1. 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 15 th, 2012

2. 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

3. 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??

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

5. 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

6. PA stimulates Akt and AMPK phosphorylation in a time- and dose-dependent manner (C2C12, L6 myotubes, myoblasts, perfused rat) L6 myoblasts Sequential activation of AMPK and Akt

7. 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

8. 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 internalization 2.) Warm cells to 37 ⁰C Positive Control C2C12 cells K: KRBH, will remove unbound PA

9. Extent of PA binding to plasma membrane: 1: Total Input 2: After 1 hr 3: After washing with KRBH 4: 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

10. Metabolism of PA during PA treatment 1: Washed with KRBH at 4⁰C 2: Washed with BSA at 4 ⁰C 3: Washed with KRBH at 4 ⁰C then incubated 10 min at 37 ⁰C 4: Washed with BSA at 4 ⁰C then incubated 10 min at 37 ⁰C Total lipids extracted and separated by TLC Data suggest that PA is the factor to induce Akt activation…? C2C12 cells 300 µM PA for 1 hr

11. Akt is involved in PA-stimulated glucose uptake API-2: Akt inhibitor L6 cells 300 µM PA, 30 min

12. 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?

13. AMPK is involved in PA-stimulated glucose uptake by regulating Akt activity AICAR: AMPK agonist Compound C: AMPK inhibitor (Dorsomorphin) 30 min 1 hr L6 cells

14. AMPK is involved in PA-mediated increase in glucose uptake AMPK-DN: AMPK dominant negative- removes the functional AMPK domainL6 cells

15. 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.

16. 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.

17. 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?

18. 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

19. 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?

20. 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

21. 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

22. Questions?

23. PA stimulates Akt phosphorylation in L6/C2C12 myotubes, L6 myoblasts, isolated rat soleus  L6 myoblasts  L6 myotubes P-Akt Total Akt PA 300 µM (min) PA 2 mM (min) Control (min)  Isolated rat soleus

24. (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