palmitic acid acutely stimulates glucose uptake via activation of akt and erk1/2 in skeletal muscle...
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Palmitic acid acutely stimulates glucose uptake via activation of Akt and ERK1/2 in skeletal muscle
cells
Jing Pu, Gong Peng, Linghai Li, Huimin Na, Yanbo Liu, and Pingsheng Liu
Journal of Lipid Research 52: 1319-1327
October 15th, 2012
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Introduction
• Skeletal muscle is the main source of glucose disposal, and therefore plays an important role in whole-body glucose homeostasis
• Chronic exposure to saturated fatty acids can cause insulin resistance
– Elevated plasma levels of FA are associated with increased incidence of IR and T2DM
– Prolonged exposure of skeletal muscle cells in vitro will reduce insulin signaling and glucose uptake
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Introduction
• Plasma levels of fatty acids fluctuate postprandially, and may have difference acute effects on glucose metabolism
• Purpose: to examine the acute effects of palmitic acid (PA) exposure on skeletal muscle– Mechanism??
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PA stimulates GLUT4 translocation to the plasma membrane, glucose uptake
L6Glut4myc cells
300 µM PA
300 µM PA or 100 nM insulin (30 min)
* p < 0.05
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PA stimulates Akt and AMPK phosphorylation in a time- and dose-dependent manner
(C2C12, L6 myotubes, myoblasts, perfused rat)
L6 myoblastsSequential activation of AMPK and Akt
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• PA stimulates acute activation of Akt and AMPK
• More rapid activation of AMPK suggests that AMPK may mediate PA-induced Akt activation.
• How does PA stimulate acute increases in glucose uptake and Akt/AMPK activation?– PA may stimulate skeletal muscle cells by binding
to the plasma membrane
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Binding of PA to plasma membrane is required for stimulation of Akt phosphorylation
1.) Incubate with 300 µM PA for 1 hr at 4⁰C to avoid PA internalization2.) Warm cells to 37 ⁰C
Positive Control
C2C12 cellsK: KRBH, will remove unbound PA
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Extent of PA binding to plasma membrane:
1: Total Input2: After 1 hr3: After washing with KRBH4: After washing with BSA
Effective amount of PA is as little as 0.43% of the amount applied[(Bar 3 – Bar 4)/Bar 1]
C2C12 cells
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Metabolism of PA during PA treatment
1: Washed with KRBH at 4⁰C2: Washed with BSA at 4 ⁰C3: Washed with KRBH at 4 ⁰C then incubated 10 min at 37 ⁰C4: Washed with BSA at 4 ⁰C then incubated 10 min at 37 ⁰C
Total lipids extracted and separated by TLCData suggest that PA is the factor to induce Akt activation…? C2C12 cells
300 µM PA for 1 hr
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Akt is involved in PA-stimulated glucose uptake
API-2: Akt inhibitor L6 cells300 µM PA, 30 min
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• PA acutely stimulates glucose uptake through activation of Akt, AMPK in a time- and dose-dependent manner
• Binding of PA to the plasma membrane is required for Akt activation
• PA induces sequential activation of AMPK and Akt. Does AMPK mediate the PA-induced activation of Akt?
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AMPK is involved in PA-stimulated glucose uptake by regulating Akt activity
AICAR: AMPK agonist Compound C: AMPK inhibitor (Dorsomorphin)
30 min1 hr
L6 cells
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AMPK is involved in PA-mediated increase in glucose uptake
AMPK-DN: AMPK dominant negative- removes the functional AMPK domain L6 cells
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• PA-stimulated AMPK phosphorylation may contribute to regulation of Akt activation, and is involved in PA-induced glucose uptake.
• PI3K is upstream of Akt in the insulin signaling pathway, and may be involved in the PA-induced stimulation of glucose uptake.
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PI3K is essential for cell response to PA
LY294002: PI3K inhibitor L6 cells
PI3K inhibition will completely inhibit PA-induced glucose uptake, reduce AMPK and Akt activation.
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• PA may acutely trigger signal transduction by binding to the plasma membrane, and stimulate glucose uptake via activation of PI3K/AMPK/Akt pathway.
• Does PA acutely stimulate activation of MEK signaling?
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Activation of ERK 1/2 is involved in PA-stimulated glucose uptake
L6 cellsPD98056, U0126: ERK1/2 inhibitors
U0126 is more efficient at reducing ERK 1/2 phosphorylation
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• PA-stimulated ERK1/2 activation, as well as PI3K/AMPK/Akt may contribute to PA-induced glucose uptake.
• What is the relationship between ERK1/2 and the PI3K/AMPK/Akt pathways?
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PI3K regulates ERK in an AMPK- and Akt-independent pathway
API-2: Akt inhibitor LY294002: PI3K inhibitor
PI3K inhibition reduces activation of ERK and Akt
Akt inhibition did not affect ERK activation.
AMPK siRNA did not affect activation of ERK
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Conclusion• Previous studies show that chronic PA exposure will
cause insulin resistance
• Acute exposure to PA will stimulate GLUT4 translocation to plasma membrane, enhance glucose uptake– Requires plasma membrane-bound PA– Acts through PI3K/AMPK/Akt and PI3K/ERK
pathways
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Questions?
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PA stimulates Akt phosphorylation in L6/C2C12 myotubes, L6 myoblasts, isolated rat soleus
L6 myoblasts
L6 myotubesP-Akt
Total Akt
PA 300 µM (min)
PA 300 µM (min)
PA 2 mM (min)Control (min)
Isolated rat soleus
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(Supplemental Data)
• Linoleic acid, oleic acid, stearic acid (and FA mixture) can acutely stimulate Akt and AMPK phosphorylation in L6 cells– Long term exposure with oleic acid will attenuate
PA-induced IR– LA will completely reverse PA-induced IR