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Gut microbiota is linked to human metabolic diseases. The previous work showed that leucine deprivation improved metabolic dysfunction, but whether leucine deprivation alters certain specific species of bacterium that brings these benefits remains unclear. Here, this work finds that leucine deprivation alters gut microbiota composition, which is sufficient and necessary for the metabolic improvements induced by leucine deprivation. Among all the affected bacteria, B. coccoides is markedly increased in the feces of leucine‐deprived mice. Moreover, gavage with B. coccoides improves insulin sensitivity and reduces body fat in high‐fat diet (HFD) mice, and singly colonization of B. coccoides increases insulin sensitivity in gnotobiotic mice. The effects of B. coccoides are mediated by metabolizing tryptophan into indole‐3‐acetic acid (I3AA) that activates the aryl hydrocarbon receptor (AhR) in the liver. Finally, this work reveals that reduced fecal B. coccoides and I3AA levels are associated with the clinical metabolic syndrome. These findings suggest that B. coccoides is a newly identified bacterium increased by leucine deprivation, which improves metabolic disorders via metabolizing tryptophan into I3AA. [ABSTRACT FROM AUTHOR]

The mechanistic target of rapamycin complex 1 (mTORC1) is involved in nutrient-induced signaling and is a master regulator of cell growth and metabolism. Amino acid-deficient conditions affect mTORC1 activity; however, its upstream regulators warrant further investigation. MicroRNAs are key regulators of nutrient-related responses; therefore, the present study aimed to assess the leucine starvation-induced microRNA profile and its impact on mTORC1 activity. Transcriptome analysis of human hepatocellular carcinoma cells (HepG2) under leucine deprivation revealed that hsa-miR-663a and hsa-miR-1469 were altered in a transcription factor 4-dependent manner. Overexpression of these microRNAs induced phosphorylation of the ribosomal protein S6 kinase beta-1, a mTORC1 downstream target. Furthermore, hsa-miR-663a downregulated proline-rich Akt1 substrate of 40 kDa (PRAS40), one of the mTORC1 components. In summary, this study provides new insights into the regulatory role of microRNAs in amino acid metabolism and demonstrates alterations in microRNA profile under leucine deprivation in human hepatocytes.

Fibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated, however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here, we show that prolonged leucine deprivation results in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation. This article has an associated First Person interview with the first author of the paper.

We have previously shown that leucine deprivation stimulates browning and lipolysis in white adipose tissue (WAT), which helps to treat obesity. Adipose tissue macrophages (ATMs) significantly influence WAT browning and lipolysis. However, it is unclear whether ATMs are involved in leucine deprivation-induced browning and lipolysis in WAT; the associated signals remain to be elucidated. Here, we investigated the role of ATMs and the possible mechanisms involved in WAT browning and lipolysis under leucine-deprivation conditions. In this study, macrophages were depleted in mice by injecting clodronate-liposomes (CLOD) into subcutaneous white adipose tissues. Then, mice lacking general control nonderepressible 2 kinase (GCN2), which is a sensor of amino acid starvation, specifically in Lyz2-expressing cells, were generated to investigate the changes in leucine deprivation-induced WAT browning and lipolysis. We found leucine deprivation decreased the accumulation and changed the polarization of ATMs. Ablation of macrophages by CLOD impaired WAT browning and lipolysis under leucine-deprivation conditions. Mechanistically, leucine deprivation activated GCN2 signals in macrophages. Myeloid-specific abrogation of GCN2 in mice blocked leucine deprivation-induced browning and lipolysis in WAT. Further analyses revealed that GCN2 activation in macrophages reduced the expression of monoamine oxidase A (MAOA), resulting in increased norepinephrine (NE) secretion from macrophages to adipocytes, and this resulted in enhanced WAT browning and lipolysis. Moreover, the injection of CL316,243, a β3-adrenergic receptor agonist, and inhibition of MAOA effectively increased the level of NE, leading to the enhancement of browning and lipolysis of WAT in myeloid GCN2 knockout mice under leucine deprivation. Collectively, our results demonstrate a novel function of GCN2 signals in macrophages, that is, regulating WAT browning and lipolysis under leucine deprivation. Our study provides important hints for possible treatment for obesity. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is an important process responsible for protein turnover and cell survival in amino acid-deprived conditions, especially for leucine (Leu). With the dramatic advances in mass spectrometry, many new post-translational modifications (PTMs) have been identified. However, whether these PTMs regulate autophagy remains unclear. Here we found global lysine crotonylation levels are significantly upregulated during Leu deprivation-induced autophagy. A comprehensive crotonylome profiling showed that YWHA/14-3-3 proteins are significantly enriched in the Leu regulated-crotonylome. The inhibition of YWHAE/14-3-3ε crotonylation by mutating two crotonylated sites to arginine, K73R K78R, significantly attenuates autophagy induced by Leu deprivation. Molecular dynamics suggest that YWHAE K73 and K78 crotonylations decrease protein conformation and thermodynamic stability. Moreover, we found crotonylation of YWHAE releases PPM1B to dephosphorylate ULK1 and consequently activate autophagy. Decrotonylation of YWHAE is mediated by HDAC7 whose activity is inhibited significantly by Leu deprivation. Taken together, our finding reveals a critical role of YWHAE crotonylation in Leu deprivation-induced autophagy. [ABSTRACT FROM AUTHOR]

Lipogenic gene expression in liver is repressed in mice upon leucine deprivation. The hormone fibroblast growth factor 21 (FGF21), which is critical to the adaptive metabolic response to starvation, is also induced under amino acid deprivation. Upon leucine deprivation, we found that FGF21 is needed to repress expression of lipogenic genes in liver and white adipose tissue, and stimulate phosphorylation of hormone-sensitive lipase in white adipose tissue. The increased expression of Ucp1 in brown adipose tissue under these circumstances is also impaired in FGF21-deficient mice. Our results demonstrate the important role of FGF21 in the regulation of lipid metabolism during amino acid starvation.

Lysine crotonylation as a protein post-translational modification regulates diverse cellular processes and functions. However, the role of crotonylation in nutrient signaling pathways remains unclear. Here, we find a positive correlation between global crotonylation levels and leucine-deprivation-induced autophagy. Crotonylome profiling identifies many crotonylated proteins regulated by leucine deprivation. Bioinformatics analysis dominates 14-3-3 proteins in leucine-mediated crotonylome. Expression of 14-3-3ε crotonylation-deficient mutant significantly inhibits leucine-deprivation-induced autophagy. Molecular dynamics analysis shows that crotonylation increases molecular instability and disrupts the 14-3-3ε amphipathic pocket through which 14-3-3ε interacts with binding partners. Leucine-deprivation-induced 14-3-3ε crotonylation leads to the release of protein phosphatase 1B (PPM1B) from 14-3-3ε interaction. Active PPM1B dephosphorylates ULK1 and subsequently initiates autophagy. We further find that 14-3-3ε crotonylation is regulated by HDAC7. Taken together, our findings demonstrate that the 14-3-3ε-PPM1B axis regulated by crotonylation may play a vital role in leucine-deprivation-induced autophagy. [Display omitted] • Global lysine crotonylation and autophagy levels are enhanced by leucine deprivation • Crotonylome profiling identifies many proteins regulated by leucine deprivation • 14-3-3ε crotonylation affects its binding to PPM1B, which is a phosphatase for ULK1 Zheng et al. find the link between lysine crotonylation and leucine-deprivation-induced autophagy. Leucine deprivation induces crotonylation of 14-3-3ε protein, which releases PPM1B to dephosphorylate ULK1 and activate autophagy. The crotonylation-regulated 14-3-3ε-PPM1B axis is critical for the initiation of autophagy. [ABSTRACT FROM AUTHOR]

Essential amino acids (EAAs) are crucial nutrients, whose levels change in rodents and patients with depression. However, how the levels of a single EAA affects depressive behaviors remains elusive. Here, we demonstrate that although deprivation of the EAA leucine has no effect in unstressed mice, it remarkably reverses the depression-like behaviors induced by chronic restraint stress (CRS). This beneficial effect is independent of feeding and is applicable to the dietary deficiency of other EAAs. Furthermore, the effect of leucine deprivation is suppressed by central injection of leucine or mimicked by central injection of leucinol. Moreover, hypothalamic agouti-related peptide (AgRP) neural activity changes during CRS and leucine deprivation, and chemogenetically inhibiting AgRP neurons eliminates the antidepressant effects of leucine deprivation. Finally, the leucine deprivation-regulated behavioral effects are mediated by amino acid sensor general control non-derepressible 2 (GCN2) in AgRP neurons. Taken together, our results suggest a new drug target and/or dietary intervention for the reduction of depressive symptoms., Competing Interests: The authors declare that no conflict of interest exists. F.G. holds the position of Editorial Board Member for Life Metabolism, and is blinded from reviewing or making decisions for the manuscript., (© The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.)

Insulin-like growth factor-1 (IGF-I) is the key regulator of fetal growth. IGF-I bioavailability is markedly diminished by IGF binding protein-1 (IGFBP-1) phosphorylation. Leucine deprivation strongly induces IGFBP-1 hyperphosphorylation, and plays an important role in fetal growth restriction (FGR). FGR is characterized by decreased amino acid availability, which activates the amino acid response (AAR) and inhibits the mechanistic target of rapamycin (mTOR) pathway. We investigated the role of AAR and mTOR in mediating IGFBP-1 secretion and phosphorylation in HepG2 cells in leucine deprivation. mTOR inhibition (rapamycin or raptor + rictor siRNA), or activation (DEPTOR siRNA) demonstrated a role of mTOR in leucine deprivation-induced IGFBP-1 secretion but not phosphorylation. When the AAR was blocked (U0126, or ERK/GCN2 siRNA), both IGFBP-1 secretion and hyperphosphorylation (pSer101/pSer119/pSer169) due to leucine deprivation were prevented. CK2 inhibition by TBB also attenuated IGFBP-1 phosphorylation in leucine deprivation. These results suggest that the AAR and mTOR independently regulate IGFBP-1 secretion and phosphorylation in response to decreased amino acid availability. [ABSTRACT FROM AUTHOR]

Abstract: Objective: We recently discovered that leucine deprivation increases hepatic insulin sensitivity via general control nondepressible (GCN) 2/mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) pathways. The goal of the present study was to investigate whether the above effects were leucine specific or were also induced by deficiency of other branched chain amino acids including valine and isoleucine. Methods: Following depletion of BCAAs, changes in metabolic parameters and the expression of genes and proteins involved in regulation of insulin sensitivity and glucose metabolism were analyzed in mice and cell lines including human HepG2 cells, primary mouse hepatocytes and a mouse myoblast cell line C2C12. Results: Valine or isoleucine deprivation for 7days has similar effect on improving insulin sensitivity as leucine, in wild type and insulin-resistant mice models. These effects are possibly mediated by decreased mTOR/S6K1 and increased AMPK signaling pathways, in a GCN2-dependent manner. Similar observations were obtained in in vitro studies. In contrast to leucine withdrawal, valine or isoleucine deprivation for 7days significantly decreased fed blood glucose levels, possibly due to reduced expression of a key gluconeogenesis gene, glucose-6-phosphatase. Finally, insulin sensitivity was rapidly improved in mice 1day following maintenance on a diet deficient for any individual BCAAs. Conclusions: Our results show that while improvement on insulin sensitivity is a general feature of BCAAs depletion, individual BCAAs have specific effects on metabolic pathways, including those that regulate glucose level. These observations provide a conceptual framework for delineating the molecular mechanisms that underlie amino acid regulation of insulin sensitivity. [Copyright &y& Elsevier]

Abstract: Autophagy is an evolutionarily conserved process responsible for degradation and recycling of cytoplasmic components through the lysosomal machinery. It has been proved to play pivotal roles in cellular homeostasis, cell growth and organism development. Moreover, abnormalities of autophagy have been linked to numerous human pathophysiologies. Emerging evidence has linked leucine deprivation induced protein breakdown to autophagy, but the underlying mechanisms controlling autophagic activity in this process are not fully understood. Here, we demonstrate that two members of the miR-17 microRNA family, miR-20a and miR-106b, may participate in regulating leucine deprivation induced autophagy via suppression of ULK1 expression in C2C12 myoblasts. We showed that leucine deprivation downregulated miR-20a and miR-106b expression via suppression of their transcription factor c-Myc. We discovered the essential autophagy gene ULK1 as cellular target of miR-20a and miR-106b. Treatment of C2C12 cells with the miR-20a or miR-106b mimic decreased the endogenous ULK1 protein levels. Dual luciferase reporter assay confirmed that the miRNA binding sequences in the 3′ UTR of ULK1 contribute to the modulation of ULK1 expression by miR-20a and miR-106b. Furthermore, inhibition of ULK1 expression by the miR-20a or miR-106b mimic blunted activation of autophagy induced by leucine deprivation, while suppression of endogenous miR-20a or miR-106b by specific antagomir in C2C12 cells showed normal autophagic activity. Altogether, our data demonstrated that miR-20a and miR-106b regulated autophagy induced by leucine deprivation in C2C12 cells via targeting ULK1. [Copyright &y& Elsevier]

Substantial studies on fatty acid synthase (FASN) have focused on its role in regulating lipid metabolism and researchers have a great interest in treating cancer with dietary manipulation of amino acids. In the current study, we found that leucine deprivation caused the FASN-dependent anticancer effect. Here we showed that leucine deprivation inhibited cell proliferation and induced apoptosis of MDA-MB-231 and MCF-7 breast cancer cells. In an in vivo tumor xenograft model, the leucine-free diet suppressed the growth of human breast cancer tumors and triggered widespread apoptosis of the cancer cells. Further study indicated that leucine deprivation decreased expression of lipogenic gene FASN in vitro and in vivo. Over-expression of FASN or supplementation of palmitic acid (the product of FASN action) blocked the effects of leucine deprivation on cell proliferation and apoptosis in vitro and in vivo. Moreover, leucine deprivation suppressed the FASN expression via regulating general control non-derepressible (GCN)2 and sterol regulatory element-binding protein 1C (SREBP1C). Taken together, our study represents proof of principle that anticancer effects can be obtained with strategies to deprive tumors of leucine via suppressing FASN expression, which provides important insights in prevention of breast cancer via metabolic intervention.

Insulin resistance is one of the major factors contributing to metabolic diseases, but the underlying mechanisms are still poorly understood. As an important cofactor, B-cell translocation gene 1 (BTG1) is involved in many physiologic processes; however, the direct effect of BTG1 on insulin sensitivity has not been described. In our study, BTG1 overexpression or knockdown improved or impaired insulin signaling in vitro, respectively. In addition, adenovirus-mediated BTG1 overexpression improved insulin sensitivity in wild-type (WT) and insulin-resistant leptin-receptor mutated (db/db) mice. In addition, transgenic BTG1-overexpressing mice were resistant to high-carbohydrate diet-induced insulin resistance. Adenovirus-mediated BTG1 knockdown consistently impaired insulin sensitivity in WT and insulin-sensitive leucine-deprived mice. Moreover, hepatic BTG1 expression was increased by leucine deprivation via the mammalian target of rapamycin/ribosomal protein S6 kinase 1 pathway. Furthermore, c-Jun expression was up-regulated by BTG1, and adenovirus-mediated c-Jun knockdown blocked BTG1-improved insulin signaling and insulin sensitivity in vitro and in vivo. Finally, BTG1 promoted c-Jun expression via stimulating c-Jun and retinoic acid receptor activities. Taken together, these results identify a novel function for BTG1 in the regulation of hepatic insulin sensitivity and provide important insights into the nutritional regulation of BTG1 expression.- Xiao, F., Deng, J., Yu, J., Guo, Y., Chen, S., Guo, F. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun., (© FASEB.)

Leptin signaling in the hypothalamus is crucial in energy homeostasis. We have previously shown that dietary deprivation of the essential amino acid leucine in mice stimulates fat loss by increasing energy expenditure. The involvement of leptin signaling in this regulation, however, has not been reported. Here, we show that leucine deprivation promotes leptin signaling in mice maintained on an otherwise normal diet and restores leptin responses in mice maintained on a high fat diet, a regimen known to induce leptin resistance. In addition, we found that leucine deprivation stimulated energy expenditure, and fat loss was largely blocked in db/db mice homozygous for a mutation in leptin receptor and a knock-in mouse line Y3F with abrogation of leptin receptor Tyr(1138)-mediated signal transducer and activator transcript 3 signaling. Overall, our studies describe a novel link between hypothalamic leptin signaling and stimulation of energy expenditure under leucine deprivation.