Role of the human concentrative nucleoside transporter (hCNT1) in the cytotoxic action of 5[Prime]-deoxy-5-fluorouridine, an active intermediate metabolite of capecitabine, a novel oral anticancer drug.

We attempt to identify the plasma membrane transporter involved in the uptake of 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5'-fluorouracil. We also examine the role of the transporter in 5'-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5'-DFUR, but not capecitabine or 5'-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5'-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5'-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a nucleoside transporter can be determined by an electrophysiological approach; 2) the hCNT1 transporter is involved in 5'-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5'-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatmen

Succinate Pathway in Head and Neck Squamous Cell Carcinoma: Potential as a Diagnostic and Prognostic Marker

Simple Summary: Emerging evidence points to succinate as an important oncometabolite in cancer development; however, the contribution of the succinate-SUCNR1 axis to cancer progression remains unclear. Head and neck squamous cell carcinoma (HNSCC) is associated with disease and treatmentrelated morbidity so there is an urgent need for innovation in treatment and diagnosis practices. Our aim was to evaluate the potential of the succinate-related pathway as a diagnostic and prognostic biomarker in HNSCC. The circulating succinate levels are increased in HNSCC, being a potential noninvasive biomarker for HNSCC diagnosis. Moreover, the succinate receptor (SUCNR1) and genes related to succinate metabolism, which are predominantly expressed in the tumoral mucosa as compared with healthy tissue, are positively associated with plasma succinate. Remarkably, we found that SUCNR1 and SDHA expression levels predict prognosis

Deficient endoplasmic reticulum-mitochondrial phosphatidylserine transfer causes liver disease

Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with nonalcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease

Adipose Tissue and Serum CCDC80 in Obesity and Its Association with Related Metabolic Disease

Abstract Coiled-coil domain-containing 80 (CCDC80) is an adipocyte-secreted protein that modulates glucose homeostasis in response to diet-induced obesity in mice. The objective of this study was to analyze the link between human CCDC80 and obesity. CCDC80 protein expression was assessed in paired visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) from 10 patients (body mass index range 22.4–38.8 kg/m2). Circulating CCDC80 levels were quantified in serum samples from two independent cross-sectional cohorts comprising 33 lean and 15 obese (cohort 1) and 32 morbidly obese (cohort 2) male patients. Insulin sensitivity, insulin secretion and blood neutrophil count were quantified in serum samples from both cohorts. Additionally, circulating free insulin-like growth factor (IGF)-1 levels and oral glucose tolerance tests were assessed in cohort 1, whereas C-reactive protein levels and degree of atherosclerosis and hepatic steatosis were studied in cohort 2. In lean patients, total CCDC80 protein content assessed by immunoblotting was lower in VAT than in SAT. In obese patients, CCDC80 was increased in VAT (P < 0.05) but equivalent in SAT compared with lean counterparts. In cohort 1, serum CCDC80 correlated negatively with the acute insulin response to glucose and IGF-1 levels and positively with blood neutrophil count independent of BMI, but not with insulin sensitivity. In cohort 2, serum CCDC80 was positively linked to the inflammatory biomarker C-reactive protein (r = 0.46; P = 0.009), atherosclerosis (carotid intima-media thickness, r = 0.62; P < 0.001) and hepatic steatosis (analysis of variance P = 0.025). Overall, these results suggest for the first time that CCDC80 may be a component of the obesity-altered secretome in VAT and could act as an adipokine whose circulant levels are linked to glucose tolerance derangements and related to inflammation-associated chronic complications

Role of the human concentrative nucleoside transporter (hCNT1) in the cytotoxic action of 5[Prime]-deoxy-5-fluorouridine, an active intermediate metabolite of capecitabine, a novel oral anticancer drug.

We attempt to identify the plasma membrane transporter involved in the uptake of 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5'-fluorouracil. We also examine the role of the transporter in 5'-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5'-DFUR, but not capecitabine or 5'-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5'-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5'-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a nucleoside transporter can be determined by an electrophysiological approach; 2) the hCNT1 transporter is involved in 5'-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5'-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatmen

G protein-coupled receptor kinase 2 plays a relevant role in insulin resistance and obesity

OBJECTIVE: Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein-coupled receptor kinase 2 (GRK2), first identified as a G protein-coupled receptor regulator, could have as a modulator of insulin responses. RESEARCH DESIGN AND METHODS: We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed. RESULTS: Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity. CONCLUSIONS: Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders

Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells

CNT2 (concentrative nucleoside cotransporter) is a plasma membrane high-affinity Na(+)-coupled adenosine transporter, also localized in intracellular structures. This transporter protein may play additional roles other than nucleoside salvage, since it has recently been shown to be under purinergic control via K(ATP) channels, by a mechanism that does not seem to involve changes in its subcellular localization. In an attempt to identify the agents that promote CNT2 trafficking, bile acids were found to increase CNT2-related transport activity in a K(ATP) channel-independent manner in both Fao hepatoma and rat liver parenchymal cells. A maximum effect was recorded after treatment with hydrophilic anions such as TCA (taurocholate). However, this effect did not involve changes in the amount of CNT2 protein, it was instead associated with a subcellular redistribution of CNT2, resulting in an accumulation of the transporter at the plasma membrane. This was deduced from subcellular fractionation studies, biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin precipitates and in vivo confocal microscopic analysis of the distribution of a YFP (yellow fluorescent protein)–CNT2 construct. The induction of CNT2 translocation, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059 and colchicine, thus suggesting the involvement of the PI3K/ERK (phosphoinositide 3-kinase/extracellular-signal related kinase) pathway in microtubule-dependent activation of recombinant CNT2. These are novel effects of bile-acid physiology and provide the first evidence for short-term regulation of CNT2 translocation into and from the plasma membrane

Effects of glucagon-like peptide-1 on the differentiation and metabolism of human adipocytes.

Glucagon-like peptide-1 (GLP-1) analogues improve glycaemic control in type 2 diabetic (T2D) patients and cause weight loss in obese subjects by as yet unknown mechanisms. We recently demonstrated that the GLP-1 receptor, which is present in adipocytes and the stromal vascular fraction of human adipose tissue (AT), is up-regulated in AT of insulin-resistant morbidly obese subjects compared with healthy lean subjects. The aim of this study was to explore the effects of in vitro and in vivo administration of GLP-1 and its analogues on AT and adipocyte functions from T2D morbidly obese subjects. We analysed the effects of GLP-1 on human AT and isolated adipocytes in vitro and the effects of GLP-1 mimetics on AT of morbidly obese T2D subjects in vivo. GLP-1 down-regulated the expression of lipogenic genes when administered during in vitro differentiation of human adipocytes from morbidly obese patients. GLP-1 also decreased the expression of adipogenic/lipogenic genes in AT explants and mature adipocytes, while increasing that of lipolytic markers and adiponectin. In 3T3-L1 adipocytes, GLP-1 decreased free cytosolic Ca2+ concentration ([Ca2+]i). GLP-1-induced responses were only partially blocked by GLP-1 receptor antagonist exendin (9–39). Moreover, administration of exenatide or liraglutide reduced adipogenic and inflammatory marker mRNA in AT of T2D obese subjects. Our data suggest that the beneficial effects of GLP-1 are associated with changes in the adipogenic potential and ability of AT to expand, via activation of the canonical GLP-1 receptor and an additional, as yet unknown, receptor