Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.
Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.
Kennedy
H J
HJ
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Pouli
A E
AE
Ainscow
E K
EK
Jouaville
L S
LS
Rizzuto
R
R
Rutter
G A
GA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D001517:Coleoptera / Q000201:enzymology; D017931:DNA Primers; D005947:Glucose / Q000494:pharmacology*; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases; D005947:Glucose; D002118:Calcium