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

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

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??

http://www.staff.ncl.ac.uk/n.j.morris/

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

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

• 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

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

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

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

Akt is involved in PA-stimulated glucose uptake

API-2: Akt inhibitor L6 cells300 µM PA, 30 min

• 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?

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

AMPK is involved in PA-mediated increase in glucose uptake

AMPK-DN: AMPK dominant negative- removes the functional AMPK domain L6 cells

• 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.

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.

• 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?

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

• 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?

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

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

Questions?

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

(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