Mariangela Melchiorre

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.

Exendin-4 Prevents c-Jun N-Terminal Protein Kinase Activation by Tumor Necrosis Factor-α (TNFα) and Inhibits TNFα-Induced Apoptosis in Insulin-Secreting Cells

Glucagon-like peptide-1 and its analogs may preserve pancreatic beta-cell mass by promoting resistance to cytokine-mediated apoptosis. The mechanisms of TNFalpha-induced apoptosis and of its inhibition by exendin-4 were investigated in insulin-secreting cells. INS-1 and MIN6 insulinoma cells were exposed to 20 ng/ml TNFalpha, with or without pretreatment with 10 nm exendin-4. Treatment with TNFalpha increased c-Jun N-terminal protein kinase (JNK) phosphorylation 2-fold, reduced inhibitor-kappaBalpha (IkappaBalpha) protein content by 50%, induced opposite changes in caspase-3 and Bcl-2 protein content, and increased cellular apoptosis. Moreover, exposure to TNFalpha resulted in increased serine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-2 and reduced basal and insulin-induced Akt phosphorylation. However, in the presence of a JNK inhibitor, TNFalpha-induced apoptosis was diminished and serine phosphorylation of IRS proteins was prevented. When cells were pretreated with exendin-4, TNFalpha-induced JNK and IRS-1/2 serine phosphorylation was markedly reduced, Akt phosphorylation was increased, caspase-3 and Bcl-2 protein levels were restored to normal, and TNFalpha-induced apoptosis was inhibited by 50%. This was associated with a 2-fold increase in IRS-2 expression levels. A similar ability of exendin-4 to prevent TNFalpha-induced JNK phosphorylation was found in isolated pancreatic human islets. The inhibitory effect of exendin-4 on TNFalpha-induced JNK phosphorylation was abrogated in the presence of the protein kinase A inhibitor H89. In conclusion, JNK activation mediates TNFalpha-induced apoptosis and impairment of the IRS/Akt signaling pathway in insulin-secreting cells. By inhibiting JNK phosphorylation in a PKA-dependent manner, exendin-4 counteracts TNFalpha-mediated apoptosis and reverses the inhibitory events in the IRS/Akt pathway, resulting in promotion of cell survival.

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.

Glucagon-Like Peptide-1 Counteracts Oxidative Stress-Dependent Apoptosis of Human Cardiac Progenitor Cells by Inhibiting the Activation of the c-Jun N-terminal Protein Kinase Signaling Pathway

Increased apoptosis of cardiac progenitor cells (CPCs) has been proposed as a mechanism of myocardial damage and dysfunction. Glucagon-like peptide-1 (GLP-1) has been shown to improve heart recovery and function after ischemia and to promote cell survival. The protective effects of GLP-1 on oxidative stress-induced apoptosis were investigated in human CPCs isolated from human heart biopsies. Mesenchymal-type cells were isolated from human heart biopsies, exhibited the marker profile of CPCs, differentiated toward the myocardiocyte, adipocyte, chondrocyte, and osteocyte lineages under appropriate culture conditions, and expressed functional GLP-1 receptors. CPCs were incubated with GLP-1 with or without hydrogen peroxide (H(2)O(2)). Phospho- and total proteins were detected by immunoblotting and immunofluorescence analysis. Gene expression was evaluated by quantitative RT-PCR. The role of the canonical GLP-1 receptor was assessed by using the receptor antagonist exendin(9-39) and receptor-specific silencer small interfering RNAs. Cell apoptosis was quantified by an ELISA assay and by flow cytometry-detected Annexin V. Exposure of CPCs to H(2)O(2) induced a 2-fold increase in cell apoptosis, mediated by activation of the c-Jun N-terminal protein kinase (JNK) pathway. Preincubation of CPCs with GLP-1 avoided H(2)O(2)-triggered JNK phosphorylation and nuclear localization, and protected CPCs from apoptosis. The GLP-1 effects were markedly reduced by coincubation with the receptor antagonist exendin(9-39), small interfering RNA-mediated silencing of the GLP-1 receptor, and pretreatment with the protein kinase A inhibitor H89. In conclusion, activation of GLP-1 receptors prevents oxidative stress-mediated apoptosis in human CPCs by interfering with JNK activation and may represent an important mechanism for the cardioprotective effects of GLP-1.

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.

Role of UBC9 in the Regulation of the Adipogenic Program in 3T3-L1 Adipocytes

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2-6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.