Cristina Caccioppoli

Fat depot-related differences in gene expression, adiponectin secretion, and insulin action and signalling in human adipocytes differentiated in vitro from precursor stromal cells

Aim/hypothesisThe distinct metabolic properties of visceral and subcutaneous adipocytes may be due to inherent characteristics of the cells that are resident in each fat depot. To test this hypothesis, human adipocytes were differentiated in vitro from precursor stromal cells obtained from visceral and subcutaneous fat depots and analysed for genetic, biochemical and metabolic endpoints.MethodsStromal cells were isolated from adipose tissue depots of nondiabetic individuals. mRNA levels of adipocyte-specific proteins were determined by real-time RT-PCR. Insulin signalling was evaluated by immunoblotting with specific antibodies. Glucose transport was measured by a 2-deoxy-glucose uptake assay. Adiponectin secretion in the adipocyte-conditioned medium was determined by a specific RIA.ResultsWith cell differentiation, mRNA levels of PPARG, C/EBPα (also known as CEBPA), AP2 (also known as GTF3A), GLUT4 (also known as SLC2A4) were markedly upregulated, whereas GLUT1 (also known as SLC2A1) mRNA did not change. However, expression of C/EBPα, AP2 and adiponectin was higher in subcutaneous than in visceral adipocytes. By contrast, adiponectin was secreted at threefold higher rates by visceral than by subcutaneous adipocytes while visceral adipocytes also showed two- to threefold higher insulin-stimulated glucose uptake. Insulin-induced phosphorylation of the insulin receptor, IRS proteins, Akt and extracellular signal-regulated kinase-1/2 was more rapid and tended to decrease at earlier time-points in visceral than in subcutaneous adipocytes.Conclusions/interpretationSubcutaneous and visceral adipocytes, also when differentiated in vitro from precursor stromal cells, retain differences in gene expression, adiponectin secretion, and insulin action and signalling. Thus, the precursor cells that reside in the visceral and subcutaneous fat depots may already possess inherent and specific metabolic characteristics that will be expressed upon completion of the differentiation programme.

Involvement of the p66Shc protein in glucose transport regulation in skeletal muscle myoblasts.

The p66(Shc) protein isoform regulates MAP kinase activity and the actin cytoskeleton turnover, which are both required for normal glucose transport responses. To investigate the role of p66(Shc) in glucose transport regulation in skeletal muscle cells, L6 myoblasts with antisense-mediated reduction (L6/p66(Shc)as) or adenovirus-mediated overexpression (L6/p66(Shc)adv) of the p66(Shc) protein were examined. L6/(Shc)as myoblasts showed constitutive activation of ERK-1/2 and disruption of the actin network, associated with an 11-fold increase in basal glucose transport. GLUT1 and GLUT3 transporter proteins were sevenfold and fourfold more abundant, respectively, and were localized throughout the cytoplasm. Conversely, in L6 myoblasts overexpressing p66(Shc), basal glucose uptake rates were reduced by 30% in parallel with a approximately 50% reduction in total GLUT1 and GLUT3 transporter levels. Inhibition of the increased ERK-1/2 activity with PD98059 in L6/(Shc)as cells had a minimal effect on increased GLUT1 and GLUT3 protein levels, but restored the actin cytoskeleton, and reduced the abnormally high basal glucose uptake by 70%. In conclusion, p66(Shc) appears to regulate the glucose transport system in skeletal muscle myoblasts by controlling, via MAP kinase, the integrity of the actin cytoskeleton and by modulating cellular expression of GLUT1 and GLUT3 transporter proteins via ERK-independent pathways.

TNFα Signals via p66Shc to Induce E-Selectin, Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66Shc on Ser36 and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66Shc in HUVEC resulted in enhanced p66Shc phosphorylation on Ser36, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66Shc protein, in which Ser36 was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66Shc on Ser36.

GLP-1 Receptor Activation Inhibits Palmitate-Induced Apoptosis via Ceramide in Human Cardiac Progenitor Cells

Context: Increased apoptosis of cardiomyocytes and cardiac progenitor cells (CPCs) in response to saturated fatty acids (SFAs) can lead to myocardial damage and dysfunction. Ceramides mediate lipotoxicity‐induced apoptosis. Glucagonlike peptide‐1 receptor (GLP1R) agonists exert beneficial effects on cardiac cells in experimental models. Objective: To investigate the protective effects of GLP1R activation on SFA‐mediated apoptotic death of human CPCs. Design: Human CPCs were isolated from cardiac appendages of nondiabetic donors and then exposed to palmitate with or without pretreatment with the GLP1R agonist exendin‐4. Ceramide accumulation was evaluated by immunofluorescence. Expression of key enzymes in de novo ceramide biosynthesis was studied by quantitative reverse‐transcription polymerase chain reaction and immunoblotting. Apoptosis was evaluated by measuring release of oligonucleosomes, caspase‐3 cleavage, caspase activity, and terminal deoxynucleotidyltransferase‐mediated dUTP nick end labeling. Results: Exposure of the CPCs to palmitate resulted in 2.3‐ and 1.9‐fold higher expression of ceramide synthase 5 (CERS5) and ceramide desaturase‐1, respectively (P < 0.05). This was associated with intracellular accumulation of ceramide and activation of c‐Jun NH2‐terminal protein kinase (JNK) signaling and apoptosis (P < 0.05). Both coincubation with fumonisin B1, a specific ceramide synthase inhibitor, and CERS5 knockdown prevented ceramide accumulation, JNK activation, and apoptosis in response to palmitate (P < 0.05). Exendin‐4 also prevented the activation of the ceramide biosynthesis and JNK in response to palmitate, inhibiting apoptosis (P < 0.05). Conclusions: Excess palmitate results in activation of ceramide biosynthesis, JNK signaling, and apoptosis in human CPCs. GLP1R activation counteracts this lipotoxic damage via inhibition of ceramide generation, and this may represent a cardioprotective mechanism.