolic diseases in humans. Furthermore, we also discuss several novel relevant strategies of therapeutic interventions, which has been attempted in rodent experiments, and possible future investigations in humans in this field.

Brown fat and beige fat are known as thermogenic fat due to their contribution to non-shivering thermogenesis in mammals following cold stimulation. Beige fat is unique due to its origin and its development in white fat. Subsequently, both brown fat and beige fat have become viable targets to combat obesity. Over the last few decades, most therapeutic strategies have been focused on the canonical pathway of thermogenic fat activation via the β3-adrenergic receptor (AR). Notwithstanding, administering β3-AR agonists often leads to side effects including hypertension and particularly cardiovascular disease. It is thus imperative to search for alternative therapeutic approaches to combat obesity. In this review, we discuss the current challenges in the field with respect to stimulating brown/beige fat thermogenesis. Additionally, we include a summary of other newly discovered pathways, including non-AR signaling- and non-UCP1-dependent mechanisms, which could be potential targets for the treatment of obesity and its related metabolic diseases.

The article presents case study of a 38-year-old male suffering from metastatic Crohn's disease (MCD). It discusses treatment of MCD by several methods including medical therapy with Salazapyrine, Glucocorticosteroids, and immunosuppressing agents such asMethotrexate or Azathioprine. It informs that the affected place's biopsy has also been done and the disease has mainly affected the gastrointestinal tract and made a inflammation.

Renal cell carcinomas (RCCs) are common cancers diagnosed in more than 350,000 people each year worldwide. Several pathways are de-regulated in RCCs, including mTORC1. However, how mTOR drives tumorigenesis in this context is unknown. The lack of faithful animal models has limited progress in understanding and targeting RCCs. Here, we generated a mouse model harboring the kidney-specific inactivation of Tsc1. These animals develop cysts that evolve into papillae, cystadenomas, and papillary carcinomas. Global profiling confirmed several metabolic derangements previously attributed to mTORC1. Notably, Tsc1 inactivation results in the accumulation of fumarate and in mTOR-dependent downregulation of the TCA cycle enzyme fumarate hydratase (FH). The re-expression of FH in cellular systems lacking Tsc1 partially rescued renal epithelial transformation. Importantly, the mTORC1-FH axis is likely conserved in human RCC specimens. We reveal a role of mTORC1 in renal tumorigenesis, which depends on the oncometabolite fumarate., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients., Methods: The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting., Results: The activation of A 3 AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1 flox/- : Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost., Conclusion: The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.

Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34 + cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease., (Copyright © 2016 by the American Society of Nephrology.)

Previous studies report a cross-talk between the polycystic kidney disease (PKD) and tuberous sclerosis complex (TSC) genes. mTOR signalling is upregulated in PKD and rapamycin slows cyst expansion, whereas renal inactivation of the Tsc genes causes cysts. Here we identify a new interplay between the PKD and TSC genes, with important implications for the pathophysiology of both diseases. Kidney-specific inactivation of either Pkd1 or Tsc1 using an identical Cre (KspCre) results in aggressive or very mild PKD, respectively. Unexpectedly, we find that mTORC1 negatively regulates the biogenesis of polycystin-1 (PC-1) and trafficking of the PC-1/2 complex to cilia. Genetic interaction studies reveal an important role for PC-1 downregulation by mTORC1 in the cystogenesis of Tsc1 mutants. Our data potentially explain the severe renal manifestations of the TSC/PKD contiguous gene syndrome and open new perspectives for the use of mTOR inhibitors in autosomal dominant PKD caused by hypomorphic or missense PKD1 mutations.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by bilateral renal cyst formation. Recent identification of signaling cascades deregulated in ADPKD has led to the initiation of several clinical trials, but an approved therapy is still lacking. Using a metabolomic approach, we identify a pathogenic pathway in this disease that can be safely targeted for therapy. We show that mutation of PKD1 results in enhanced glycolysis in cells in a mouse model of PKD and in kidneys from humans with ADPKD. Glucose deprivation resulted in lower proliferation and higher apoptotic rates in PKD1-mutant cells than in nondeprived cells. Notably, two distinct PKD mouse models treated with 2-deoxyglucose (2DG), to inhibit glycolysis, had lower kidney weight, volume, cystic index and proliferation rates as compared to nontreated mice. These metabolic alterations depend on the extracellular signal-related kinase (ERK) pathway acting in a dual manner by inhibiting the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) axis on the one hand while activating the mTOR complex 1 (mTORC1)-glycolytic cascade on the other. Enhanced metabolic rates further inhibit AMPK. Forced activation of AMPK acts in a negative feedback loop, restoring normal ERK activity. Taken together, these data indicate that defective glucose metabolism is intimately involved in the pathobiology of ADPKD. Our findings provide a strong rationale for a new therapeutic strategy using existing drugs, either individually or in combination.

Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.

Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (approximately 520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.