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R. HERNÁNDEZ, T. TERUEL Y M. LORENZO CERAMIDA Y RESISTENCIA A LA INSULINA

defosforilar Akt, como se desprende de las observaciones siguientes: 1) el tratamiento
con ceramida o con TNF-alfa aumenta la actividad fosfatasa PP2A, 2) el tratamiento con
ácido okadaico junto con ceramida restaura completamente la fosforilación de Akt por
insulina y 3) la transfección transitoria de una forma constitutivamente activa de Akt no
restablece la fosforilación de Akt. Estos resultados indican que la ceramida producida
por el TNF-alfa induce un estado de resistencia a insulina en los adipocitos marrones,
manteniendo Akt en un estado defosforilado e inactivo.
Palabras clave: Transporte de glucosa-TNF-ceramida-adipocito-Resistencia a Insulina

SUMMARY

Ceramide mediates Insulin resistance by Tumor Necrosis Factor alpha in brown
adipocytes

Tumor necrosis factor (TNF) alpha caused insulin resistance on glucose uptake
in fetal brown adipocytes. We have explored the hypothesis that some effects of TNF-
alpha could be mediated by the generation of ceramide, since TNF-alpha treatment in-
duced the production of ceramide in these primary cells. A short-chain ceramide ana-
logue, C2-ceramide, completely precluded insulin-stimulated glucose uptake and insu-
lin-induced GLUT4 translocation to plasma membrane, either determined by Western
blot or by immunofluorescent localization of GLUT4. These effects were not produced
in the presence of a biologically inactive ceramide analogue, C2-dihydroceramide.
Analysis of the phosphatidylinositol (PI) 3-kinase signaling pathway indicated that C2-
ceramide was precluding insulin stimulation of Akt kinase activity, but neither PI3-
kinase nor PKCzeta activities. C2-ceramide completely abolished insulin-stimulated
Akt/PKB phosphorylation on both regulatory residues Thr308 and Ser473 as TNF-alpha
did, as well as inhibited insulin-induced mobility shift in Akt1 and Akt2 separated in
PAGE. Moreover, C2-ceramide seems to be activating a phosphatase involved in
dephosphorylating Akt since 1) a PP2A activity was increased in C2-ceramide and
TNF-alpha-treated cells, 2) treatment with okadaic acid concomitantly with C2-ceramide
completely restored Akt phosphorylation by insulin and 3) transient transfection of a
constitutively active form of Akt did not restore Akt activity. Our results indicate that
ceramide produced by TNF-alpha induced insulin resistance in brown adipocytes by
maintaining Akt in an inactive dephosphorylated state.
Key words: Glucose transport-TNF-ceramide-adipocyte-Insulin resistance

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