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VOL. 76 (3), 357-377, 2010 TSC1-TSC2 COMPLEX ON THE CROSSROAD OF PANCREATIC...

3.2. Insulin stimulates TSC/mTORC1 signaling pathway through
PI3K/Akt in pancreatic ß cells

To assess the signaling pathway specificity of insulin or glucose-

mediated TSC2 phosphorylation, we used selective inhibitors of the

class I PI3K (wortmannin 40 nM), and MEK 1/2 (U0126 5 µM). Use
of wortmannin blocked insulin-stimulated Akt Ser473 phosphorylation
and subsequently TSC2 Ser939 and Thr1462 phosphorylation and

mTORC1 activity. Inhibition of MEK/ERK pathway did not affect ei-

ther Akt or TSC2 phosphorylation levels. Surprisingly, although both

reconstituted cell lines express similar levels of IR (Figure 1A), wort-

mannin 40 nM only partially impaired Akt/TSC2 phosphorylation in

IRA expressing ß cell line. However, this phosphorylation was totally
blunted in IR +/+ or IRB expressing ß cells (Figure 2). Time-course
stimulation also revealed a more sustained phosphorylation of

Akt/TSC2 in Rec A cells versus Rec B cells (data not shown). In IR -

/- cells, glucose did not stimulate either Akt or TSC2 phosphorylation
in these residues. However, glucose stimulated p70S6K Thr389 phos-

phorylation (Figure 2).

3.3. Glucose activates TSC/mTORC1 signaling via ERK 1/2
independently from insulin signaling in pancreatic
ß cells

To test whether glucose by itself was capable to stimulate TSC2
Ser664 phosphorylation in pancreatic ß cells, we submitted IR -/- ß cells
to 5 mM glucose stimulation. We found a glucose-mediated activation
of TSC2 Ser664 phosphorylation by Western-blot analysis (Figure 3A).
This phosphorylation was completely blunted by the addition of
U0126. TSC2 Ser664 phosphorylation has been described as a marker
of hyperactivation of MEK/ERK signaling in tuberous sclerosis, breast
and colon human cancer (28).

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