Paloma Navarro

Brown adipose tissue–specific insulin receptor knockout shows diabetic phenotype without insulin resistance

Although insulin regulates metabolism in both brown and white adipocytes, the role of these tissues in energy storage and utilization is quite different. Recombination technology using the Cre-loxP approach allows inactivation of the insulin receptor in a tissue-specific manner. Mice lacking insulin receptors in brown adipocytes show an age-dependent loss of interscapular brown fat but increased expression of uncoupling protein-1 and -2. In parallel, these mice develop an insulin-secretion defect resulting in a progressive glucose intolerance, without insulin resistance. This model provides direct evidence for not only a role for the insulin receptors in brown fat adipogenesis, the data also suggest a novel role of brown adipose tissue in the regulation of insulin secretion and glucose homeostasis.

The opposing roles of the Akt and c-Myc signalling pathways in survival from CD95-mediated apoptosis

Expression of the proto-oncogene c-myc stimulates cell proliferation in the presence of the appropriate survival factors and triggers apoptosis in their absence; this dual capacity ensures that cell growth is restricted to the correct paracrine environment and is thereby strictly controlled. Recently our laboratory demonstrated that c-Myc-induced apoptosis requires the CD95 death receptor pathway and that insulin-like growth factor (IGF-1) signalling suppresses this killing. To investigate further the links between c-Myc and IGF-1 pathways in CD95-induced apoptosis, we examined the effects of c-Myc and a downstream IGF-1 survival kinase, Akt, on killing mediated by CD95 and its recruited effector proteins (FADD and caspase-8). Here, we show that c-Myc activation does not exacerbate killing induced by FADD or pro-caspase-8, which narrows the point at which c-Myc exerts its action downstream of the interaction of CD95 with its ligand and upstream of FADD. We show further that activated Akt suppresses CD95-induced apoptosis and that Akt exerts its activity at a point downstream of FADD but upstream of caspase-8. These results restrict the possible mechanisms by which CD95-induced apoptosis is modulated by death signals and survival factors.

Tumor Necrosis Factor-α Causes Insulin Receptor Substrate-2-Mediated Insulin Resistance and Inhibits Insulin-Induced Adipogenesis in Fetal Brown Adipocytes1

Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-alpha for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-alpha. The antiadipogenic effect of TNF-alpha was accompanied by a down-regulation of CCAAT/enhancer-binding protein-alpha and beta mRNAs and up-regulation of CCAAT/enhancer-binding protein-delta, with the expression of peroxisome proliferator-activated receptor-gamma remaining essentially unmodified. Moreover, TNF-alpha caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-alpha resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-alpha pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of alpha-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-alpha induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.

Inhibition of PI 3-kinase and RAS blocks IGF-I and insulin-induced uncoupling protein 1 gene expression in brown adipocytes.

Fetal brown adipocytes expressed uncoupling protein 1 (UCP1) mRNA, this expression being blunted throughout culture for 24 h in a serum‐free medium. At physiological doses, either insulin‐like growth factor I (IGF‐I) or insulin turned out to be as potent as dibutyryl cAMP (dbcAMP) in increasing UCP1 gene transcription rate (1 h) and also UCP1 mRNA accumulation (3 h), their maximal effect (15‐fold increase) reached upon treatment for 24 h. Upon treatment with either IGF‐I or insulin for 48 h, a 7‐fold increase in the UCP1 protein content relative to levels in the control cells was found, this induction being abolished in the presence of cycloheximide. Moreover, either IGF‐I or insulin transactivates the UCP1‐chloramphenicol acetyl transferase (CAT) fusion gene after transient transfection of primary brown adipocytes, these effects being tissue‐specific. Transient transfection of dominant‐negative form of phosphatidylinositol (PI) 3‐kinase completely blocked the transactivation of the fusion gene UCP1‐CAT induced by either IGF‐I or insulin, although inhibition of p70S6kinase with rapamycin does not preclude transactivation of the UCP1 promoter by insulin. Furthermore, transient transfection of dominant‐negative form of p21‐ras or treatment of cells with a mitogen‐activated protein kinase kinase (MEK‐1) inhibitor (PD098059) completely abolished insulin‐induced UCP1‐CAT transactivation. Cotransfectionwith dominant‐negative p85 or with dominant‐negative Ras also produced down‐regulation of the insulin or IGF‐I‐induced 12‐O‐tetradecanoylphorbol‐13‐acetate response element (TRE)‐CAT (five AP‐1, activating protein‐1, binding sites arranged in tandem) transactivation. In addition, insulin induced AP‐1 DNA binding activity, this effect being totally prevented in the presence of MEK‐1 inhibitor. These results strongly suggest that either IGF‐I or insulin induced thermogenic‐differentiation through AP‐1 activity in a PI 3‐kinase and Ras/MAPK dependent manner in brown adipocytes. J. Cell. Physiol. 176:99–109, 1998.

Activated Ha-ras Induces Apoptosis by Association with Phosphorylated Bcl-2 in a Mitogen-activated Protein Kinase-independent Manner

Serum deprivation of Ha-ras-transformed brown adipocyte cell line resulted in a dramatic apoptotic cell death, as detected either by DNA laddering or by an increase in the percentage of hypodiploid cells or by nuclei condensation and fragmentation, as compared with immortalized cell line or primary fetal brown adipocytes. Moreover, transient transfection of immortalized brown adipocytes with a constitutively activeras gene (Ha-ras lys12) mimics the high rate of apoptosis detected in the transformed cell line. On the other hand, transient transfection of the dominant-negative construct of raf-1 rescued serum-deprived Ha-ras-transformed brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the external display of phosphatidylserine, and the DNA laddering. However, inhibition of mitogen-activated protein kinase with PD098059 did not preclude apoptosis and in fact increased the rate of apoptosis observed in serum-deprived Ha-ras-transformed cells, indicating that the Ras/Raf-1 pathway induced apoptosis throughout a mitogen-activated protein kinase kinase 1 (MEK-1)-independent pathway. Furthermore, apoptosis in Ha-ras-transformed brown adipocytes is concurrent with an up-regulation in the expression of the pro-apoptotic protein Bcl-xS, the expression of the anti-apoptotic protein Bcl-2 being down-regulated. Finally, an association of Ras and Raf with phosphorylated Bcl-2 protein was demonstrated in immunoprecipitates from apoptotic cells. Thus, we propose a mechanism of apoptosis in Ha-ras-transformed adipocytes under serum deprivation involving Raf-1 association with phosphorylated Bcl-2, down-regulation of Bcl-2 expression, and up-regulation of Bcl-xS expression.

Okadaic acid inhibits insulin-induced glucose transport in fetal brown adipocytes in an Akt-independent and protein kinase C ζ-dependent manner

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates‐1 and ‐2 (IRS‐1 and IRS‐2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS‐1 and IRS‐2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate–polyacrylamide gel electrophoresis mobility. IRS‐1/IRS‐2‐associated p85α and phosphatidylinositol (PI) 3‐kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin‐induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin‐induced protein kinase C (PKC) ζ activity. However, activation of Akt/PKB or p70 S6 kinase (p70s6k) by insulin remained unaltered. Our results suggest that downstream of PI 3‐kinase, insulin signalling diverges into at least two independent pathways through Akt/PKB and PKC ζ, the PKC ζ pathway contributing to glucose transport induced by insulin in fetal brown adipocytes.