M1-derived extracellular vesicles polarize recipient macrophages into M2-like macrophages and alter skeletal muscle homeostasis in a hyper-glucose environment.

BACKGROUND: Macrophages release not only cytokines but also extracellular vesicles (EVs). which are small membrane-derived nanovesicles with virus-like properties transferring cellular material between cells. Until now, the consequences of macrophage plasticity on the release and the composition of EVs have been poorly explored. In this study, we determined the impact of high-glucose (HG) concentrations on macrophage metabolism, and characterized their derived-EV subpopulations. Finally, we determined whether HG-treated macrophage-derived EVs participate in immune responses and in metabolic alterations of skeletal muscle cells. METHODS: THP1-macrophages were treated with 15mM (MG15) or 30mM (MG30) glucose. Then, M1/M2 canonical markers, pro- and anti-inflammatory cytokines, activities of proteins involved in glycolysis or oxidative phosphorylation were evaluated. Macrophage-derived EVs were characterized by TEM, NTA, MRSP, and 1H-Nuclear magnetic resonance spectroscopy for lipid composition. Macrophages or C2C12 muscle cells were used as recipients of MG15 and MG30-derived EVs. The lipid profiles of recipient cells were determined, as well as proteins and mRNA levels of relevant genes for macrophage polarization or muscle metabolism. RESULTS: Untreated macrophages released small and large EVs (sEVs, lEVs) with different lipid distributions. Proportionally to the glucose concentration, glycolysis was induced in macrophages, associated to mitochondrial dysfunction, triacylglycerol and cholesterol accumulation. In addition, MG15 and MG30 macrophages had increased level of CD86 and increase release of pro-inflammatory cytokines. HG also affected macrophage sphingolipid and phospholipid compositions. The differences in the lipid profiles between sEVs and lEVs were abolished and reflected the lipid alterations in MG15 and MG30 macrophages. Interestingly, MG15 and MG30 macrophages EVs induced the expression of CD163, Il-10 and increased the contents of triacylglycerol and cholesterol in recipient macrophages. MG15 lEVs and sEVs induced insulin-induced AKT hyper-phosphorylation and accumulation of triacylglycerol in myotubes, a state observed in pre-diabetes. Conversely, MG30 lEVs and sEVs induced insulin-resistance in myotubes. CONCLUSIONS: As inflammation involves first M1 macrophages, then the activation of M2 macrophages to resolve inflammation, this study demonstrates that the dialog between macrophages through the EV route is an intrinsic part of the inflammatory response. In a hyperglycemic context, EV macrophages could participate in the development of muscle insulin-resistance and chronic inflammation.

Exploring mitochondrial metabolism of wild-type and diabetic mice skin explants using the Seahorse technology.

BACKGROUND: Skin wound healing is a complex mechanism which requires a lot of energy, mainly provided by mitochondrial respiration. However, little is known about the mitochondrial bioenergetics of mice skin. We sought to develop a microplate-based assay to directly measure oxygen consumption in whole mice skin with the goal of identifying mitochondrial dysfunction in diabetic skin using an extracellular flux. MATERIALS AND METHODS: Different parameters were optimized to efficiently measure the oxygen consumption rate (OCR). First, the most pertinent skin side of wild-type mice was first determined. Then, concentrations of mitochondrial inhibitors were then optimized to get the best efficacy. Finally, punch sizes were modulated to get the best OCR profile. RESULTS: Dermis had the best metabolic activity side of the skin. Unlike the increased concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A, which showed no improvement of these drugs' effects, varying the skin punch size was successful. Finally, type II diabetic (T2D) skin produced less ATP through mitochondrial metabolism and had a greater non-mitochondrial oxygen consumption than wild-type or type I diabetic (T1D) skin. CONCLUSION: Here we designed, for the first time, a reliable protocol to measure mitochondria function in whole mouse skin. Our optimized protocol was valuable in assessing alterations associated with diabetes and could be applied to future studies of pathological human skin metabolism.

Neuronal Blockade of Thyroid Hormone Signaling Increases Sensitivity to Diet-Induced Obesity in Adult Male Mice.

Thyroid hormone increases energy expenditure. Its action is mediated by TR, nuclear receptors present in peripheral tissues and in the central nervous system, particularly in hypothalamic neurons. Here, we address the importance of thyroid hormone signaling in neurons, in general for the regulation of energy expenditure. We generated mice devoid of functional TR in neurons using the Cre/LoxP system. In hypothalamus, which is the center for metabolic regulation, mutations were present in 20% to 42% of the neurons. Phenotyping was performed under physiological conditions that trigger adaptive thermogenesis: cold and high-fat diet (HFD) feeding. Mutant mice displayed impaired thermogenic potential in brown and inguinal white adipose tissues and were more prone to diet-induced obesity. They showed a decreased energy expenditure on chow diet and gained more weight on HFD. This higher sensitivity to obesity disappeared at thermoneutrality. Concomitantly, the AMPK pathway was activated in the ventromedial hypothalamus of the mutants as compared with the controls. In agreement, sympathetic nervous system (SNS) output, visualized by tyrosine hydroxylase expression, was lower in the brown adipose tissue of the mutants. In contrast, absence of TR signaling in the mutants did not affect their ability to respond to cold exposure. This study provides the first genetic evidence that thyroid hormone signaling exerts a significant influence in neurons to stimulate energy expenditure in some physiological context of adaptive thermogenesis. TR function in neurons to limit weight gain in response to HFD and this effect is associated with a potentiation of SNS output.

Brown adipocytes local response to thyroid hormone is required for adaptive thermogenesis in adult male mice.

Thyroid hormone (T3) and its nuclear receptors (TR) are important regulators of energy expenditure and adaptive thermogenesis, notably through their action in the brown adipose tissue (BAT). However, T3 acts in many other peripheral and central tissues which are also involved in energy expenditure. The general picture of how T3 regulates BAT thermogenesis is currently not fully established, notably due to the absence of extensive omics analyses and the lack of specific mice model. Here, we first used transcriptome and cistrome analyses to establish the list of T3/TR direct target genes in brown adipocytes. We then developed a novel model of transgenic mice, in which T3 signaling is specifically suppressed in brown adipocytes at adult stage. We addressed the capacity of these mice to mount a thermogenic response when challenged by either a cold exposure or a high-fat diet, and analyzed the associated changes in BAT transcriptome. We conclude that T3 plays a crucial role in the thermogenic response of the BAT, controlling the expression of genes involved in lipid and glucose metabolism and regulating BAT proliferation. The resulting picture provides an unprecedented view on the pathways by which T3 activates energy expenditure through an efficient adaptive thermogenesis in the BAT.

TRα inhibits arterial renin-angiotensin system expression and prevents cholesterol accumulation in vascular smooth muscle cells.

OBJECTIVES: The tissue renin-angiotensin system (tRAS) plays a key role in the maintenance of cellular homeostasis but is also implicated in atherosclerosis. Thyroid hormone (TH) contributes, via genomic effects, to control of tRAS gene expression in the arterial wall and vascular smooth muscle cells (VSMCs). We investigated the specific functions of TH receptors-α and -ß (TRα and TRß) on tRAS gene expression in the aorta and VSMCs, and the potential protective effect of TRα against atherosclerosis. MATERIAL AND METHODS: Using aorta and cultured aortic VSMCs from TRα and TRß deficient mice, tRAS gene expression was analyzed by determining mRNA levels on real-time PCR. Gene regulation under cholesterol loading mimicking atherosclerosis conditions was also examined in VSMCs in vitro. RESULTS: TRα deletion significantly increased expression of angiotensinogen (AGT) and angiotensin II receptor type 1 subtype a (AT1Ra) at transcriptional level in aorta, a tissue with high TRα expression level. TRα activity thus seems to be required for maintenance of physiological levels of AGTand AT1Raexpression in the arterial wall. In addition, during cholesterol loading, TRα deletion significantly increased cholesterol content in VSMCs, with a weaker decrease in AGTexpression. CONCLUSION: TRα seems to have an inhibitory impact on AGTand AT1Raexpression, and loss of TRα function in TRα0/0 mice increases tRAS expression in the aortic wall. More importantly, TRα deletion significantly increases VSMC cholesterol content. Our results are consistent with a protective role of TRα against atherosclerosis.

Thyroid Hormone Receptor Alpha Deletion in ApoE-/- Mice Alters the Arterial Renin-Angiotensin System and Vascular Smooth Muscular Cell Cholesterol Metabolism.

Thyroid hormone (TH) regulates gene transcription by binding to TH receptors (TRs). TRs regulate the genes of lipid metabolism and the renin-angiotensin system (RAS). We examined the effect of TRα deletion in ApoE-/- mice (DKO mice) on the following: (i) the expression of genes controlling cholesterol metabolism and tissue (t)RAS in the liver and aorta and (ii) the expression of these genes and the regulation of cholesterol content in cultured vascular smooth muscle cells (VSMCs). TRα deletion in ApoE-/- mice led to the repression of genes involved in the synthesis and influx of cholesterol in the liver. However, TRα deletion in the arterial wall suppressed the expression of genes involved in the esterification and excretion of cholesterol and enhanced the expression of angiotensinogen (AGT). The VSMCs of the ApoE-/- and DKO mice increased their cholesterol content during cholesterol loading, but failed to increase the expression of ATP-binding cassette transporter A1 (ABCA1). T3 addition partially corrected these abnormalities in the cells of the ApoE-/- mice but not those of the DKO mice. In conclusion, TRα deletion in ApoE-/- mice slightly increases the expression of tRAS in the aorta and aggravates the dysregulation of cholesterol content in the VSMCs.

Maternal eating behavior is a major synchronizer of fetal and postnatal peripheral clocks in mice.

Most living organisms show circadian rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. To study peripheral clocks in vivo, we developed an original model, the Rev-Luc mouse to follow noninvasively and longitudinally Rev-Luc oscillations in peripheral clocks using in vivo bioluminescence imaging. We found in vitro and in vivo a robust diurnal rhythm of Rev-Luc, mainly in liver, intestine, kidney and adipose tissues. We further confirmed in vivo that Rev-Luc peripheral tissues are food-entrainable oscillators, not affected by age or sex. These data strongly support the relevance of the Rev-Luc model for circadian studies, especially to investigate in vivo the establishment and the entrainment of the rhythm throughout ontogenesis. We then showed that Rev-Luc expression develops dynamically and gradually, both in amplitude and in phase, during fetal and postnatal development. We also demonstrate for the first time that the immature peripheral circadian system of offspring in utero is mainly entrained by maternal cues from feeding regimen. The prenatal entrainment will also differentially determine the Rev-Luc expression in pups before weaning underlining the importance of the maternal chrononutrition on the circadian system entrainment of the offspring.

In vivo MRS for the assessment of mouse colon using a dedicated endorectal coil: initial findings.

Inflammatory bowel disease is a common group of inflammation conditions that can affect the colon and the rectum. These pathologies require a careful follow-up of patients to prevent the development of colorectal cancer. Currently, conventional endoscopy is used to depict alterations of the intestinal walls, and biopsies are performed on suspicious lesions for further analysis (histology). MRS enables the in vivo analysis of biochemical content of tissues (i.e. without removing any samples). Combined with dedicated endorectal coils (ERCs), MRS provides new ways of characterizing alterations of tissues. An MRS in vivo protocol was specifically set up on healthy mice and on mice chemically treated to induce colitis. Acquisitions were performed on a 4.7 T system using a linear volume birdcage coil for the transmission of the B1 magnetic field, and a dedicated ERC was used for signal reception. Colon-wall complex, lumen and visceral fat were assessed on healthy and treated mice with voxel sizes ranging from 0.125 µL to 2 µL while keeping acquisition times below 3 min. The acquired spectra show various biochemical contents such as α- and ß-methylene but also glycerol backbone and diacyl. Choline was detected in tumoral regions. Visceral fat regions display a high lipid content with no water, whereas colon-wall complex exhibits both high lipid and high water contents. To the best of our knowledge, this is the first time that in vivo MRS using an ERC has been performed in the assessment of colon walls and surrounding structures. It provides keys for the in vivo characterization of small local suspicious lesions and offers complementary solutions to biopsies.

Endoluminal high-resolution MR imaging protocol for colon walls analysis in a mouse model of colitis.

OBJECTIVE: An endoluminal magnetic resonance (MR) imaging protocol including the design of an endoluminal coil (EC) was defined for high-spatial-resolution MR imaging of mice gastrointestinal walls at 4.7 T. MATERIALS AND METHODS: A receive-only radiofrequency single-loop coil was developed for mice colon wall imaging. Combined with a specific protocol, the prototype was first characterized in vitro on phantoms and on vegetables. Signal-to-noise ratio (SNR) profiles were compared with a quadrature volume birdcage coil (QVBC). Endoluminal MR imaging protocol combined with the EC was assessed in vivo on mice. RESULTS: The SNR measured close to the coil is significantly higher (10 times and up to 3 mm of the EC center) than the SNR measured with the QVBC. The gain in SNR can be used to reduce the in-plane pixel size up to 39 × 39 µm(2) (234 µm slice thickness) without time penalty. The different colon wall layers can only be distinguished on images acquired with the EC. CONCLUSION: Dedicated EC provides suitable images for the assessment of mice colon wall layers. This proof of concept provides gains in spatial resolution and leads to adequate protocols for the assessment of human colorectal cancer, and can now be used as a new imaging tool for a better understanding of the pathology.

Thyroid hormone receptor-α deletion decreases heart function and exercise performance in apolipoprotein E-deficient mice.

The deletion of thyroid hormone receptor-α (TRα) in atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice (ApoE(-/-)TRα(0/0)) accelerates the formation of atherosclerotic plaques without aggravation of hypercholesterolemia. To evaluate other predisposition risk factors to atherosclerosis in this model, we studied blood pressure (BP) and cardiac and vascular functions, as well as exercise tolerance in young adult ApoE(-/-)TRα(0/0) mice before the development of atherosclerotic plaques. Telemetric BP recorded for 4 consecutive days showed that the spontaneous systolic BP was slightly decreased in ApoE(-/-)TRα(0/0) compared with ApoE(-/-) mice associated with a reduced locomotor activity. The percentage of animals that completed endurance (57% vs. 89%) and maximal running (0% vs. 89% at 46 cm/s speed in ApoE(-/-)TRα(0/0) and ApoE(-/-) mice, respectively) tests was lower in ApoE(-/-)TRα(0/0) mice. Moreover, during the maximal running test, both maximal running speed and running distance were significantly reduced in ApoE(-/-)TRα(0/0) mice, associated with a blunted BP response to exercise. Transthoracic echocardiography revealed a decreased interventricular septum thickness and an increased end-systolic left ventricular volume in ApoE(-/-)TRα(0/0) mice. Accordingly, left ventricular fractional shortening, ejection fraction, and stroke volume were all significantly decreased in ApoE(-/-)TRα(0/0) mice with a concomitant blunted cardiac output. No interstrain difference was observed in vascular reactivity, except that ApoE(-/-)TRα(0/0) mice exhibited an enhanced acetylcholine-induced relaxation in mesenteric and distal femoral arteries. In conclusion, the deletion of TRα in ApoE(-/-) mice alters cardiac structure and contractility; both could contribute to blunted BP response to physical exercise and impaired exercise performance.

In vivo imaging of the spatiotemporal activity of the eIF2α-ATF4 signaling pathway: Insights into stress and related disorders.

The eIF2α-ATF4 pathway is involved in cellular adaptation to stress and is dysregulated in numerous diseases. Activation of this pathway leads to phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and the recruitment of the transcription factor ATF4 (activating transcription factor 4) to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of target genes. To monitor the spatiotemporal modulation of this pathway in living animals, we generated a novel CARE-driven luciferase mouse model (CARE-LUC). These transgenic mice enable the investigation of the eIF2α-ATF4 pathway activity in the whole organism and at the tissue and cellular levels by combining imaging, luciferase assays, and immunochemistry. Using this mouse line, we showed the tissue-specific activation pattern of this pathway in response to amino acid deficiency or endoplasmic reticulum stress and the hepatic induction of this pathway in a stress-related pathology model of liver fibrosis. The CARE-LUC mouse model represents an innovative tool to investigate the eIF2α-ATF4 axis and to develop drugs targeting this important pathway in the remediation of related pathologies.

Vascular function of the mesenteric artery isolated from thyroid hormone receptor-α knockout mice.

OBJECTIVE: This study evaluated the consequences of thyroid hormone receptor-α (TRα) disruption on vascular reactivity. METHODS: The activity of superior mesenteric arteries isolated from TRα knockout mice generated in the SV129 background (TRα(0/0)SV) or in a pure C57BL/6 background (TRα(0/0)C57) was compared to that of their corresponding wild-type strains (SV129 or C57BL/6 mice). RESULTS: The wild-type SV129 mice exhibited an impaired acetylcholine (Ach)-induced mesenteric artery relaxation compared to C57BL/6 mice, associated with greater responses to angiotensin II (AII) and phenylephrine (PE). The disruption of TRα decreased the vascular response to sodium nitroprusside and PE in both the SV129 and C57BL/6 genetic backgrounds. Responses to Ach and AII were also blunted, but only in TRα(0/0)C57 mice. The administration of 3,3'5-triiodo-L-thyronine sodium salt (T3) elicited a vasodilatation in C57BL/6 mice even at the lowest concentration (10(-9)M); a maximal relaxation of more than 50% was observed with the concentrations between 10(-9) and 10(-8)M. However, the response to T3 was nearly absent in TRα(0/0)C57 mice. CONCLUSION: TRα is essential for the control of vascular tone, particularly in thyroid hormone-mediated relaxation. The difference in response to Ach observed between the two wild-type mice should be taken into account for interpreting the vascular responses of genetically engineered mice.

Tagging live cells that express specific peptidase activity with solid-state fluorescence.

A three-component probe harnesses the extraordinary properties of a solid-state fluorophore for the detection of living cells exhibiting a particular peptidase activity. The off-on mode by which the probe operates, the bright fluorescence of the resulting precipitate, and the rapid response allow an exceptional signal-to-background ratio during microscopic imaging. A tertiary carbamate link between the spacer and phenolic fluorophore is at the heart of the probe's long-term stability. The degree of chlorination of the probe determines its response time and thus its suitability for live-cell analysis. Our probe also allows highly resolved localization of peptidase activity during gel analysis or on agar. In comparison, probes releasing soluble fluorophores demonstrate complete diffusion of the fluorescent signal. These results demonstrate the probe's potential for diverse biomedical applications, including high-fidelity flow cytometry and sensitive colony assays.

TRα protects against atherosclerosis in male mice: identification of a novel anti-inflammatory property for TRα in mice.

Hypothyroidism is associated with an increased occurrence of atherosclerosis, suggesting some protective role for thyroid hormones (THs). Hypercholesterolemia is one of the major risk factor to develop this disease. Here, we show that the well-known TH cholesterol lowering effect was dependent on TH nuclear receptor (TR)ß liver activity. But most importantly, TRα was also shown to contribute of slowing down atherosclerosis progression via an independent mechanism. Introduction of TRα(0/0) deletion in the ApoE(-/-) background accelerated the appearance of plaques. Earlier cholesterol accumulation was detected in aorta macrophages, likely due to impaired cholesterol efflux. The IL-1ß inflammatory cytokine was elevated in serum and macrophages in correlation with an activation of the AKT/nuclear factor κB pathway in these cells. Inhibition of AKT prevented inflammation and restored normal cholesterol efflux. Similar low-grade inflammation was identified in TRα(0/0) male mice. Thus, the mere absence of TRα is associated with elevated levels of cytokines likely responsible for cholesterol accumulation and atherosclerosis. This TRα protective activity should be relevant for other inflammatory pathologies.

Awakening of a ferrous complex's electronic spin in an aqueous solution induced by a chemical stimulus.

A low-spin, macrocyclic iron(II) complex in an aqueous solution responds to the addition of a chemical reactant (dithionite) by transformation into a high-spin complex, detectable by measurement of the longitudinal relaxation time (T(1)) of surrounding water hydrogen nuclear spins. The initial compound does not modify T(1) of pure water at concentrations as high as 4 mM. The response is pH-dependent, and the complex is robust at a variety of conditions.

An electroneutral macrocyclic iron(II) complex that enhances MRI contrast in vivo.

The first example of a macrocyclic ferrous complex, where two tetrazolyl pendent arms compensate the charge of the metal center, is synthesized and examined for its capacity to enhance MRI contrast in vitro and in vivo in the mouse.

Fast screening of paramagnetic molecules in zebrafish embryos by MRI.

Zebrafish embryo is a well-established model used in many fields of modern experimental biology. We demonstrate that it provides a promising model platform for exploring fundamental MR aspects that can be used to screen and study active MR molecules before progressing to more complex living systems. Setting up a dedicated MRI methodology, we arrayed a large number of living embryos, which were microinjected at very early stages of development with different contrast agents. We also showed that MRI signal intensity correlates with the gadolinium content of zebrafish embryos. This allowed us to validate a new approach for MR compound screening. Using a specific surface coil of 5 mm inner diameter, we obtained for the first time high-spatial-resolution images at 7 T of living zebrafish embryos with a 47 microm isotropic voxel size with an acquisition time of 39 min. Finally, we discuss potential applications of this development: a viable in vivo assay for screening small pharmacological compounds; assessment of and tracking the action of molecules over time. Exploring in vivo biological activity, gene function analysis, and detailed characterization of disease processes in fish are natural extensions of these preliminary studies.

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock.

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.

In vivo activation of PPAR target genes by RXR homodimers.

The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR:RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wild-type cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha-/- mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.

The days and nights of cancer cells.

Biological clocks are intrinsic time-keeping systems that regulate behavior and physiological functions in most living organisms. Recent works in this area have addressed possible molecular links between the endogenous circadian clock and cell cycle regulation. In this review, by addressing how circadian clocks can interfere with the cell cycle and how the disruption of the circadian rhythm may cause defects in regulation of cell proliferation, we highlight this potential connection between circadian rhythm and cell cycle. We also discuss how the acquisition of recent data in circadian clock mechanism may help chronotherapy, which takes into account the biological time to improve cancer treatments, and may open new therapeutic avenues for treating circadian-related diseases.