Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells.
Tight coupling between cytosolic and mitochondrial metabolism is key for GSIS (glucose-stimulated insulin secretion). In the present study we examined the regulatory contribution of PDH (pyruvate dehydrogenase) kinase 1, a negative regulator of PDH, to metabolic coupling in 832/13 clonal beta-cells. Knockdown of PDH kinase 1 with siRNA (small interfering RNA) reduced its mRNA (>80%) and protein level (>40%) after 72 h. PDH activity, glucose-stimulated cellular oxygen consumption and pyruvate-stimulated mitochondrial oxygen consumption increased 1.7- (P<0.05), 1.6- (P<0.05) and 1.6-fold (P<0.05) respectively. Gas chromatography/MS revealed an altered metabolite profile upon silencing of PDH kinase 1, determined by increased levels of the tricarboxylic acid cycle intermediates malate, fumarate and alpha-ketoglutarate. These metabolic alterations were associated with exaggerated GSIS (5-fold compared with 3.1-fold in control cells; P<0.01). Insulin secretion, provoked by leucine and dimethylsuccinate, which feed into the tricarboxylic acid cycle bypassing PDH, was unaffected. The oxygen consumption and metabolic data strongly suggest that knockdown of PDH kinase 1 in beta-cells permits increased metabolic flux of glucose-derived carbons into the tricarboxylic acid cycle via PDH. Enhanced insulin secretion is probably caused by increased generation of tricarboxylic acid cycle-derived reducing equivalents for mitochondrial electron transport to generate ATP and/or stimulatory metabolic intermediates. On the basis of these findings, we suggest that PDH kinase 1 is an important regulator of PDH in clonal beta-cells and that PDH kinase 1 and PDH are important for efficient metabolic coupling. Maintaining low PDH kinase 1 expression/activity, keeping PDH in a dephosphorylated and active state, may be important for beta-cells to achieve the metabolic flux rates necessary for maximal GSIS.
Krus
Ulrika
U
Department of Clinical Sciences, Unit of Molecular Metabolism, Lund University Diabetes Centre, Malmö University Hospital, 20502 Malmö, Sweden.
Kotova
Olga
O
Spégel
Peter
P
Hallgard
Elna
E
Sharoyko
Vladimir V
VV
Vedin
Anna
A
Moritz
Thomas
T
Sugden
Mary C
MC
Koeck
Thomas
T
Mulder
Hindrik
H
D002460:Cell Line; D002999:Clone Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000201:enzymology / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D010101:Oxygen Consumption / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D007328:Insulin; D024101:Mitochondrial Proteins; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D034741:RNA, Small Interfering; D017346:Protein Serine-Threonine Kinases