The Final Pathway of Carbohydrate Oxidation: Revisiting the mLOC

Abstract

The confirmation of the Intracellular Lactate Shuttle proposed by Brooks and colleagues in 1998 revolutionized the way lactate metabolism was studied in the field of exercise physiology, nutrition, metabolism, and medicine. Previous investigations identified the structure of the mitochondrial Lactate Oxidation Complex (mLOC) which includes MCT1, LDH, COX4, and CD147. While the predominant view in research and textbooks was that pyruvate was imported into the mitochondria to be oxidized it wasn’t until 2009 that Jiang and Colleagues elucidated the mitochondrial pyruvate carrier (mPC). Knowing this, many researchers developed their questions around either pyruvate or lactate metabolism, which may have led to confusion in the field since both substrates play an important role in carbohydrate oxidation. This dilemma led us to the hypothesis that the mPC is in fact an extended member of the mLOC. Mixed skeletal muscle and liver were excised and whole blood was collected post-mortem from (n=5) 4-month-old C57B/l6 mice. Tissues were homogenized in an isotonic solution supplemented with a protease and phosphatase inhibitor. Blood was resuspended in H2O to rupture the cell membranes and the lysates (RBC) were used as negative controls. Mitochondria were isolated by differential centrifugation and resuspended in RIPA buffer. Protein concentrations of tissues were determined using a BCA standard. Western blotting was performed by loading 20 ug of each lysate, separated on an SDS-PAGE gel, and transferred to a PVDF membrane. Membranes were blocked and probed for MCT1(1:1000), mPC1 (1:500), COX4(1:1000), LDHA (1:1000). An SDS-PAGE was repeated and the mPC was excised from the gel. A trypsin digest was performed to isolate the peptides and then analyzed by one-dimensional LCMS/MS at the UC Berkeley QB3 proteomics/mass spectrometry lab. For cDNA preparation, RNA was extracted from 15 mg of skeletal muscle using the Qiagen RNeasy Fibrous tissue kit, reverse transcribed, and concentrated using Quibit ssDNA kit. Quantitative PCR was run on a QuantStudio3 using Power SYBR Green chemistry to confirm the presence of mPC1 in skeletal muscle. Our results revealed that mPC1 was expressed in skeletal muscle along with other genes associated with the mLOC. Immunoblots of mitochondrial lysates contained MCT1, mPC1, LDH, and COX4 in both the skeletal muscle and liver mitochondrial fractions. As expected, MCT1 was also present in RBC lysates as the MCTs are present in both the sarcolemma and mitochondrial membrane. Moreover, LCMS/MS analysis confirmed the presence of mPC1 corroborating our immunoblots. Colocalization using immunocytochemistry confirms the revised mLOC structure. Accordingly, both the pyruvate and lactate transporters assist in maintaining the redox balance and varying rates of carbohydrate oxidation during changing physiological conditions. This promising data has implications for a variety of fields ranging from cancer biology to exercise physiology. NIH Grant #1 R01 AG059715-01 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.