Multiomics reveals multilevel control of renal and systemic metabolism by the renal tubular circadian clock.

Circadian rhythmicity in renal function suggests rhythmic adaptations in renal metabolism. To decipher the role of the circadian clock in renal metabolism, we studied diurnal changes in renal metabolic pathways using integrated transcriptomic, proteomic, and metabolomic analysis performed on control mice and mice with an inducible deletion of the circadian clock regulator Bmal1 in the renal tubule (cKOt). With this unique resource, we demonstrated that approximately 30% of RNAs, approximately 20% of proteins, and approximately 20% of metabolites are rhythmic in the kidneys of control mice. Several key metabolic pathways, including NAD+ biosynthesis, fatty acid transport, carnitine shuttle, and ß-oxidation, displayed impairments in kidneys of cKOt mice, resulting in perturbed mitochondrial activity. Carnitine reabsorption from primary urine was one of the most affected processes with an approximately 50% reduction in plasma carnitine levels and a parallel systemic decrease in tissue carnitine content. This suggests that the circadian clock in the renal tubule controls both kidney and systemic physiology.

Methodological advancements in organ-specific ectopic lipid quantitative characterization: Effects of high fat diet on muscle and liver intracellular lipids.

OBJECTIVE: Ectopic lipid accumulation is a hallmark of metabolic diseases, linking obesity to non-alcoholic fatty liver disease, insulin resistance and diabetes. The use of zebrafish as a model of obesity and diabetes is raising due to the conserved properties of fat metabolism between humans and zebrafish, the homologous genes regulating lipid uptake and transport, the implementation of the '3R's principle and their cost-effectiveness. To date, a method allowing the conservation of lipid droplets (LDs) and organs in zebrafish larvae to image ectopic lipids is not available. Our objectives were to develop a novel methodology to quantitatively evaluate organ-specific LDs, in skeletal muscle and liver, in response to a nutritional perturbation. METHODS: We developed a novel embedding and cryosectioning protocol allowing the conservation of LDs and organs in zebrafish larvae. To establish the quantitative measures, we used a three-arm parallel nutritional intervention design. Zebrafish larvae were fed a control diet containing 14% of nutritional fat or two high fat diets (HFDs) containing 25 and 36% of dietary fats. In muscle and liver, LDs were characterized using immunofluorescence confocal microscopy. In liver, intrahepatocellular lipids were discriminated from intrasinusoid lipids. To complete liver characteristics, fibrosis was identified with Masson's Trichrome staining. Finally, to confirm the conservation and effect of HFD, molecular players of fat metabolism were evaluated by RT-qPCR. RESULTS: The cryosections obtained after setting up the embedding and cryopreservation method were of high quality, preserving tissue morphology and allowing the visualization of ectopic lipids. Both HFDs were obesogenic, without modifying larvae survival or development. Neutral lipid content increased with time and augmented dietary fat. Intramuscular LD volume density increased and was explained by an increase in LDs size but not in numbers. Intrahepatocellular LD volume density increased and was explained by an increased number of LDs, not by their increased size. Sinusoid area and lipid content were both increased. Hepatic fibrosis appeared with both HFDs. We observed alterations in the expression of genes associated with LD coating proteins, LD dynamics, lipogenesis, lipolysis and fatty acid oxidation. CONCLUSIONS: In this study, we propose a reproducible and fast method to image zebrafish larvae without losing LD quality and organ morphology. We demonstrate the impact of HFD on LD characteristics in liver and skeletal muscle accompanied by alterations of key players of fat metabolism. Our observations confirm the evolutionarily conserved mechanisms in lipid metabolism and reveal organ specific adaptations. The methodological advancements proposed in this work open the doors to study organelle adaptations in obesity and diabetes related research such as lipotoxicity, organelle contacts and specific lipid depositions.

Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues.

This study investigated the role of CDK4 in the oxidative metabolism of brown adipose tissue (BAT). BAT from Cdk4-/- mice exhibited fewer lipids and increased mitochondrial volume and expression of canonical thermogenic genes, rendering these mice more resistant to cold exposure. Interestingly, these effects were not BAT cell-autonomous but rather driven by increased sympathetic innervation. In particular, the ventromedial hypothalamus (VMH) is known to modulate BAT activation via the sympathetic nervous system. We thus examined the effects of VMH neuron-specific Cdk4 deletion. These mice display increased sympathetic innervation and enhanced cold tolerance, similar to Cdk4-/- mice, in addition to browning of scWAT. Overall, we provide evidence showing that CDK4 modulates thermogenesis by regulating sympathetic innervation of adipose tissue depots through hypothalamic nuclei, including the VMH. This demonstrates that CDK4 not only negatively regulates oxidative pathways, but also modulates the central regulation of metabolism through its action in the brain.

Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish.

The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with super-assembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here, we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2  + IV2 , I + IV, and I + III2  + IV2 . To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (scaf1). scaf1-/- fish displayed altered OXPHOS activity due to the disrupted interaction of complexes III and IV. scaf1-/- fish were smaller in size and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by Scaf1 modulates OXPHOS efficiency and allows the optimization of metabolic resources.

A novel approach to measure mitochondrial respiration in frozen biological samples.

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

Spastin mutations impair coordination between lipid droplet dispersion and reticulum.

Lipid droplets (LD) are affected in multiple human disorders. These highly dynamic organelles are involved in many cellular roles. While their intracellular dispersion is crucial to ensure their function and other organelles-contact, underlying mechanisms are still unclear. Here we show that Spastin, one of the major proteins involved in Hereditary Spastic Paraplegia (HSP), controls LD dispersion. Spastin depletion in zebrafish affects metabolic properties and organelle dynamics. These functions are ensured by a conserved complex set of splice variants. M1 isoforms determine LD dispersion in the cell by orchestrating endoplasmic reticulum (ER) shape along microtubules (MTs). To further impact LD fate, Spastin modulates transcripts levels and subcellular location of other HSP key players, notably Seipin and REEP1. In pathological conditions, mutations in human Spastin M1 disrupt this mechanism and impacts LD network. Spastin depletion influences not only other key proteins but also modulates specific neutral lipids and phospholipids, revealing an impact on membrane and organelle components. Altogether our results show that Spastin and its partners converge in a common machinery that coordinates LD dispersion and ER shape along MTs. Any alteration of this system results in HSP clinical features and impacts lipids profile, thus opening new avenues for novel biomarkers of HSP.

Renal tubular arginase-2 participates in the formation of the corticomedullary urea gradient and attenuates kidney damage in ischemia-reperfusion injury in mice.

AIM: Arginase 2 (ARG2) is a mitochondrial enzyme that catalyses hydrolysis of l-arginine into urea and l-ornithine. In the kidney, ARG2 is localized to the S3 segment of the proximal tubule. It has been shown that expression and activity of this enzyme are upregulated in a variety of renal pathologies, including ischemia-reperfusion (IR) injury. However, the (patho)physiological role of ARG2 in the renal tubule remains largely unknown. METHODS: We addressed this question in mice with conditional knockout of Arg2 in renal tubular cells (Arg2lox/lox /Pax8-rtTA/LC1 or, cKO mice). RESULTS: We demonstrate that cKO mice exhibit impaired urea concentration and osmolality gradients along the corticomedullary axis. In a model of unilateral ischemia-reperfusion injury (UIRI) with an intact contralateral kidney, ischemia followed by 24 hours of reperfusion resulted in significantly more pronounced histological damage in ischemic kidneys from cKO mice compared to control and sham-operated mice. In parallel, UIRI-subjected cKO mice exhibited a broad range of renal functional abnormalities, including albuminuria and aminoaciduria. Fourteen days after UIRI, the cKO mice exhibited complex phenotype characterized by significantly lower body weight, increased plasma levels of early predictive markers of kidney disease progression (asymmetric dimethylarginine and symmetric dimethylarginine), impaired mitochondrial function in the ischemic kidney but no difference in kidney fibrosis as compared to control mice. CONCLUSION: Collectively, these results establish the role of ARG2 in the formation of corticomedullary urea and osmolality gradients and suggest that this enzyme attenuates kidney damage in ischemia-reperfusion injury.

Mitochondria in Embryogenesis: An Organellogenesis Perspective.

Organogenesis is well characterized in vertebrates. However, the anatomical and functional development of intracellular compartments during this phase of development remains unknown. Taking an organellogenesis point of view, we characterize the spatiotemporal adaptations of the mitochondrial network during zebrafish embryogenesis. Using state of the art microscopy approaches, we find that mitochondrial network follows three distinct distribution patterns during embryonic development. Despite of this constant morphological change of the mitochondrial network, electron transport chain supercomplexes occur at early stages of embryonic development and conserve a stable organization throughout development. The remodeling of the mitochondrial network and the conservation of its structural components go hand-in-hand with somite maturation; for example, genetic disruption of myoblast fusion impairs mitochondrial network maturation. Reciprocally, mitochondria quality represents a key factor to determine embryonic progression. Alteration of mitochondrial polarization and electron transport chain halts embryonic development in a reversible manner suggesting developmental checkpoints that depend on mitochondrial integrity. Our findings establish the subtle dialogue and co-dependence between organogenesis and mitochondria in early vertebrate development. They also suggest the importance of adopting subcellular perspectives to understand organelle-organ communications during embryogenesis.

Tanycytes Regulate Lipid Homeostasis by Sensing Free Fatty Acids and Signaling to Key Hypothalamic Neuronal Populations via FGF21 Secretion.

The hypothalamus plays a key role in the detection of energy substrates to regulate energy homeostasis. Tanycytes, the hypothalamic ependymo-glia, are located at a privileged position to integrate multiple peripheral inputs. We observed that tanycytes produce and secrete Fgf21 and are located close to Fgf21-sensitive neurons. Fasting, likely via the increase in circulating fatty acids, regulates this central Fgf21 production. Tanycytes store palmitate in lipid droplets and oxidize it, leading to the activation of a reactive oxygen species (ROS)/p38-MAPK signaling pathway, which is essential for tanycytic Fgf21 expression upon palmitate exposure. Tanycytic Fgf21 deletion triggers an increase in lipolysis, likely due to impaired inhibition of key neurons during fasting. Mice deleted for tanycytic Fgf21 exhibit increased energy expenditure and a reduction in fat mass gain, reminiscent of a browning phenotype. Our results suggest that tanycytes sense free fatty acids to maintain body lipid homeostasis through Fgf21 signaling within the hypothalamus.

Distinct patterns of skeletal muscle mitochondria fusion, fission and mitophagy upon duration of exercise training.

AIM: Healthy ageing interventions encompass regular exercise to prevent mitochondrial dysfunction, key player in sarcopenia pathogenesis. Mitochondrial biogenesis has been well documented, but mitochondrial remodelling in response to exercise training is poorly understood. Here we investigated fusion, fission and mitophagy before and after an exercise intervention in older adults. METHODS: Skeletal muscle biopsies were collected from 22 healthy sedentary men and women before and after 4 months of supervised training. Eight lifelong trained age- and gender-matched volunteers served as positive controls. Transmission electron microscopy was used to estimate mitochondrial content. Western blotting and qRT-PCR were used to detect changes in specific proteins and transcripts. RESULTS: After intervention, mitochondrial content increased to levels of controls. While enhancement of fusion was prevalent after intervention, inhibition of fission and increased mitophagy were dominant in controls. Similarly to PARKIN, BCL2L13 content was higher in controls. The observed molecular adaptations paralleled long-term effects of training on physical fitness, exercise efficiency and oxidative capacity. CONCLUSIONS: This study describes distinct patterns of molecular adaptations in human skeletal muscle under chronic exercise training. After 16 weeks of exercise, the pattern was dominated by fusion to increase mitochondrial content to the metabolic demands of exercise. In lifelong exercise, the pattern was dominated by mitophagy synchronized with increased fusion and decreased fission, indicating an increased mitochondrial turnover. In addition to these temporally distinct adaptive mechanisms, this study suggests for the first time a specific role of BCL2L13 in chronic exercise that requires constant maintenance of mitochondrial quality.

CDK4 Phosphorylates AMPKα2 to Inhibit Its Activity and Repress Fatty Acid Oxidation.

The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4-/- mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK.

Enhanced Respiratory Chain Supercomplex Formation in Response to Exercise in Human Skeletal Muscle.

Mitochondrial dysfunction is a hallmark of multiple metabolic complications. Physical activity is known to increase mitochondrial content in skeletal muscle, counteracting age-related decline in muscle function and protecting against metabolic and cardiovascular complications. Here, we investigated the effect of 4 months of exercise training on skeletal muscle mitochondria electron transport chain complexes and supercomplexes in 26 healthy, sedentary older adults. Exercise differentially modulated respiratory complexes. Complex I was the most upregulated complex and not stoichiometrically associated to the other complexes. In contrast to the other complexes, complex I was almost exclusively found assembled in supercomplexes in muscle mitochondria. Overall, supercomplex content was increased after exercise. In particular, complexes I, III, and IV were redistributed to supercomplexes in the form of I+III2+IV. Taken together, our results provide the first evidence that exercise affects the stoichiometry of supercomplex formation in humans and thus reveal a novel adaptive mechanism for increased energy demand.

CDK4 is an essential insulin effector in adipocytes.

Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4(R24C)). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.

E2F transcription factor-1 regulates oxidative metabolism.

Cells respond to stress by coordinating proliferative and metabolic pathways. Starvation restricts cell proliferative (glycolytic) and activates energy productive (oxidative) pathways. Conversely, cell growth and proliferation require increased glycolytic and decreased oxidative metabolism levels. E2F transcription factors regulate both proliferative and metabolic genes. E2Fs have been implicated in the G1/S cell-cycle transition, DNA repair, apoptosis, development and differentiation. In pancreatic ß-cells, E2F1 gene regulation facilitated glucose-stimulated insulin secretion. Moreover, mice lacking E2F1 (E2f1(-/-)) were resistant to diet-induced obesity. Here, we show that E2F1 coordinates cellular responses by acting as a regulatory switch between cell proliferation and metabolism. In basal conditions, E2F1 repressed key genes that regulate energy homeostasis and mitochondrial functions in muscle and brown adipose tissue. Consequently, E2f1(-/-) mice had a marked oxidative phenotype. An association between E2F1 and pRB was required for repression of genes implicated in oxidative metabolism. This repression was alleviated in a constitutively active CDK4 (CDK4(R24C)) mouse model or when adaptation to energy demand was required. Thus, E2F1 represents a metabolic switch from oxidative to glycolytic metabolism that responds to stressful conditions.

E4F1 deficiency results in oxidative stress-mediated cell death of leukemic cells.

The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA-mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest.

[Emerging key role of cell cycle regulators in cell metabolism]. / Le double jeu des régulateurs du cycle cellulaire: contrôle de la prolifération et du métabolisme.

The role of cell cycle regulators in the control of cell proliferation has been extensively studied, but independently of these functions in cell proliferation, it now appears that these proteins are also key to the adapted metabolic response of the cells. This has some logic since cell cycle is linked to metabolic control. This review focusses on the involvment of cyclins, cyclin dependent kinases or E2F factor in the control of adipogenesis, glucidic homeostasis, and energy consumption. Murine models in which genes encoding these regulators have been invalidated have been key to unravel these novel functions of cell cycle regulators in cell metabolism. Furthermore, these findings may also have some relevance for metabolic disorders such as obesity or diabetes.

CXC ligand 5 is an adipose-tissue derived factor that links obesity to insulin resistance.

We show here high levels of expression and secretion of the chemokine CXC ligand 5 (CXCL5) in the macrophage fraction of white adipose tissue (WAT). Moreover, we find that CXCL5 is dramatically increased in serum of human obese compared to lean subjects. Conversely, CXCL5 concentration is decreased in obese subjects after a weight reduction program, or in obese non-insulin-resistant, compared to insulin-resistant, subjects. Most importantly we demonstrate that treatment with recombinant CXCL5 blocks insulin-stimulated glucose uptake in muscle in mice. CXCL5 blocks insulin signaling by activating the Jak2/STAT5/SOCS2 pathway. Finally, by treating obese, insulin-resistant mice with either anti-CXCL5 neutralizing antibodies or antagonists of CXCR2, which is the CXCL5 receptor, we demonstrate that CXCL5 mediates insulin resistance. Furthermore CXCR2-/- mice are protected against obesity-induced insulin resistance. Taken together, these results show that secretion of CXCL5 by WAT resident macrophages represents a link between obesity, inflammation, and insulin resistance.