AMPK Methods and Protocols

7. Add 2.2 volumes of ethanol. Vortex.


8. Incubate the tubes at RT for 10 min or for a longer (20 min–d-
   ays) period of time on ice or at
   20 C(seeNote 18).


9. Spin the tube at 12,000gfor 15 min at 4C.


10. Wash the pellet with 850μl of 70% (v/v) ethanol, spinning at
    12,000 rcf for 5 min at 4C.


11. Remove ethanol, dry, and resuspend in ultrapure RNase-free
    H 2 O.


12. Optional DNase treatment (seeNote 19).

3.5 Assessing
the Impact of AMPK
on Glucose-Stimulated
Insulin Secretion
in Isolated Islets

The effects of AMPK on insulin secretion and pancreaticβ-cell

function could be studied by static conditions or by perfusing islets

to measure the kinetics of insulin release in response to stimulating

factors including glucose, amino acids, or chemical compounds

(Fig.1, Reproduced from Kone et al. 2014 with permission from

FASEB). Here we describe the static glucose-stimulated insulin

secretion (GSIS). Typically, glucose is used in various ranges of

concentration and potassium chloride (KCl). The media used is a

Krebs-Ringer buffer, with a basal low glucose concentration, and

then the islets are stimulated with a higher glucose concentration.

1. Each condition is performed in triplicates (seeNote 20).


2. Wash the isolated islets by picking the islets (seeNote 9) into a
   new 10 cm petri dish containing 8 ml of KRBH solution
   (without glucose).


3. Preincubate the islets in unstimulated condition with a “low”
   glucose KRBH-3 mM Glu solution for 1 h at 37C, CO 2 5%,
   and O 2 95% (seeNote 21).


4. After 1 h, stimulate the islets with different concentrations of
   glucose in KRBH (KRBH-3 mM Glu, KRBH-17 mM Glu) and
   KCl (KRBH-30 mM KCl). The KCl is used as a control,
   because it acts as non-specific membrane depolarizer.


5. Under the microscope, pick 10 size-matched islets for each
   condition into each well a 12-well plate (seeNote 22).


6. Add 500μl of KRBHþglucose or KCl into specific wells
   containing islets.


7. Incubate in a water bath for 30 min with gentle shaking
   (70 rpm/min).


8. Transfer the content of each well (islets and KRBH solution)
   into 1.5 ml standard microfuge tubes and place it on ice (see
   Note 23).


9. Centrifuge at 350gfor 3 min at 4C.


10. Collect supernatant containing the secreted insulin into new
    tubes.

Manipulation and Measurement in Islets 425