Maria Guitart

Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for &bgr;-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

Plasma PTX3 protein levels inversely correlate with insulin secretion and obesity, whereas visceral adipose tissue PTX3 gene expression is increased in obesity

Plasma acutephase protein pentraxin 3 (PTX3) concentration is dysregulated in human obesity and metabolic syndrome. Here, we explore its relationship with insulin secretion and sensitivity, obesity markers, and adipose tissue PTX3 gene expression. Plasma PTX3 protein levels were analyzed in a cohort composed of 27 lean [body mass index (BMI) ≤ 25 kg/m(2)] and 48 overweight (BMI 25-30 kg/m(2)) men (cohort 1). In this cohort, plasma PTX3 was negatively correlated with fasting triglyceride levels and insulin secretion after intravenous and oral glucose administration. Plasma PTX3 protein and PTX3 gene expression in visceral (VAT) and subcutaneous (SAT) whole adipose tissue and adipocyte and stromovascular fractions were analyzed in cohort 2, which was composed of 19 lean, 28 overweight, and 15 obese subjects (BMI >30 kg/m(2)). An inverse association with body weight and waist/hip ratio was observed in cohort 2. In VAT depots, PTX3 mRNA levels were higher in subjects with BMI >25 kg/m(2) than in lean subjects, positively correlated with IL-1β mRNA levels, and higher in the adipocyte than stromovascular fraction. Human preadipocyte SGBS cell line was used to study PTX3 production in response to factors that obesity entails. In SGBS adipocytes, PTX3 gene expression was enhanced by IL-1β and TNFα but not IL-6 or insulin. In conclusion, the negative correlation between PTX3 and glucose-stimulated insulin secretion suggests a role for PTX3 in metabolic control. PTX3 gene expression is upregulated in VAT depots in obesity, despite lower plasma PTX3 protein, and by some proinflammatory cytokines in cultured adipocytes.

FATP1 localizes to mitochondria and enhances pyruvate dehydrogenase activity in skeletal myotubes.

Fatty acid transport protein 1 (FATP1) has been previously immunolocalized in intracellular compartments. Here we show that FATP1 localizes to the mitochondria in cultured myotubes, by immunoblots of subcellular fractions and immunocytology of the fusion protein FATP1-GFP. FATP1 strongly stimulates CO(2) production from glucose whereas nonmitochondrial metabolism of glucose is only slightly enhanced. FATP1 raises the activity and activates the pyruvate dehydrogenase (PDH) complex and the pyruvate decarboxylase PDH-E1 catalytic subunit, without changing E2, E3BP or E1alpha and increasing E1beta protein content. These data reveals the localization and points to a regulatory function of FATP1 in myotube mitochondria.

Expression and glycogenic effect of glycogen-targeting protein phosphatase 1 regulatory subunit GL in cultured human muscle

Glycogen-targeting PP1 (protein phosphatase 1) subunit G(L) (coded for by the PPP1R3B gene) is expressed in human, but not rodent, skeletal muscle. Its effects on muscle glycogen metabolism are unknown. We show that G(L) mRNA levels in primary cultured human myotubes are similar to those in freshly excised muscle, unlike subunits G(M) (gene PPP1R3A) or PTG (protein targeting to glycogen; gene PPP1R3C), which decrease strikingly. In cultured myotubes, expression of the genes coding for G(L), G(M) and PTG is not regulated by glucose or insulin. Overexpression of G(L) activates myotube GS (glycogen synthase), glycogenesis in glucose-replete and -depleted cells and glycogen accumulation. Compared with overexpressed G(M), G(L) has a more potent activating effect on glycogenesis, while marked enhancement of their combined action is only observed in glucose-replete cells. G(L) does not affect GP (glycogen phosphorylase) activity, while co-overexpression with muscle GP impairs G(L) activation of GS in glucose-replete cells. G(L) enhances long-term glycogenesis additively to glucose depletion and insulin, although G(L) does not change the phosphorylation of GSK3 (GS kinase 3) on Ser9 or its upstream regulator kinase Akt/protein kinase B on Ser473, nor its response to insulin. In conclusion, in cultured human myotubes, the G(L) gene is expressed as in muscle tissue and is unresponsive to glucose or insulin, as are G(M) and PTG genes. G(L) activates GS regardless of glucose, does not regulate GP and stimulates glycogenesis in combination with insulin and glucose depletion.

Differential pattern of glycogen accumulation after protein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression, compared to PPP1R3C and PPP1R3A, in skeletal muscle cells

BackgroundPPP1R6 is a protein phosphatase 1 glycogen-targeting subunit (PP1-GTS) abundant in skeletal muscle with an undefined metabolic control role. Here PPP1R6 effects on myotube glycogen metabolism, particle size and subcellular distribution are examined and compared with PPP1R3C/PTG and PPP1R3A/GM.ResultsPPP1R6 overexpression activates glycogen synthase (GS), reduces its phosphorylation at Ser-641/0 and increases the extracted and cytochemically-stained glycogen content, less than PTG but more than GM. PPP1R6 does not change glycogen phosphorylase activity. All tested PP1-GTS-cells have more glycogen particles than controls as found by electron microscopy of myotube sections. Glycogen particle size is distributed for all cell-types in a continuous range, but PPP1R6 forms smaller particles (mean diameter 14.4 nm) than PTG (36.9 nm) and GM (28.3 nm) or those in control cells (29.2 nm). Both PPP1R6- and GM-derived glycogen particles are in cytosol associated with cellular structures; PTG-derived glycogen is found in membrane- and organelle-devoid cytosolic glycogen-rich areas; and glycogen particles are dispersed in the cytosol in control cells. A tagged PPP1R6 protein at the C-terminus with EGFP shows a diffuse cytosol pattern in glucose-replete and -depleted cells and a punctuate pattern surrounding the nucleus in glucose-depleted cells, which colocates with RFP tagged with the Golgi targeting domain of β-1,4-galactosyltransferase, according to a computational prediction for PPP1R6 Golgi location.ConclusionsPPP1R6 exerts a powerful glycogenic effect in cultured muscle cells, more than GM and less than PTG. PPP1R6 protein translocates from a Golgi to cytosolic location in response to glucose. The molecular size and subcellular location of myotube glycogen particles is determined by the PPP1R6, PTG and GM scaffolding.

Design and Implementation of a Microelectrode Assembly for Use on Noncontact In Situ Electroporation of Adherent Cells

In situ electroporation of adherent cells provides significant advantages with respect to electroporation systems for suspension cells, such as causing minimal stress to cultured cells and simplifying and saving several steps within the process. In this study, a new electrode assembly design is shown and applied to in situ electroporate adherent cell lines growing in standard multiwell plates. We designed an interdigitated array of electrodes patterned on copper with printed circuit board technology and covered with nickel/gold. Small interelectrode distances were used to achieve effective electroporation with low voltages. Epoxy-based microseparators were constructed to avoid direct contact with the cells and to create more uniform electric fields. The device was successful in the electropermeabilization of two different adherent cell lines, C2C12 and HEK 293, as assessed by the intracellular delivery of the fluorescent dextran FD20S. Additionally, as a collateral effect, we observed cell electrofusion in HEK 293 cells, thus making this device also useful for performing cell fusion. In summary, we show the effectiveness of this minimally invasive device for electroporation of adherent cells cultured in standard multiwell plates. The cheap technologies used in the fabrication process of the electrode assembly indicate potential use as a low-cost, disposable device.

Fatty Acid Transport Protein 1 (FATP1) Localizes in Mitochondria in Mouse Skeletal Muscle and Regulates Lipid and Ketone Body Disposal

FATP1 mediates skeletal muscle cell fatty acid import, yet its intracellular localization and metabolic control role are not completely defined. Here, we examine FATP1 localization and metabolic effects of its overexpression in mouse skeletal muscle. The FATP1 protein was detected in mitochondrial and plasma membrane fractions, obtained by differential centrifugation, of mouse gastrocnemius muscle. FATP1 was most abundant in purified mitochondria, and in the outer membrane and soluble intermembrane, but not in the inner membrane plus matrix, enriched subfractions of purified mitochondria. Immunogold electron microscopy localized FATP1-GFP in mitochondria of transfected C2C12 myotubes. FATP1 was overexpressed in gastrocnemius mouse muscle, by adenovirus-mediated delivery of the gene into hindlimb muscles of newborn mice, fed after weaning a chow or high-fat diet. Compared to GFP delivery, FATP1 did not alter body weight, serum fed glucose, insulin and triglyceride levels, and whole-body glucose tolerance, in either diet. However, fatty acid levels were lower and β-hydroxybutyrate levels were higher in FATP1- than GFP-mice, irrespective of diet. Moreover, intramuscular triglyceride content was lower in FATP1- versus GFP-mice regardless of diet, and β-hydroxybutyrate content was unchanged in high-fat-fed mice. Electroporation-mediated FATP1 overexpression enhanced palmitate oxidation to CO2, but not to acid-soluble intermediate metabolites, while CO2 production from β-hydroxybutyrate was inhibited and that from glucose unchanged, in isolated mouse gastrocnemius strips. In summary, FATP1 was localized in mitochondria, in the outer membrane and intermembrane parts, of mouse skeletal muscle, what may be crucial for its metabolic effects. Overexpressed FATP1 enhanced disposal of both systemic fatty acids and intramuscular triglycerides. Consistently, it did not contribute to the high-fat diet-induced metabolic dysregulation. However, FATP1 lead to hyperketonemia, likely secondary to the sparing of ketone body oxidation by the enhanced oxidation of fatty acids.

A new spiral microelectrode assembly for electroporation and impedance measurements of adherent cell monolayers

In this study, a new microelectrode assembly based on spiral geometry applicable to in situ electroporation of adherent cell monolayers on standard multiwell plates is presented. Furthermore, the structure is specially conceived to perform electrical impedance spectroscopy (EIS) measurements during electroporation. Its performance for cell membrane permeabilization is tested with a fluorescent probe. Gene electrotransfer is also assayed using a plasmid DNA encoding GFP in four different cell lines (CHO, HEK293, 3T3-L1 and FTO2B). Additionally, siRNA α-GFP electrotransfection is tested in GFP gene-expressing CHO cells. Our data show considerable differences between permeabilization and gene transfer results and cell line dependence on gene expression rates. Successful siRNA electro-mediated delivery is also achieved. We demonstrate the applicability of our device for electroporation-mediated gene transfer of adherent cells in standard laboratory conditions. Finally, electrical impedance measurements during electroporation of CHO and 3T3-L1 cells are also given.

Automatic system for electroporation of adherent cells growing in standard multi-well plates

In this study an automatic system is presented to perform electroporation, also known as electropermeabilization, on adherent cells. It is an intention of this system to apply electric field pulses directly to cells growing in standard multi-well plates as a step forward to include this technique in standard laboratory protocols. An interdigitated microelectrode assembly constructed with Printed Circuit Board (PCB) is placed closely above the cell monolayer, and in order to avoid direct contact with cells, small micro-separators were included in the structure. Additionally, distribution of current density was modified by filling the gap between adjacent electrodes with a non conductive material as predicted by electric field simulations. This modification helps to concentrate the electric field intensity in the region where cells are present. The device was tested using C2C12 cell line growing adhered in 24 multi-well plates and fluorescent labeled dextran FD20S as the molecule to be delivered. Successful transfection was observed with minimal invasiveness of the operation reducing the stress caused to cells.

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

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.