Paradoxical Effect of Mitochondrial Respiratory Chain Impairment on Insulin Signaling and Glucose Transport in Adipose Cells

Juerg Straubhaar,Alison Burkart,Xiarong Shi,Michael P Czech,Silvia Corvera,Sarah M Nicoloro

doi:10.1074/jbc.m800510200

Juerg Straubhaar, Alison Burkart + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m800510200

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Abstract

Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.

Highlights

That signal to other tissues [5, 6]
We have found that differentiation of 3T3-L1 adipocytes is accompanied by increased mitochondrial biogenesis, assessed primarily by mass spectrometry of peptides increased during adipogenesis [9]
A possible relationship between mitochondrial levels and adipose tissue function has been inferred from results in animals and humans indicating that alterations in mitochondrial levels correlate with changes in insulin sensitivity [9, 10, 15,16,17,18,19,20,21,22,23]

Summary

EXPERIMENTAL PROCEDURES

Materials—Real-time PCR primers were designed using the Primer Bank (pga.mgh.harvard.edu/primerbank/index.html). At day 2 of differentiation, medium was collected and cells were trypsinized and transfected with Tfam or scrambled siRNA by electroporation. After three washes with ice-cold phosphate-buffered saline (PBS), the cells were lysed with 0.4 ml of 1% Triton X-100. Flow Cytometry—Cells were trypsinized and incubated in KRH buffer without or with 100 nM MitoTracker௡ GreenFM for 30 min at 37 °C. 1 ϫ 106 cells were resuspended in 1 ml of ice-cold phosphate-buffered saline with 0.5% BSA and analyzed on an LSR II cytometer analyzer (BD Biosciences). To measure ATP synthesis dynamically, cells were first suspended in respiration buffer (0.1% BSA, 75 mM mannitol, 25 mM sucrose 100 mM KCl, 10 mM KH2PO4, 5 mM MgCl2, 20 mM, Tris, pH 7.5, 106 cells/ ml), and 10 ␮g/ml digitonin was added for 5 min.

Mito Probes unchanged

RESULTS

Biosynthesis of steroids

DISCUSSION