Glucagon Diabetes mellitus Islet microcirculation

Isolation of islet cells
Quantitative RT-PCR
Fluorescence-activated
cell sorter (FACS)
purifications
1 st sort, by FAD (flavine
adenine dinucleotide)
content
ß- & α-cells
2 nd sort, by NAD(P)H
content of the α-cell
fraction
pure α- & non α-cells
• Total RNA was extracted from α- and ß-
cell fractions using RNeasy Mini kit ®
• Converted into cDNA using Superscript
reverse transcriptase
• Primers were designed to amplify the
insulin receptor, Kir6.2, and
sulfonylurea receptor (SUR)1; glucagon,
insulin and cyclophilin transcripts
Pipeleers et al.,1985
13
• At least 4 independent experiments
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• Zinc secretion assay
– atomic absorption spectrophotometer
• Hormone secretion assays
– static incubation (triplicate)
• FACS-isolated cells : 15-30 min
• Dispersed islet : 60 min
– radioimmunoassay
• Glucagon : anti-glucagon
• Insulin : anti-insulin
• C-peptide : commercial kit
Electrophysiology
Isolated α-cell
Perforated-patch whole-cell configuration
Patch perforation Amphotericin B
Pipette solution
(in mM): K 2
SO 4
76,
NaCl 10, KCl 10,
MgCl 2
1, HEPES 5
(pH 7.35 with KOH)
Extracellular solution
(in mM): NaCl 138, KCl
5.6, MgCl 2
1, CaCl 2
2.6,
HEPES 5 (pH 7.40 with
NaOH), glucose 0
+ additional agents
15
16
Electrophysiology
Isolated α-cell
whole-cell patch-clamp recordings of K ATP channel current activity
Pipette resistance 2-6 MOhm
Statistical analyses
• Two-tailed, Student’s t test
Pipette solution
(in mM):KCl 125,
KOH 30, EGTA 10,
HEPES 5, MgCl 2
1,
Mg-ATP 0.3,
K-ADP 0.3
(pH 7.15)
Extracellular solution
(in mM):NaCl 138, KCl
5.6, MgCl 2
1, CaCl 2
2.6, HEPES 5 (pH 7.40
with NaOH), glucose
0 + additional agents
-60
-70
-80
200 ms
2 s
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18