1.1 Isolation
and Maintenance
of Pancreatic Islets
We provide a brief description of the method based in collagenase
injection into the pancreatic duct, which includes all the minor
modifications currently applied by our group to result in a high
yield and quality of isolated islets. We also detail the conditions at
which we maintain both mouse and human islets in culture.1.2 Approaches
to AMPK Modulation
in Islets
We describe the main methods we use to modulate islet AMPK
activity in vitro that are pharmacological activators and virus expres-
sing a dominant-negative or a constitutively active form of AMPK.1.3 Measurement
of AMPK Activity
in Isolated Islets
In this section, we detail the procedures of SAMS peptide assay and
Western blot for phosphorylated AMPK and its targets ACC and
Raptor, as methods to determine AMPK activity in islets.1.4 Preparation
of Islet RNA for RNA-
seq (or qPCR)
We provide a detailed description of RNA isolation using TRIzol,
which provides high purity/quality RNA for downstream applica-
tions such as RNA sequencing or RT-qPCR.1.5 Assessing
the Impact of AMPK
on Glucose-Stimulated
Insulin Secretion
in Isolated Islets
Healthy β-cells in the pancreatic islet respond to high glucose
concentrations by secreting insulin. Therefore, we have chosen to
describe in detail an assay to evaluate insulin secretion in response
to glucose as a direct method to evaluate islet function.Fig. 1Glucose-stimulated insulin secretion is increased in AMPK α1 and
α2 double knockout (dKO) islets, compared to wild type (WT). After generating
mutant mice lacking AMPKα1 andα2 specifically in pancreatic beta cells, 8–10
size-matched islets were isolated and stimulated with low concentration of
glucose (3 mM) and high concentration of glucose (17 mM) during 30 min in
static incubation. Data normalized to total insulin.*p<0.05,**p<0.01,
***p<0.001. Reproduced from Kone et al. 2014 with permission from FASEB [7]Manipulation and Measurement in Islets 415