MTFP1 controls mitochondrial fusion to regulate inner membrane quality control and maintain mtDNA levels.

Mitochondrial dynamics play a critical role in cell fate decisions and in controlling mtDNA levels and distribution. However, the molecular mechanisms linking mitochondrial membrane remodeling and quality control to mtDNA copy number (CN) regulation remain elusive. Here, we demonstrate that the inner mitochondrial membrane (IMM) protein mitochondrial fission process 1 (MTFP1) negatively regulates IMM fusion. Moreover, manipulation of mitochondrial fusion through the regulation of MTFP1 levels results in mtDNA CN modulation. Mechanistically, we found that MTFP1 inhibits mitochondrial fusion to isolate and exclude damaged IMM subdomains from the rest of the network. Subsequently, peripheral fission ensures their segregation into small MTFP1-enriched mitochondria (SMEM) that are targeted for degradation in an autophagic-dependent manner. Remarkably, MTFP1-dependent IMM quality control is essential for basal nucleoid recycling and therefore to maintain adequate mtDNA levels within the cell.

Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.

The dynamin-related guanosine triphosphatase, Drp1 (encoded by Dnm1l), plays a central role in mitochondrial fission and is requisite for numerous cellular processes; however, its role in muscle metabolism remains unclear. Here, we show that, among human tissues, the highest number of gene correlations with DNM1L is in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

Spatial mapping of mitochondrial networks and bioenergetics in lung cancer.

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

Parkin regulates adiposity by coordinating mitophagy with mitochondrial biogenesis in white adipocytes.

Parkin, an E3 ubiquitin ligase, plays an essential role in mitochondrial quality control. However, the mechanisms by which Parkin connects mitochondrial homeostasis with cellular metabolism in adipose tissue remain unclear. Here, we demonstrate that Park2 gene (encodes Parkin) deletion specifically from adipose tissue protects mice against high-fat diet and aging-induced obesity. Despite a mild reduction in mitophagy, mitochondrial DNA content and mitochondrial function are increased in Park2 deficient white adipocytes. Moreover, Park2 gene deletion elevates mitochondrial biogenesis by increasing Pgc1α protein stability through mitochondrial superoxide-activated NAD(P)H quinone dehydrogenase 1 (Nqo1). Both in vitro and in vivo studies show that Nqo1 overexpression elevates Pgc1α protein level and mitochondrial DNA content and enhances mitochondrial activity in mouse and human adipocytes. Taken together, our findings indicate that Parkin regulates mitochondrial homeostasis by balancing mitophagy and Pgc1α-mediated mitochondrial biogenesis in white adipocytes, suggesting a potential therapeutic target in adipocytes to combat obesity and obesity-associated disorders.

AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology.

Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane-localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.

Deletion of ABCB10 in beta-cells protects from high-fat diet induced insulin resistance.

OBJECTIVE: The contribution of beta-cell dysfunction to type 2 diabetes (T2D) is not restricted to insulinopenia in the late stages of the disease. Elevated fasting insulinemia in normoglycemic humans is a major factor predicting the onset of insulin resistance and T2D, demonstrating an early alteration of beta-cell function in T2D. Moreover, an early and chronic increase in fasting insulinemia contributes to insulin resistance in high-fat diet (HFD)-fed mice. However, whether there are genetic factors that promote beta-cell-initiated insulin resistance remains undefined. Human variants of the mitochondrial transporter ABCB10, which regulates redox by increasing bilirubin synthesis, have been associated with an elevated risk of T2D. The effects of T2D ABCB10 variants on ABCB10 expression and the actions of ABCB10 in beta-cells are unknown. METHODS: The expression of beta-cell ABCB10 was analyzed in published transcriptome datasets from human beta-cells carrying the T2D-risk ABCB10 variant. Insulin sensitivity, beta-cell proliferation, and secretory function were measured in beta-cell-specific ABCB10 KO mice (Ins1Cre-Abcb10flox/flox). The short-term role of beta-cell ABCB10 activity on glucose-stimulated insulin secretion (GSIS) was determined in isolated islets. RESULTS: Carrying the T2Drisk allele G of ABCB10 rs348330 variant was associated with increased ABCB10 expression in human beta-cells. Constitutive deletion of Abcb10 in beta-cells protected mice from hyperinsulinemia and insulin resistance by limiting HFD-induced beta-cell expansion. An early limitation in GSIS and H2O2-mediated signaling caused by elevated ABCB10 activity can initiate an over-compensatory expansion of beta-cell mass in response to HFD. Accordingly, increasing ABCB10 expression was sufficient to limit GSIS capacity. In health, ABCB10 protein was decreased during islet maturation, with maturation restricting beta-cell proliferation and elevating GSIS. Finally, ex-vivo and short-term deletion of ABCB10 in islets isolated from HFD-fed mice increased H2O2 and GSIS, which was reversed by bilirubin treatments. CONCLUSIONS: Beta-cell ABCB10 is required for HFD to induce insulin resistance in mice by amplifying beta-cell mass expansion to maladaptive levels that cause fasting hyperinsulinemia.

High-Throughput Image Analysis of Lipid-Droplet-Bound Mitochondria.

Changes to mitochondrial architecture are associated with various adaptive and pathogenic processes. However, quantification of changes to mitochondrial structures is limited by the yet unmet challenge of defining the borders of each individual mitochondrion within an image. Here, we describe a novel method for segmenting primary brown adipocyte (BA) mitochondria images. We describe a granular approach to quantifying subcellular structures, particularly mitochondria in close proximity to lipid droplets: peridroplet mitochondria. In addition, we lay out a novel machine-learning-based mitochondrial segmentation method that eliminates the bias of manual mitochondrial segmentation and improves object recognition compared to conventional thresholding analyses. By applying these methods, we discovered a significant difference between cytosolic and peridroplet BA mitochondrial H2O2 production and validated the machine-learning algorithm in BA via norepinephrine-induced mitochondrial fragmentation and comparing manual analyses to the automated analysis. This approach provides a high-throughput analysis protocol to quantify ratiometric probes in subpopulations of mitochondria in adipocytes.

ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

Estrogen receptor α controls metabolism in white and brown adipocytes by regulating Polg1 and mitochondrial remodeling.

Obesity is heightened during aging, and although the estrogen receptor α (ERα) has been implicated in the prevention of obesity, its molecular actions in adipocytes remain inadequately understood. Here, we show that adipose tissue ESR1/Esr1 expression inversely associated with adiposity and positively associated with genes involved in mitochondrial metabolism and markers of metabolic health in 700 Finnish men and 100 strains of inbred mice from the UCLA Hybrid Mouse Diversity Panel. To determine the anti-obesity actions of ERα in fat, we selectively deleted Esr1 from white and brown adipocytes in mice. In white adipose tissue, Esr1 controlled oxidative metabolism by restraining the targeted elimination of mitochondria via the E3 ubiquitin ligase parkin. mtDNA content was elevated, and adipose tissue mass was reduced in adipose-selective parkin knockout mice. In brown fat centrally involved in body temperature maintenance, Esr1 was requisite for both mitochondrial remodeling by dynamin-related protein 1 (Drp1) and uncoupled respiration thermogenesis by uncoupled protein 1 (Ucp1). In both white and brown fat of female mice and adipocytes in culture, mitochondrial dysfunction in the context of Esr1 deletion was paralleled by a reduction in the expression of the mtDNA polymerase γ subunit Polg1 We identified Polg1 as an ERα target gene by showing that ERα binds the Polg1 promoter to control its expression in 3T3L1 adipocytes. These findings support strategies leveraging ERα action on mitochondrial function in adipocytes to combat obesity and metabolic dysfunction.

Quantification of cristae architecture reveals time-dependent characteristics of individual mitochondria.

Recent breakthroughs in live-cell imaging have enabled visualization of cristae, making it feasible to investigate the structure-function relationship of cristae in real time. However, quantifying live-cell images of cristae in an unbiased way remains challenging. Here, we present a novel, semi-automated approach to quantify cristae, using the machine-learning Trainable Weka Segmentation tool. Compared with standard techniques, our approach not only avoids the bias associated with manual thresholding but more efficiently segments cristae from Airyscan and structured illumination microscopy images. Using a cardiolipin-deficient cell line, as well as FCCP, we show that our approach is sufficiently sensitive to detect perturbations in cristae density, size, and shape. This approach, moreover, reveals that cristae are not uniformly distributed within the mitochondrion, and sites of mitochondrial fission are localized to areas of decreased cristae density. After a fusion event, individual cristae from the two mitochondria, at the site of fusion, merge into one object with distinct architectural values. Overall, our study shows that machine learning represents a compelling new strategy for quantifying cristae in living cells.

Method for live-cell super-resolution imaging of mitochondrial cristae and quantification of submitochondrial membrane potentials.

The emergence of diffraction-unlimited live-cell imaging technologies has enabled the examination of mitochondrial form and function in unprecedented detail. We recently developed an approach for visualizing the inner mitochondrial membrane and determined that cristae membranes possess distinct mitochondrial membrane potentials, representing unique bioenergetic subdomains within the same organelle. Here, we outline a methodology for resolving cristae and inner boundary membranes using the LSM880 with Airyscan. Furthermore, we demonstrate how to analyze TMRE fluorescence intensity using the Nernst equation to calculate membrane potentials of individual cristae. Altogether, using these new techniques to study the electrochemical properties of the cristae can help to gain deeper insight into the still elusive nature of the mitochondrion.

Cristae undergo continuous cycles of membrane remodelling in a MICOS-dependent manner.

The mitochondrial inner membrane can reshape under different physiological conditions. How, at which frequency this occurs in living cells, and the molecular players involved are unknown. Here, we show using state-of-the-art live-cell stimulated emission depletion (STED) super-resolution nanoscopy that neighbouring crista junctions (CJs) dynamically appose and separate from each other in a reversible and balanced manner in human cells. Staining of cristae membranes (CM), using various protein markers or two lipophilic inner membrane-specific dyes, further revealed that cristae undergo continuous cycles of membrane remodelling. These events are accompanied by fluctuations of the membrane potential within distinct cristae over time. Both CJ and CM dynamics depended on MIC13 and occurred at similar timescales in the range of seconds. Our data further suggest that MIC60 acts as a docking platform promoting CJ and contact site formation. Overall, by employing advanced imaging techniques including fluorescence recovery after photobleaching (FRAP), single-particle tracking (SPT), live-cell STED and high-resolution Airyscan microscopy, we propose a model of CJ dynamics being mechanistically linked to CM remodelling representing cristae membrane fission and fusion events occurring within individual mitochondria.

Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent.

The mitochondrial membrane potential (ΔΨm ) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨm . However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨm at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨm , corresponding to cristae and IBM. ΔΨm was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨm heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨm . Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.

Elamipretide Promotes Mitophagosome Formation and Prevents Its Reduction Induced by Nutrient Excess in INS1 ß-cells.

Elamipretide is a tetrapeptide that restores defects in mitochondrial function, binds to cardiolipin, and is being tested in clinical trials for mitochondria-related diseases. However, whether elamipretide modulates mitochondrial quality control and dynamics, processes essential to preserve mitochondrial function, is unclear. Thus, we tested the effects of elamipretide on mitochondrial morphology, mitophagosome formation, and their early disruption induced by excess nutrients in INS1 ß-cells. Elamipretide treatment was sufficient to increase engulfment of mitochondria into autophagosomes in control INS1 ß-cells, without inducing widespread changes in mitochondrial morphology or membrane potential. In an early pathogenic context mimicked by short-term exposure to nutrient excess, elamipretide treatment prevented both mitochondrial fragmentation and defects in the engulfment of mitochondria into autophagosomes. On the other hand, elamipretide did not prevent lysosomal defects induced by nutrient excess. Accordingly, elamipretide treatment did not entail benefits on pathogenic p62 and LC3II accumulation or on insulin secretory function. In conclusion, our data show that elamipretide selectively stimulates the engulfment of mitochondria into autophagosomes and prevents its defects induced by nutrient excess. Thus, we propose that improved selectivity of mitochondrial quality control processes might contribute to the benefits stemming from elamipretide treatments in other disease models.

Potential role of chitinase 3-like-1 in inflammation-associated carcinogenic changes of epithelial cells.

The family of mammalian chitinases includes members both with and without glycohydrolase enzymatic activity against chitin, a polymer of N-acetylglucosamine. Chitin is the structural component of fungi, crustaceans, insects and parasitic nematodes, but is completely absent in mammals. Exposure to antigens containing chitin- or chitin-like structures sometimes induces strong T helper type-I responses in mammals, which may be associated with the induction of mammalian chitinases. Chitinase 3-like-1 (CHI3L1), a member of the mammalian chitinase family, is induced specifically during the course of inflammation in such disorders as inflammatory bowel disease, hepatitis and asthma. In addition, CHI3L1 is expressed and secreted by several types of solid tumors including glioblastoma, colon cancer, breast cancer and malignant melanoma. Although the exact function of CHI3L1 in inflammation and cancer is still largely unknown, CHI3L1 plays a pivotal role in exacerbating the inflammatory processes and in promoting angiogenesis and remodeling of the extracellular matrix. CHI3L1 may be highly involved in the chronic engagement of inflammation which potentiates development of epithelial tumorigenesis presumably by activating the mitogen-activated protein kinase and the protein kinase B signaling pathways. Anti-CHI3L1 antibodies or pan-chitinase inhibitors may have the potential to suppress CHI3L1-mediated chronic inflammation and the subsequent carcinogenic change in epithelial cells.