An imbalance in energy homeostasis leads to increased lipid storage and obesity. Mechanisms that regulate cellular lipid storage or utilization are thought to play a key role in maintaining energy balance and overall metabolic health. However the mechanisms that regulate energy expenditure and fuel utilization are not well understood. Here we address these gaps in knowledge by first describing a novel mechanism to increase energy expenditure by regulating mitochondrial fuel utilization. We find that blocking pyruvate entry into the mitochondria increases energy expenditure by activating an ATP-demanding lipid cycle of LD breakdown and buildup that is fueled by lipid oxidation. Moreover, recent studies have demonstrated heterogeneity in mitochondrial function, and identified a unique population of BAT mitochondria that support lipid storage by anchoring themselves to LD and facilitating TG synthesis and LD expansion. However, it remains unknown if these unique mitochondria are conserved in other tissues. Here, I describe the development of the first approach to isolate PDM from WAT. Using this approach I show that PDM in WAT have a unique function that is distinct from BAT. Future research understanding the mechanisms that control subpopulations of mitochondria and their roles in lipid homeostasis can provide novel methods of altering energy metabolism through mitochondria.

Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are both characterized by altered mitochondrial respiratory capacity, steatosis, increased oxidative stress, and inflammation. ATP-binding cassette B10 (ABCB10) is an inner mitochondrial membrane protein that has been shown to participate in redox protection in hematopoietic cells and cardiomyocytes. While it is highly expressed in the liver, it is unknown if ABCB10 has a role in pathogenesis of NAFLD or ALD. Here I address these gaps in knowledge by first describing ABCB10 transport cargo. We find that ABCB10 exports the heme by-product biliverdin (BV) for subsequent reduction to lipophilic antioxidant bilirubin (BR), confirming the mechanism by which ABCB10 exerts antioxidant activity. Moreover, we find that ABCB10 content is differentially altered in NAFLD versus ALD. Here, I describe how increased hepatic ABCB10 promotes NAFLD by decreasing mitochondrial respiration and promoting redox-mediated insulin resistance, both of which lead to hepatosteatosis. Conversely, severe alcoholic hepatitis in mice and patients results in decreased hepatic ABCB10 content. However, hepatic ABCB10 in both mild alcoholic steatohepatitis (ASH) and severe AH does not exert similar effects on mitochondrial respiration and steatosis as in NAFLD. Here I demonstrate that hepatic ABCB10 is not the pivot point which determines progression of ALD nor is it determinative of hepatic mitochondrial respiratory function in ASH or AH but rather mitigates the formation of hepatotoxic neutrophil extracellular traps (NETs) in severe ALD. Future research understanding the role of ABCB10-mediated BR generation and its role redox signaling/homeostasis can provide novel therapeutic approaches to treating liver disease.

ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesityWhile the impact of hydrophilic antioxidants on hepatic insulin resistance and non-alcoholic fatty liver disease (NAFLD) is well understood, the function of lipophilic antioxidants in these conditions is not as clear. Bilirubin, a lipophilic antioxidant known for scavenging hydrogen peroxide (H2O2), undergoes oxidation to biliverdin within mitochondria. This biliverdin is then exported to the cytosol and reduced back to bilirubin by cytosolic biliverdin reductase (BLVRA). The process of biliverdin export from mitochondria is crucial for the regeneration of bilirubin and its role in intracellular H2O2 scavenging. Our research identified ABCB10 as the transporter responsible for exporting biliverdin from mitochondria. We observed that ABCB10 assists in transporting biliverdin and that its deletion leads to biliverdin build-up inside mitochondria. In mice, obesity and insulin resistance were found to increase ABCB10 expression in the liver, boosting both cytosolic and mitochondrial bilirubin levels in an ABCB10-dependent way. Surprisingly, deleting ABCB10 in the livers of obese mice led to protection against steatosis and high blood sugar. This was accompanied by improved insulin regulation of glucose production and reduced expression of the lipogenic enzyme SREBP-1c. This protective effect correlated with better mitochondrial performance and increased deactivation of PTP1B, an enzyme that interferes with insulin signaling and boosts SREBP-1c expression. Reintroducing bilirubin in ABCB10 knockout hepatocytes negated these beneficial effects, highlighting bilirubin's detrimental role associated with ABCB10 activity. Therefore, we've uncovered a vital transport mechanism that magnifies the effects of intracellular bilirubin in insulin resistance and steatosis during obesity.

The mitochondrial biliverdin exporter ABCB10 mitigates the formation of neutrophil extracellular traps in alcoholic hepatitisEffective treatment for acute liver failure due to alcoholic hepatitis (AH) is currently limited to liver transplantation. AH-affected livers exhibit a distinct molecular pattern, marked by impaired redox metabolism in hepatocytes and the presence of neutrophils that produce myeloperoxidase (MPO) and create neutrophil extracellular traps (NETs). Enhanced NET formation and MPO activity are known to aggravate liver damage in AH, both in mice and as a predictor of poor outcomes in human patients. Understanding the mechanisms that inappropriately trigger NET formation in the liver could lead to new treatments for AH. It remains uncertain if the redox imbalances in AH hepatocytes directly promote NET formation. Our research indicates that in AH, both human and mouse livers show reduced levels of the mitochondrial biliverdin exporter ABCB10, which is essential for producing the ROS-neutralizing agent bilirubin within hepatocytes. We found that increasing ABCB10 expression in the hepatocytes of mice with AH effectively reduced MPO gene expression and histone H3 citrullination, an indicator of NET formation. This anti-inflammatory action appears to be due to ABCB10’s role in lessening ROS-induced effects in the liver. Specifically, increasing ABCB10 activity led to a decrease in hepatic 4-HNE protein adducts without notably impacting mitochondrial fat metabolism or reducing steatosis and hepatocyte death. Therefore, our findings suggest that ABCB10's role in managing ROS within hepatocytes can alleviate the harmful activation of neutrophils to form NETs in AH. Enhancing ABCB10 function in hepatocytes might thus reduce acute liver failure in humans by curbing the inflammatory response triggered by excessive NET formation.

Proper regulation of cellular lipid storage and oxidation is indispensable for the maintenance of cellular energy homeostasis and health. Mitochondrial function has been shown to be a main determinant of functional lipid storage and oxidation, which is of particular interest for the adipose tissue as it is the main site of triacylglyceride storage in lipid droplets (LDs). Obesity stems from an imbalance between energy intake and energy expenditure, leading to increased lipid storage. Therefore, a better understanding of the mechanisms by which LD break-down and build-up are regulated can be crucial in the development of treatments against metabolic diseases.

Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cyclingCombined fatty acid esterification and lipolysis, termed lipid cycling, is an ATP consuming process that contributes to energy expenditure. Therefore, interventions that stimulate energy expenditure through lipid cycling are of great interest. Here we find that pharmacological and genetic inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes activates lipid cycling and energy expenditure, even in the absence of adrenergic stimulation. We show that the resulting increase in ATP demand elevates mitochondrial respiration coupled to ATP synthesis and fueled by lipid oxidation. We identify that glutamine consumption and the Malate-Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). We thus demonstrate that energy expenditure through enhanced lipid cycling can be activated in brown adipocytes by decreasing mitochondrial pyruvate availability. We here present a new mechanism to increase energy expenditure and fat oxidation in brown adipocytes, which does not require adrenergic stimulation of mitochondrial uncoupling.

The role of mitochondria attached to Lipid droplets in cellular energy and lipid metabolismRecent studies have identified a subpopulation of mitochondria attached to LDs, peridroplet mitochondria (PDM) that can be separated from cytoplasmic mitochondria (CM) by centrifugation. PDM have distinct bioenergetics, proteome, cristae organization and dynamics that support LD build-up. However, most of our knowledge on PDM function is based on PDM isolated from brown adipose tissue (BAT), mostly due to a lack of available protocols to isolate PDM from other tissues such as white adipose tissue (WAT). Here we show an optimized protocol to isolate PDM from BAT and WAT with high yield and purity by adding a proteolytic treatment. Using this protocol, we show that PDM attached to differently sized LDs have unique bioenergetics characteristics. Thus, our new isolation protocol has the potential to improve our understating of PDM function in WAT and BAT in different populations of LDs.

Though mitochondrial research has become more prominent in the last forty years, most methodologies to study mitochondria have been focused on cell and animal models. Respirometry, the assessment of mitochondrial function measured as oxygen consumption by Complex IV of the electron transport chain, is the “gold standard” for mitochondrial assessment, yet it is rarely utilized in human samples. This is due to two fundamental methodological challenges: (1) samples for respirometry must be processed and ran within hours of acquisition on specialized and complex equipment, and (2) human sampling for respirometry must be acquired using invasive tissue biopsies. While we as a field realize the importance of oxygen consumption as a measurement of mitochondrial function, the current methodologies are now limiting the types of biological and clinical questions we can ask and answer. Addressing the first challenge preventing widespread adoption of respirometry, the recently developed RIFS (Respirometry In previously-Frozen Specimen) assay allows for the measurement of maximal respiratory capacity from cells or tissues which have been previously frozen. This allows samples to be processed and then stored or biobanked for later respirometry assessment, ultimately making respirometry methodologies significantly easier, cheaper, and more accessible. To the second challenge though, there has been minimal breakthrough.

In this thesis we will cover novel methodologies overcoming challenges which are paramount to the expansion of respirometry methodologies for clinical applications. We will discuss the RIFS protocol in detail, describe the methodologies developed to perform a first-of-its-kind high-throughput and high-resolution respirometry study of the human brain, and will detail the development of a technique to measure mitochondrial respiration in cells human salivary leukocytes. Together, these chapters present novel methodologies designed to overcome the primary limitations of performing respirometry in human studies.

Mitochondrial architecture has long been associated with metabolic flexibility, although the precise causalities and the underlying mechanisms are still enigmatic. We showed that mitochondrial fragmentation is highly associated with fatty acid oxidation (FAO) rates. Furthermore, forced mitochondrial elongation using Mfn2 over-expression or Drp1 depletion significantly decreased FAO rates, while Mfn2 knockout lead to enhanced FAO rates, suggesting that mitochondrial fragmentation is functionally coupled with fatty acid utilization. Notably, the increased FAO rates upon mitochondrial fragmentation is attributed to decreased CPT1 sensitivity to malonyl-CoA, the endogenous CPT1 inhibitor, consistent with the observation that mitochondrial fragmentation specifically facilitates long chain-fatty acid oxidation (LCFAO), but not short chain fatty acid oxidation. Furthermore, we show pharmacological activators of CPT1 enhance FAO of elongate dmitochondria but not in the presence of malonyl-CoA binding. Suggesting mitochondrial morphology influences CPT1’s binding to a CoA moiety. Taken together, our study provides a biochemical and mechanistic explanation of how mitochondrial architecture links cellular metabolic needs to differential fuel utilization, such as LCFAO, which may be implicated in a myriad of human physiologies and pathologies. In non-alcoholic steatohepatitis (NASH), increased Drp1 mediated mitochondrial fragmentation has been observed in hepatocytes. NASH is characterized by liver inflammation, increased hepatocyte swelling, and some degree of fibrosis. Hepatic steatosis can occur when glucose and fatty acid availability exceeds the energy demand of the liver. This presents the question, is increased fission in NASH a compensatory or a pathogenic mechanism? To address this question, we decreased Drp1 function in liver from adult mice with established NASH (26 week-GAN diet) using GalNAc-siRNA mediated delivery. While NASH alone only elevated circulating AST and ALT levels, markers of hepatic injury, Drp1KD in the NASH liver engaged the mitochondrial integrated stress response (ISR) by OMA1 activation and ER stress. The fibrosis and hepatocyte necrosis observed only in Drp1KD of NASH mice can be explained by reduced metabolic adaptability as a result of inhibiting fission-mediated fragmentation, reducing FAO capacity, and enhancing accumulation of NEFA. Overall, our data suggests Drp1-mediated mitochondrial fragmentation is an adaptive mechanism in NASH that prevents lipotoxicity, overactivation of the integrated stress response, and ER stress to limit fibrosis, inflammation, and necrosis.