Tools for Mitochondrial Metabolism

Dysregulation of the pyruvate dehydogenase (PDH) complex (PDC) in skeletal muscle has been implicated in type 2 diabetes and various mitochondrial diseases. The activity of the PDC complex is mainly regulated by the phosphorylation state of Ser²⁹³, Ser²³², Ser³⁰⁰ on the E1α subunit. Phosphorylation inactivates the complex, while removal of the phosphate activates the complex.

We have developed a novel and specific antibodies to measure phosphorylation against all three sites on E1α. These antibodies detect only the phosphorylated form of PDH-E1α in rat brain, heart, liver mitochondrial homogenates, human tissue culture whole cell lysates, as well as in intact cells using fluorescent immunocytochemistry. These unique antibodies will allow the facile measurement of flux through the glycolytic pathway by monitoring PDH phosphorylation as a readout of activity. These reagents should prove a useful tool in models of diabetes and neuronal pathogenesis for assessing the signals required for coordinating mitochondrial metabolic responses to a large array of stimuli including glucose levels, reactive oxygen species, apoptotic responses, and extracellular growth signals.

Figure 1: Detection of PDH-E1α, phosphorylated at Ser²⁹³, Ser³⁰⁰, and Ser²³² by immunocytochemistry. Samples: COS7 cells incubated in presence dichloroacetate (DCA; 5 mM for 2 h). All samples were incubated with MitoTracker^® Red stain (100 nM for 20 min; to visualize mitochondria) and fixed in 3.7% formaldehyde, followed by methanol (3 min at -20°C), and permeabilization in PBS, 6% BSA, 0.3% Triton™ X-100 detergent, 0.1% Tween^® 20 detergent. Primary antibodies: PhosphoDetect™ Anti-PDH-E1α (pSer²⁹³) Rabbit pAb (Cat. No. AP1062) (panel B), PhosphoDetect™ Anti-PDH-E1α (pSer³⁰⁰) Rabbit pAb (Cat. No. AP1064) (panel C), PhosphoDetect™ Anti-PDH-E1α (pSer²³²) Rabbit pAb (Cat. No. AP1063) (panel D) (0.5 µg/ml, green fluorescence) and anti-PDH-E1α antibody (green fluorescence, top 3 panels) (panel A). Detection: fluorescence (Alexa Fluor^® 488 secondary antibody) with DAPI counterstain (300 nM, 1 min, blue fluorescence). Data courtesy of Matthew Rardin, University of California San Diego.

Figure 2: Detection of human PDH-E1α, phosphorylated at Ser²⁹³, Ser³⁰⁰, and Ser²³² by immunoblotting. Samples: Whole tissue extract from mouse liver (20 µg) left untreated (lane 1) or treated with dichloroacetate (DCA, 5 mM for 4 h) (lane 2). Primary antibody: PhosphoDetect™ Anti PDH-E1α (pSer²⁹³) Rabbit pAb (Cat. No. AP1062) PhosphoDetect™ Anti PDH-E1α (pSer³⁰⁰) Rabbit pAb (Cat. No. AP1064) PhosphoDetect™ Anti PDH-E1α (pSer²³²) Rabbit pAb (Cat. No. AP1063) all used at 0.25 µg/ml and Anti-PDHE-1α. Detection: chemiluminescence. Data courtesy of Matthew Rardin, University of California San Diego.