Circadian transcriptome processing and analysis: a workflow for muscle stem cells.

Circadian rhythms coordinate biological processes with Earth's 24-h daily light/dark cycle. In the last years, efforts in the field of chronobiology have sought to understand the ways in which the circadian clock controls transcription across tissues and cells. This has been supported by the development of different bioinformatic approaches that allow the identification of 24-h oscillating transcripts. This workflow aims to describe how to isolate muscle stem cells for RNA sequencing analysis from a typical circadian experiment and introduces bioinformatic tools suitable for the analysis of circadian transcriptomes.

Liver and muscle circadian clocks cooperate to support glucose tolerance in mice.

Physiology is regulated by interconnected cell and tissue circadian clocks. Disruption of the rhythms generated by the concerted activity of these clocks is associated with metabolic disease. Here we tested the interactions between clocks in two critical components of organismal metabolism, liver and skeletal muscle, by rescuing clock function either in each organ separately or in both organs simultaneously in otherwise clock-less mice. Experiments showed that individual clocks are partially sufficient for tissue glucose metabolism, yet the connections between both tissue clocks coupled to daily feeding rhythms support systemic glucose tolerance. This synergy relies in part on local transcriptional control of the glucose machinery, feeding-responsive signals such as insulin, and metabolic cycles that connect the muscle and liver. We posit that spatiotemporal mechanisms of muscle and liver play an essential role in the maintenance of systemic glucose homeostasis and that disrupting this diurnal coordination can contribute to metabolic disease.

Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration.

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life.

An obesogenic feedforward loop involving PPARγ, acyl-CoA binding protein and GABAA receptor.

Acyl-coenzyme-A-binding protein (ACBP), also known as a diazepam-binding inhibitor (DBI), is a potent stimulator of appetite and lipogenesis. Bioinformatic analyses combined with systematic screens revealed that peroxisome proliferator-activated receptor gamma (PPARγ) is the transcription factor that best explains the ACBP/DBI upregulation in metabolically active organs including the liver and adipose tissue. The PPARγ agonist rosiglitazone-induced ACBP/DBI upregulation, as well as weight gain, that could be prevented by knockout of Acbp/Dbi in mice. Moreover, liver-specific knockdown of Pparg prevented the high-fat diet (HFD)-induced upregulation of circulating ACBP/DBI levels and reduced body weight gain. Conversely, knockout of Acbp/Dbi prevented the HFD-induced upregulation of PPARγ. Notably, a single amino acid substitution (F77I) in the γ2 subunit of gamma-aminobutyric acid A receptor (GABAAR), which abolishes ACBP/DBI binding to this receptor, prevented the HFD-induced weight gain, as well as the HFD-induced upregulation of ACBP/DBI, GABAAR γ2, and PPARγ. Based on these results, we postulate the existence of an obesogenic feedforward loop relying on ACBP/DBI, GABAAR, and PPARγ. Interruption of this vicious cycle, at any level, indistinguishably mitigates HFD-induced weight gain, hepatosteatosis, and hyperglycemia.

Running skeletal muscle clocks on time- the determining factors.

Circadian rhythms generate 24 h-long oscillations, which are key regulators of many aspects of behavior and physiology. Recent circadian transcriptome studies have discovered rhythmicity at the transcriptional level of hundreds of skeletal muscle genes, with roles in skeletal muscle growth, maintenance, and metabolic functions. These rhythms allow this tissue to perform molecular functions at the appropriate time of the day in order to anticipate environmental changes. However, while the last decade of research has characterized several aspects of the skeletal muscle molecular clock, many still are unexplored, including its functions, regulatory mechanisms, and interactions with other tissues. The central clock is believed to be located in the suprachiasmatic nucleus (SCN) of the brain hypothalamus, providing entrainment to peripheral organs through humoral and neuronal signals. However, these mechanisms of action are still unknown. Conversely, muscle tissue can be entrained through extrinsic, SCN-independent factors, such as feeding and physical activity. In this review, we provide an overview of the recent research about the extrinsic and intrinsic factors required for skeletal muscle clock regulation. Furthermore, we discuss the need for future studies to elucidate the mechanisms behind this regulation, which will in turn help dissect the role of circadian disruption at the onset of aging and diseases.

Quantification of intracellular ACBP/DBI levels.

Acyl-CoA binding protein (ACBP), also called diazepam-binding inhibitor (DBI), is a ubiquitous protein that can be secreted from cells by an unconventional pathway. Depending on its levels and on its subcellular localization, ACBP/DBI can regulate lipid metabolism. Several studies have shown that ACBP/DBI is secreted by an autophagy-dependent mechanism, positioning this catabolic pathway as the mechanism that controls lipid metabolism through the intracellular modulation of the levels of this protein. Autophagy is activated, among other stimuli, when cells have increased energy requirements; this causes a drop in the intracellular ACBP/DBI levels due to its release into the extracellular space and triggers an increase in the lipid catabolism. Conversely, when autophagy is inhibited, during pathological (obesity) or physiological (after-meal) situations, the intracellular levels of ACBP/DBI increase resulting in the activation of lipid anabolism, this effect has been demonstrated to be the link between obesity and autophagy inhibition. Here, we detail three different protocols for the detection of the ACBP/DBI levels by immunofluorescence, image flow cytometry or immunoblot techniques, which allow the quantification of ACBP/DBI levels and, indirectly, its autophagy-dependent release.

Autophagy-mediated metabolic effects of aspirin.

Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b-/- or Bcln1+/-) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.

A bidirectional crosstalk between autophagy and TP53 determines the pace of aging.

When the orthologue of tumor suppressor protein p53 (TP53), cep-1, is inactivated in Caenorhabditis elegans, the nematodes manifest an autophagy-dependent increase in lifespan. A recent paper by Yang et al. demonstrates that accelerated aging phenotype of autophagy-deficient mice can be reduced by the knockout (KO) of Trp53. These findings point to a complex bidirectional crosstalk between autophagy and TP53 that has vast implications for the aging process.

Metabolic and psychiatric effects of acyl coenzyme A binding protein (ACBP)/diazepam binding inhibitor (DBI).

Acyl coenzyme A binding protein (ACBP), also known as diazepam binding inhibitor (DBI) is a multifunctional protein with an intracellular action (as ACBP), as well as with an extracellular role (as DBI). The plasma levels of soluble ACBP/DBI are elevated in human obesity and reduced in anorexia nervosa. Accumulating evidence indicates that genetic or antibody-mediated neutralization of ACBP/DBI has anorexigenic effects, thus inhibiting food intake and inducing lipo-catabolic reactions in mice. A number of anorexiants have been withdrawn from clinical development because of their side effects including an increase in depression and suicide. For this reason, we investigated the psychiatric impact of ACBP/DBI in mouse models and patient cohorts. Intravenously (i.v.) injected ACBP/DBI protein conserved its orexigenic function when the protein was mutated to abolish acyl coenzyme A binding, but lost its appetite-stimulatory effect in mice bearing a mutation in the γ2 subunit of the γ-aminobutyric acid (GABA) A receptor (GABAAR). ACBP/DBI neutralization by intraperitoneal (i.p.) injection of a specific mAb blunted excessive food intake in starved and leptin-deficient mice, but not in ghrelin-treated animals. Neither i.v. nor i.p. injected anti-ACBP/DBI antibody affected the behavior of mice in the dark-light box and open-field test. In contrast, ACBP/DBI increased immobility in the forced swim test, while anti-ACBP/DBI antibody counteracted this sign of depression. In patients diagnosed with therapy-resistant bipolar disorder or schizophrenia, ACBP/DBI similarly correlated with body mass index (BMI), not with the psychiatric diagnosis. Patients with high levels of ACBP/DBI were at risk of dyslipidemia and this effect was independent from BMI, as indicated by multivariate analysis. In summary, it appears that ACBP/DBI neutralization has no negative impact on mood and that human depression is not associated with alterations in ACBP/DBI concentrations.

Antibody-mediated neutralization of ACBP/DBI has anorexigenic and lipolytic effects.

We recently identified acyl coenzyme A-binding protein (ACBP)/diazepam binding inhibitor (DBI) as a novel 'hunger factor': a protein that is upregulated in human or murine obesity and that, if administered to mice, causes hyperphagy, adipogenesis and obesity. Conversely, neutralization of ACBP/DBI by systemic injection of neutralizing monoclonal antibodies or autoantibodies produced after auto-immunization against ACBP/DBI has anorexigenic and lipolytic effects. Thus, neutralization of ACBP/DBI results in reduced food intake subsequent to the activation of anorexigenic neurons and the inactivation of orexigenic neurons in the hypothalamus. Moreover, ACBP/DBI neutralization results into enhanced triglyceride lipolysis in white fat, a surge in free fatty acids in the plasma, enhanced incorporation of glycerol-derived carbon atoms into glucose, as well as an increase in ß-oxidation, resulting in a net reduction of fat mass. Importantly, ACBP/DBI neutralization also stimulated an increase in autophagy in various organs, suggesting that it might mediate anti-ageing effects.

Acyl-CoA-binding protein (ACBP): a phylogenetically conserved appetite stimulator.

Recently, we reported that, in mice, hunger causes the autophagy-dependent release of a protein called "acyl-CoA-binding protein" or "diazepam binding inhibitor" (ACBP/DBI) from cells, resulting in an increase in plasma ACBP concentrations. Administration of extra ACBP is orexigenic and obesogenic, while its neutralization is anorexigenic in mice, suggesting that ACBP is a major stimulator of appetite and lipo-anabolism. Accordingly, obese persons have higher circulating ACBP levels than lean individuals, and anorexia nervosa is associated with subnormal ACBP plasma concentrations. Here, we investigated whether ACBP might play a phylogenetically conserved role in appetite stimulation. We found that extracellular ACBP favors sporulation in Saccharomyces cerevisiae, knowing that sporulation is a strategy for yeast to seek new food sources. Moreover, in the nematode Caenorhabditis elegans, ACBP increased the ingestion of bacteria as well as the frequency pharyngeal pumping. These observations indicate that ACBP has a phylogenetically ancient role as a 'hunger factor' that favors food intake.

Oxidative phosphorylation as a potential therapeutic target for cancer therapy.

In contrast to prior belief, cancer cells require oxidative phosphorylation (OXPHOS) to strive, and exacerbated OXPHOS dependency frequently characterizes cancer stem cells, as well as primary or acquired resistance against chemotherapy or tyrosine kinase inhibitors. A growing arsenal of therapeutic agents is being designed to suppress the transfer of mitochondria from stromal to malignant cells, to interfere with mitochondrial biogenesis, to directly inhibit respiratory chain complexes, or to disrupt mitochondrial function in other ways. For the experimental treatment of cancers, OXPHOS inhibitors can be advantageously combined with tyrosine kinase inhibitors, as well as with other strategies to inhibit glycolysis, thereby causing a lethal energy crisis. Unfortunately, most of the preclinical data arguing in favor of OXPHOS inhibition have been obtained in xenograft models, in which human cancer cells are implanted in immunodeficient mice. Future studies on OXPHOS inhibitors should elaborate optimal treatment schedules and combination regimens that stimulate-or at least are compatible with-anticancer immune responses for long-term tumor control.

The elusive "hunger protein": an appetite-stimulatory factor that is overabundant in human obesity.

Paradoxically, most if not all previously known appetite-stimulatory hormones are downregulated in human obesity, reflecting failing homeostatic circuitries. Recently, we discovered that acyl-coenzyme-A binding protein/diazepam-binding inhibitor (ACBP/DBI) acts as a lipogenic and appetite stimulator, when systemically injected into mice. ACBP/DBI plasma levels are also elevated in obese subjects, supporting the notion that it may represent the elusive "hunger protein" that explains overeating in human obesity.

Pseudodiabetes-not a contraindication for metabolic interventions.

Type-2 diabetes is characterized by glycosuria, hyperglycemia, glucose intolerance, hyperinsulinemia, and insulin resistance. One or several among these alterations are also found after starvation, ketogenic diet, and pharmacological treatment with rapamycin or antibody-mediated neutralization of the obesogenic factor ACBP/DBI. Thus, a variety of metabolic interventions that improve metabolic health can induce a transient state of "pseudo-diabetes".

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

3,4-Dimethoxychalcone induces autophagy through activation of the transcription factors TFE3 and TFEB.

Caloric restriction mimetics (CRMs) are natural or synthetic compounds that mimic the health-promoting and longevity-extending effects of caloric restriction. CRMs provoke the deacetylation of cellular proteins coupled to an increase in autophagic flux in the absence of toxicity. Here, we report the identification of a novel candidate CRM, namely 3,4-dimethoxychalcone (3,4-DC), among a library of polyphenols. When added to several different human cell lines, 3,4-DC induced the deacetylation of cytoplasmic proteins and stimulated autophagic flux. At difference with other well-characterized CRMs, 3,4-DC, however, required transcription factor EB (TFEB)- and E3 (TFE3)-dependent gene transcription and mRNA translation to trigger autophagy. 3,4-DC stimulated the translocation of TFEB and TFE3 into nuclei both in vitro and in vivo, in hepatocytes and cardiomyocytes. 3,4-DC induced autophagy in vitro and in mouse organs, mediated autophagy-dependent cardioprotective effects, and improved the efficacy of anticancer chemotherapy in vivo. Altogether, our results suggest that 3,4-DC is a novel CRM with a previously unrecognized mode of action.

Acyl-CoA-binding protein (ACBP): the elusive 'hunger factor' linking autophagy to food intake.

The best-known appetite-regulating factors identified in rodents are leptin, an appetite inhibitor, and ghrelin, an appetite stimulator. Rare cases of loss-of-functions mutations affecting leptin and its receptor, as well as polymorphisms concerning ghrelin and its receptor, have been documented in human obesity, apparently validating the relevance of leptin and ghrelin for human physiology. Paradoxically, however, the overwhelming majority of obese individuals manifest high leptin and low ghrelin plasma levels, suggesting that both factors are not directly disease-relevant. We recently discovered that acyl-CoA-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), acts as an efficient lipogenic and appetite stimulator in mice. Indeed, in response to starvation, ACBP/DBI is released from tissues in an autophagy-dependent fashion and increases in the plasma. Intravenous injection of ACBP/DBI stimulates feeding behavior through a reduction of circulating glucose levels, and consequent activation of orexigenic neurons in the hypothalamus. In contrast, neutralization of ACBP/DBI abolishes the hyperphagia observed after starvation of mice. Of note, ACBP/DBI is increased in the plasma of obese persons and mice, pointing to a convergence (rather than divergence) between its role in appetite stimulation and human obesity. Based on our results, we postulate a novel 'hunger reflex' in which starvation induces a surge in extracellular ACBP/DBI, which in turn stimulates feeding behavior. Thus, ACBP/DBI might be the elusive 'hunger factor' that explains increased food uptake in obesity.