Abstract Lactate and Mitochondrial Reticular Connections: Enteric and Systemic Shuttles, Oxidation, and Aging by Robert G. Leija Doctor of Philosophy in Integrative Biology University of California, Berkeley Professor George A. Brooks, Chair The history of discoveries on the pathway of carbohydrate disposal and anaerobic glycolysis can be traced to studies of isolated amphibian muscles studied without circulation or oxygenation. At the same time, results that utilized techniques in isotopic tracer studies have clarified concerns regarding metabolic regulation during exercise and nourishment. Considering these notions, the purpose of this research was to interrogate the many facets of whole-body inter- and intra-lactate shuttling, mitochondrial lactate oxidation, and changes in overall mitochondrial reticulum architecture that occur with age. By utilizing a mixed model investigation, we illustrated whole-body lactate kinetics following an Oral Glucose Tolerance Test (OGTT) and, more importantly, the discovery of a Postprandial Lactate Shuttle in humans. To do this we recruited 15 young (21-35 yr; 7 men and 8 women) participants, had the forearm vein catheterized for primed-continuous infusions of [3-13C] Lactate and [6,6-2H]-Glucose. A contralateral warmed hand vein was catheterized for arterialized blood sampling. Indirect calorimetry was performed simultaneously to determine the total oxidation rates (Rox) of lactate. In our participants, lactate rate of appearance (Ra) increased from baseline 5 min post-consumption and rose before the concentration of glucose, thus providing evidence of an enteric PLS. Secondary increments in the concentration of blood lactate and its rate of appearance coincided with those of glucose, which indicates the presence of a more significant, secondary, systemic PLS phase driven by hepatic glucose release. Related to this novel finding on systemic lactate circulation and the carbon distribution from different metabolic substrates, we believed it was necessary to revisit the structure of the mitochondrial Lactate Oxidation Complex (mLOC). To interrogate this, we used classic and contemporary techniques in experiments utilizing isolated mitochondrial fragments from the skeletal muscle of mice. Specifically, we performed techniques including immunoblotting, co-immunoprecipitation, immunofluorescence and colocalization, and in situ proximity ligation assays and provided compelling evidence for a tight association between the mitochondrial Pyruvate Carrier (MPC) and the mLOC. These unique observations provide a metabolic map for the final systemic uptake of carbohydrate-derived lactate and pyruvate and, more importantly, its subsequent distribution by the mitochondrial reticulum. Ultimately, the functional distribution and subsequent catabolism of glucose to lactate and pyruvate via enteric, systemic, and intracellular shuttling are collectively reliant upon a morphologically robust and organized mitochondrial reticulum. Correspondingly, to determine the relationship between mitochondrial morphology and function, we utilized isolated fragments of mitochondria from the skeletal muscle of young and old male and female mice to evaluate the changes in substrate oxidation and, in conjunction, analyze the 3-dimensional (3D) structure of the mitochondrial reticulum. Our results revealed that isolated mitochondrial respiration from older mice retained overall P:O (ADP:Oxygen) efficiency when titrations of both carbohydrate and fatty-acid-derived substrates were used. At the same time, there was a reduction in the respiratory control ratios indicative of mitochondrial membrane uncoupling. Interestingly, our immunoblot and enzymatic activity assays on Cytochrome Oxidase (COx) and Citrate Synthase (CS) used to quantify mitochondrial content did not present significant age-related differences. Instead, experiments used to quantify enzymatic abundances of the mitochondrial dynamic proteins (FISS1, DRP1, and MFN1) were found to be lower in the older group. More importantly, 2 and 3-D renderings of the mitochondrial reticulum from both transverse and longitudinal muscle sections illustrated that while mitochondrial reticular density was similar between young and old mice, the size of the diameter was significantly smaller, and the shape of the reticulum illustrated overall reductions in connectivity. The compilation of data presented here represents a metabolic map of lactate partitioning between and within tissues and utilizes young and old rodents as a model to study changes in metabolism that accompany age. Moreover, we suggest that changes in the pathway of carbohydrate distribution are likely associated with age-related mitochondrial membrane fragility and disorganization.