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Abstract Sporadic Parkinson’s Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD.

Objective: Enhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. Method: NAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). Result: NAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. Conclusion: NAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications.

Objective: Uncoupling protein 1 (UCP1) catalyses mitochondrial proton leak in brown adipose tissue to facilitate nutrient oxidation for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we set out to characterise activator binding to purified UCP1 to clarify the activation process, discern novel activators and the potential to target UCP1. Methods: We assessed ligand binding to purified UCP1 by protein thermostability shift analysis, which unlike many conventional approaches can inform on the binding of hydrophobic ligands to membrane proteins. A detailed activator interaction analysis and screening approach was carried out, supported by investigations of UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1 expression-controlled cell lines. Results: We reveal that fatty acids and other activators influence UCP1 through a specific destabilising interaction, behaving as transport substrates that shift the protein to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the over-the-counter drug ibuprofen, where ligand analysis indicates that UCP1 has a relatively wide structural specificity for interacting molecules. Ibuprofen successfully induced UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1-expressing HEK293 cells but not in cultured brown adipocytes, suggesting drug delivery differs in each cell type. Conclusions: These findings clarify the nature of the activator-UCP1 interaction and demonstrate that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.

Beta-adrenergic stimulation of brown adipose tissue leads to thermogenesis via the activating transcription factor 2 (ATF2) mediated expression of the thermogenic genes Ucp1 and Pgc-1α. Here, the authors show that the scaffold protein p62 regulates brown adipose tissue function through modifying ATF2 genomic binding and subsequent Ucp1 and Pgc-1α induction.

Brown adipose thermogenesis increases energy expenditure and relies on uncoupling protein 1 (UCP1), however, UCP1 knock-out mice show resistance to diet-induced obesity at room temperature. Here, the authors show that this resistance relies on FGF21-signaling, inducing the browning of white adipose tissue.

Oxygen consumption allows measuring the metabolic activity of organisms. Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (Danio rerio) in situ. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and penetration of various mitochondrial inhibitors, and confirm that sedation reduces the variability of OCR. At 48h post-fertilization, embryos (maintained at a routine temperature of 28°C) were exposed to different medium temperatures ranging from 18°C to 37°C for 20h prior OCR measurement. Measurement temperatures from 18°C to 45°C in the XF96 were achieved by lowering the room temperature and active in-built heating. At 18°C assay temperature, basal OCR was low due to decreased ATP-linked respiration, which was not limited by mitochondrial power, as seen in substantial spare respiratory capacity. Basal OCR of the embryos increased with assay temperature and were stable up to 37°C assay temperature, with pre-exposure of 37°C resulting in more thermo-resistant basal OCR measured at 41°C. Adverse effects of the mitochondrial inhibitor oligomycin were seen at 37°C and chemical uncouplers disrupted substrate oxidation gradually with increasing assay temperature. Proton leak respiration increased at assay temperatures above 28°C and compromised the efficiency of ATP production, calculated as coupling efficiency. Thus, temperature impacts mitochondrial respiration by reduced cellular ATP turnover at lower temperatures and by increased proton leak at higher temperatures. This conclusion is coherent with the assessment of heart rate, an independent indicator of systemic metabolic rate, which increased with exposure temperature, peaking at 28°C, and decreased at higher temperatures. Collectively, plate-based respirometry allows assessing distinct parts of mitochondrial energy transduction in zebrafish embryos and investigating the effect of temperature and temperature acclimation on mitochondrial bioenergetics in situ.

Background/Objectives: Although the prevalence of obesity and its associated metabolic disorders is increasing in both sexes, the clinical phenotype differs between men and women, highlighting the need for individual treatment options. Mitochondrial dysfunction in various tissues, including white adipose tissue (WAT), has been accepted as a key factor for obesity-associated comorbidities such as diabetes. Given higher expression of mitochondria-related genes in the WAT of women, we hypothesized that gender differences in the bioenergetic profile of white (pre-) adipocytes from obese (age- and BMI-matched) donors must exist. Subjects/Methods: Using Seahorse technology, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of (pre-)adipocytes from male (n = 10) and female (n = 10) deeply-phenotyped obese donors under hypo-, normo- and hyperglycemic (0, 5 and 25 mM glucose) and insulin-stimulated conditions. Additionally, expression levels (mRNA/protein) of mitochondria-related genes (e.g. UQCRC2) and glycolytic enzymes (e.g. PKM2) were determined. Results: Dissecting cellular OCR and ECAR into different functional modules revealed that preadipocytes from female donors show significantly higher mitochondrial to glycolytic activity (higher OCR/ECAR ratio, p = 0.036), which is supported by a higher ratio of UQCRC2 to PKM2 mRNA levels (p = 0.021). However, no major gender differences are detectable in in vitro differentiated adipocytes (e.g. OCR/ECAR, p = 0.248). Importantly, glucose and insulin suppress mitochondrial activity (i.e. ATP-linked respiration) significantly only in preadipocytes of female donors, reflecting their trends towards higher insulin sensitivity. Conclusions: Collectively, we show that preadipocytes, but not in vitro differentiated adipocytes, represent a model system to reveal gender differences with clinical importance for metabolic disease status. In particular preadipocytes of females maintain enhanced mitochondrial flexibility, as demonstrated by pronounced responses of ATP-linked respiration to glucose. Keywords: Oxidative phosphorylation, Glycolysis, Cellular metabolism

Tobacco smoking and cold exposure are environmental modulators of human energy metabolism suppressing appetite and increasing energy expenditure, respectively. Here, the authors develop a novel pharmacological strategy in which they simultaneously mimic the metabolic benefits of both phenomena through small-molecule combination therapy, and show that this treatment improves metabolic health of obese mice.

Objective: Energy metabolism is challenged upon nutrient stress, eventually leading to a variety of metabolic diseases that represent a major global health burden. Methods: Here, we combine quantitative mitochondrial respirometry (Seahorse technology) and proteomics (LC-MS/MS-based total protein approach) to understand how molecular changes translate to changes in mitochondrial energy transduction during diet-induced obesity (DIO) in the liver. Results: The integrative analysis reveals that significantly increased palmitoyl-carnitine respiration is supported by an array of proteins enriching lipid metabolism pathways. Upstream of the respiratory chain, the increased capacity for ATP synthesis during DIO associates strongest to mitochondrial uptake of pyruvate, which is routed towards carboxylation. At the respiratory chain, robust increases of complex I are uncovered by cumulative analysis of single subunit concentrations. Specifically, nuclear-encoded accessory subunits, but not mitochondrial-encoded or core units, appear to be permissive for enhanced lipid oxidation. Conclusion: Our integrative analysis, that we dubbed “respiromics”, represents an effective tool to link molecular changes to functional mechanisms in liver energy metabolism, and, more generally, can be applied for mitochondrial analysis in a variety of metabolic and mitochondrial disease models. Keywords: Mitochondria, Respirometry, Proteomics, Mitochondrial pyruvate carrier, Liver disease, Bioenergetics, Obesity, Diabetes

Background & Aims: Although nearly half of pancreatic ductal adenocarcinoma (PDAC) patients have diabetes mellitus with episodes of hyperglycemia, its tumor microenvironment is hypoglycemic. Thus, it is crucial for PDAC cells to develop adaptive mechanisms dealing with oscillating glucose levels. So far, the biological impact of such glycemic variability on PDAC biology remains unknown. Methods: Murine PDAC cells were cultured in low- and high-glucose medium to investigate the molecular, biochemical, and metabolic influence of glycemic variability on tumor behavior. A set of in vivo functional assays including orthotopic implantation and portal and tail vein injection were used. Results were further confirmed on tissues from PDAC patients. Results: Glycemic variability has no significant effect on PDAC cell proliferation. Hypoglycemia is associated with local invasion and angiogenesis, whereas hyperglycemia promotes metastatic colonization. Increased metastatic colonization under hyperglycemia is due to increased expression of runt related transcription factor 3 (Runx3), which further activates expression of collagen, type VI, alpha 1 (Col6a1), forming a glycemic pro-metastatic pathway. Through epigenetic machinery, retinoic acid receptor beta (Rarb) expression fluctuates according to glycemic variability, acting as a critical sensor relaying the glycemic signal to Runx3/Col6a1. Moreover, the signal axis of Rarb/Runx3/Col6a1 is pharmaceutically accessible to a widely used antidiabetic substance, metformin, and Rar modulator. Finally, PDAC tissues from patients with diabetes show an increased expression of COL6A1. Conclusions: Glycemic variability promotes both local invasion and metastatic colonization of PDAC. A pro-metastatic signal axis Rarb/Runx3/Col6a1 whose activity is controlled by glycemic variability is identified. The therapeutic relevance of this pathway needs to be explored in PDAC patients, especially in those with diabetes. Keywords: Pancreatic Cancer, Glucose Metabolism, Metastasis, Retinoic Acid

Background: Tissue-resident macrophages have mixed developmental origins. They derive in variable extent from yolk sac (YS) hematopoiesis during embryonic development. Bone marrow (BM) hematopoietic progenitors give rise to tissue macrophages in postnatal life, and their contribution increases upon organ injury. Since the phenotype and functions of macrophages are modulated by the tissue of residence, the impact of their origin and developmental paths has remained incompletely understood. Methods: In order to decipher cell-intrinsic macrophage programs, we immortalized hematopoietic progenitors from YS and BM using conditional HoxB8, and carried out an in-depth functional and molecular analysis of differentiated macrophages. Results: While YS and BM macrophages demonstrate close similarities in terms of cellular growth, differentiation, cell death susceptibility and phagocytic properties, they display differences in cell metabolism, expression of inflammatory markers and inflammasome activation. Reduced abundance of PYCARD (ASC) and CASPASE-1 proteins in YS macrophages abrogated interleukin-1β production in response to canonical and non-canonical inflammasome activation. Conclusions: Macrophage ontogeny is associated with distinct cellular programs and immune response. Our findings contribute to the understanding of the regulation and programming of macrophage functions.

The data presented in this article describe the fatty acid composition of chow, liver tissue and isolated liver mitochondria from mice fed for 6â24 weeks with a high caloric western diet (WD) in comparison to control diet (normal diet, ND). The fatty acid composition was measured via gas chromatography flame ionization detection (GC-FID). Moreover, WD-induced mitochondrial protein changes are presented in this work and were analyzed by mass spectrometry (LCâMS/MS). For further interpretation and discussion of the presented data please refer to the research article entitled âMitochondrial adaptation in steatotic miceâ (Einer et al., 2017) [1].

Objective: Obesity-associated WAT inflammation is characterized by the accumulation and local activation of macrophages (MΦs), and recent data from mouse studies suggest that macrophages are modifiers of adipocyte energy metabolism and mitochondrial function. As mitochondrial dysfunction has been associated with obesity and the metabolic syndrome in humans, herein we aimed to delineate how human macrophages may affect energy metabolism of white adipocytes. Methods: Human adipose tissue gene expression analysis for markers of macrophage activation and tissue inflammation (CD11c, CD40, CD163, CD206, CD80, MCP1, TNFα) in relationship to mitochondrial complex I (NDUFB8) and complex III (UQCRC2) was performed on subcutaneous WAT of 24 women (BMI 20–61 kg/m2). Guided by these results, the impact of secreted factors of LPS/IFNγ- and IL10/TGFβ-activated human macrophages (THP1, primary blood-derived) on mitochondrial function in human subcutaneous white adipocytes (SGBS, primary) was determined by extracellular flux analysis (Seahorse technology) and gene/protein expression. Results: Stepwise regression analysis of human WAT gene expression data revealed that a linear combination of CD40 and CD163 was the strongest predictor for mitochondrial complex I (NDUFB8) and complex III (UQCRC2) levels, independent of BMI. IL10/TGFβ-activated MΦs displayed high CD163 and low CD40 expression and secreted factors that decreased UQCRC2 gene/protein expression and ATP-linked respiration in human white adipocytes. In contrast, LPS/IFNγ-activated MΦs showed high CD40 and low CD163 expression and secreted factors that enhanced adipocyte mitochondrial activity resulting in a total difference of 37% in ATP-linked respiration of white adipocytes (p = 0.0024) when comparing the effect of LPS/IFNγ- vs IL10/TGFβ-activated MΦs. Conclusion: Our data demonstrate that macrophages modulate human adipocyte energy metabolism via an activation-dependent paracrine mechanism.

Abstract Brown adipocytes are important in regulating non-shivering thermogenesis, whole body glucose and lipid homeostasis. Increasing evidence supports an important role of metabolites as well as macro- and micronutrients in brown adipocyte differentiation and function. Calcium is one of the most abundant ions in the body regulating multiple cellular processes. We observed that increasing extracellular calcium concentration during brown adipocyte differentiation blocks lipid accumulation and suppresses induction of major adipogenic transcription factors such as PPARγ and C/EBPα. In contrast, the depletion of calcium in the medium enhances adipogenesis and expression of brown adipocyte selective genes, such as UCP1. Mechanistically, we show that elevated extracellular calcium inhibits C/EBPβ activity through hyperactivation of ERK, a process that is independent of intracellular calcium levels and reversibly halts differentiation. Moreover, increased extracellular calcium solely after the induction phase of differentiation specifically suppresses gene expression of UCP1, PRDM16 and PGC1-α. Notably, depleting extracellular calcium provokes opposite effects. Together, we show that modulating extracellular calcium concentration controls brown adipocyte differentiation and thermogenic gene expression, highlighting the importance of tissue microenvironment on brown adipocyte heterogeneity and function.

Long-term consumption of a calorie-rich diet persistently activates brain microglia. Here, the authors show that microglial activity in mouse brains oscillates daily in conjunction with feeding, and that TNFα, secreted by activated microglia, induces mitochondrial stress in satiety-promoting POMC neurons.

The development of obesity and type 2 diabetes (T2D) has been associated with impaired mitochondrial function. In pancreatic beta (β) cells, mitochondrial energy metabolism plays a central role in triggering and controlling glucose-stimulated insulin secretion (GSIS). Here, we have explored whether mitochondrial bioenergetic parameters assessed with Seahorse extracellular flux technology can quantitatively predict insulin secretion. We metabolically stressed male C57BL/6 mice by high-fat feeding (HFD) and measured the glucose sensitivity of islet respiration and insulin secretion. The diet-induced obese (DIO) mice developed hyperinsulinemia, but no pathological secretory differences were apparent between isolated DIO and chow islets. Real-time extracellular flux analysis, however, revealed a lower respiratory sensitivity to glucose in DIO islets. Correlation of insulin secretion with respiratory parameters uncovers compromised insulin secretion in DIO islets by oxidative power. Normalization to increased insulin contents during DIO improves the quantitative relation between GSIS and respiration, allowing to classify dysfunctional properties of pancreatic insulin secretion, and thereby serving as valuable biomarker for pancreatic islet glucose responsiveness and health.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22119-x

Objective: Central melanocortin pathways are well-established regulators of energy balance. However, scant data exist about the role of systemic melanocortin peptides. We set out to determine if peripheral α-melanocyte stimulating hormone (α-MSH) plays a role in glucose homeostasis and tested the hypothesis that the pituitary is able to sense a physiological increase in circulating glucose and responds by secreting α-MSH. Methods: We established glucose-stimulated α-MSH secretion using humans, non-human primates, and mouse models. Continuous α-MSH infusions were performed during glucose tolerance tests and hyperinsulinemic-euglycemic clamps to evaluate the systemic effect of α-MSH in glucose regulation. Complementary ex vivo and in vitro techniques were employed to delineate the direct action of α-MSH via the melanocortin 5 receptor (MC5R)–PKA axis in skeletal muscles. Combined treatment of non-selective/selective phosphodiesterase inhibitor and α-MSH was adopted to restore glucose tolerance in obese mice. Results: Here we demonstrate that pituitary secretion of α-MSH is increased by glucose. Peripheral α-MSH increases temperature in skeletal muscles, acts directly on soleus and gastrocnemius muscles to significantly increase glucose uptake, and enhances whole-body glucose clearance via the activation of muscle MC5R and protein kinase A. These actions are absent in obese mice, accompanied by a blunting of α-MSH-induced cAMP levels in skeletal muscles of obese mice. Both selective and non-selective phosphodiesterase inhibition restores α-MSH induced skeletal muscle glucose uptake and improves glucose disposal in obese mice. Conclusion: These data describe a novel endocrine circuit that modulates glucose homeostasis by pituitary α-MSH, which increases muscle glucose uptake and thermogenesis through the activation of a MC5R-PKA-pathway, which is disrupted in obesity. Keywords: α-MSH, Pituitary, Skeletal muscles, MC5R, PKA, Glucose homeostasis

Objective: Fibroblast growth factor 21 (FGF21) was recently discovered as stress-induced myokine during mitochondrial disease and proposed as key metabolic mediator of the integrated stress response (ISR) presumably causing systemic metabolic improvements. Curiously, the precise cell-non-autonomous and cell-autonomous relevance of endogenous FGF21 action remained poorly understood. Methods: We made use of the established UCP1 transgenic (TG) mouse, a model of metabolic perturbations made by a specific decrease in muscle mitochondrial efficiency through increased respiratory uncoupling and robust metabolic adaptation and muscle ISR-driven FGF21 induction. In a cross of TG with Fgf21-knockout (FGF21−/−) mice, we determined the functional role of FGF21 as a muscle stress-induced myokine under low and high fat feeding conditions. Results: Here we uncovered that FGF21 signaling is dispensable for metabolic improvements evoked by compromised mitochondrial function in skeletal muscle. Strikingly, genetic ablation of FGF21 fully counteracted the cell-non-autonomous metabolic remodeling and browning of subcutaneous white adipose tissue (WAT), together with the reduction of circulating triglycerides and cholesterol. Brown adipose tissue activity was similar in all groups. Remarkably, we found that FGF21 played a negligible role in muscle mitochondrial stress-related improved obesity resistance, glycemic control and hepatic lipid homeostasis. Furthermore, the protective cell-autonomous muscle mitohormesis and metabolic stress adaptation, including an increased muscle proteostasis via mitochondrial unfolded protein response (UPRmt) and amino acid biosynthetic pathways did not require the presence of FGF21. Conclusions: Here we demonstrate that although FGF21 drives WAT remodeling, the adaptive pseudo-starvation response under elevated muscle mitochondrial stress conditions operates independently of both WAT browning and FGF21 action. Thus, our findings challenge FGF21 as key metabolic mediator of the mitochondrial stress adaptation and powerful therapeutic target during muscle mitochondrial disease. Author Video: Author Video Watch what authors say about their articles Keywords: Browning, FGF21, GDF15, Myokine, Mitochondrial disease, Muscle mitohormesis

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, but a substantial proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 °C, mitochondria were more than 10 °C warmer when the respiratory chain (RC) was fully functional, both in human embryonic kidney (HEK) 293 cells and primary skin fibroblasts. This differential was abolished in cells depleted of mitochondrial DNA or treated with respiratory inhibitors but preserved or enhanced by expressing thermogenic enzymes, such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at or slightly above 50 °C. In view of their potential consequences, these observations need to be further validated and explored by independent methods. Our study prompts a critical re-examination of the literature on mitochondria.

Objective: Circulating fibroblast growth factor 21 (FGF21) is an important auto- and endocrine player with beneficial metabolic effects on obesity and diabetes. In humans, thermogenic brown adipose tissue (BAT) was recently suggested as a source of FGF21 secretion during cold exposure. Here, we aim to clarify the role of UCP1 and ambient temperature in the regulation of FGF21 in mice. Methods: Wildtype (WT) and UCP1-knockout (UCP1 KO) mice, the latter being devoid of BAT-derived non-shivering thermogenesis, were exposed to different housing temperatures. Plasma metabolites and FGF21 levels were determined, gene expression was analyzed by qPCR, and tissue histology was performed with adipose tissue. Results: At thermoneutrality, FGF21 gene expression and serum levels were not different between WT and UCP1 KO mice. Cold exposure led to highly increased FGF21 serum levels in UCP1 KO mice, which were reflected in increased FGF21 gene expression in adipose tissues but not in liver and skeletal muscle. Ex vivo secretion assays revealed FGF21 release only from BAT, progressively increasing with decreasing ambient temperatures. In association with increased FGF21 serum levels in the UCP1 KO mouse, typical FGF21-related serum metabolites and inguinal white adipose tissue morphology and thermogenic gene expression were altered. Conclusions: Here we show that the genetic ablation of UCP1 increases FGF21 gene expression in adipose tissue. The removal of adaptive nonshivering thermogenesis renders BAT a significant source of endogenous FGF21 under thermal stress. Thus, the thermogenic competence of BAT is not a requirement for FGF21 secretion. Notably, high endogenous FGF21 levels in UCP1-deficient models and subjects may confound pharmacological FGF21 treatments.

Brown and beige adipose tissue may represent important therapeutic targets for the treatment of diabetes and obesity as these organs dissipate nutrient energy as heat through the thermogenic uncoupling protein 1 (UCP1). While mice are commonly used to mimic the potential effects of brown/beige adipose tissue that may act in human metabolism, new animal models are edging into the market for translational medicine. Pigs reflect human metabolism better than mice in multiple parameters such as obesity-induced hyperglycemia, cholesterol profiles and energy metabolism. Recently, it was reported that energy expenditure and body temperature in pigs is induced by the hormone leptin, and that leptin's action is mediated by UCP1 in adipose tissue. Given the tremendous importance of identifying molecular mechanisms for targeting therapeutics, we critically examine the evidence supporting the presence of UCP1 in pigs and conclude that methodological shortcomings prevent an unequivocal claim for the presence of UCP1 in pigs. Despite this, we believe that leptin's effects on energy expenditure in pigs are potentially more transformative to human medicine in the absence of UCP1, as adult and obese humans possess only minor amounts of UCP1. In general, we propose that the biology of new animal models requires attention to comparative studies with humans given the increasing amount of genomic information for various animal species.

Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells synthesize CoQ mostly endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, which tissues depend on exogenous CoQ uptake as well as the mechanism by which CoQ is taken up by cells and the role of this process in BAT function are not well understood. Here, we report that the scavenger receptor CD36 drives the uptake of CoQ by BAT and is required for normal BAT function. BAT from mice lacking CD36 displays CoQ deficiency, impaired CoQ uptake, hypertrophy, altered lipid metabolism, mitochondrial dysfunction, and defective nonshivering thermogenesis. Together, these data reveal an important new role for the systemic transport of CoQ to BAT and its function in thermogenesis.

Recently we demonstrated that the capacity of isolated muscle mitochondria to produce reactive oxygen species, measured as H2O2 efflux, is temperature-sensitive in isolated muscle mitochondria of ectothermic fish and the rat, a representative endothermic mammal. However, at physiological temperatures (15° and 37°C for the fish and rat, respectively), the fraction of total mitochondrial electron flux that generated H2O2, the fractional electron leak (FEL), was far lower in the rat than in fish. Those results suggested that the elevated body temperatures associated with endothermy may lead to a compensatory decrease in mitochondrial ROS production relative to respiratory capacity. To test this hypothesis we compare slow twitch (red) muscle mitochondria from the endothermic Pacific bluefin tuna (Thunnus orientalis) with mitochondria from three ectothermic fishes [rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), and the lake sturgeon (Acipenser fulvescens)] and the rat. At a common assay temperature (25°C) rates of mitochondrial respiration and H2O2 efflux were similar in tuna and the other fishes. The thermal sensitivity of fish mitochondria was similar irrespective of ectothermy or endothermy. Comparing tuna to the rat at a common temperature, respiration rates were similar, or lower depending on mitochondrial substrates. FEL was not different across fish species at a common assay temperature (25°C) but was markedly higher in fishes than in rat. Overall, endothermy and warming of Pacific Bluefin tuna red muscle may increase the potential for ROS production by muscle mitochondria but the evolution of endothermy in this species is not necessarily associated with a compensatory reduction of ROS production relative to the respiratory capacity of mitochondria.

The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.

In the original PDF version of this article, affiliation 1, ‘Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Muenchen & German Center for Diabetes Research (DZD), Neuherberg, Germany’, was incorrectly given as ‘Institute of Diabetes and Regeneration Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germany ‘. This has now been corrected in the PDF version of the article; the HTML version was correct at the time of publication.