Dimerization of hub protein DYNLL1 and bZIP transcription factor CREB3L1 enhances transcriptional activation of CREB3L1 target genes like arginine vasopressin.

bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.

Decorin and/or biglycan knockdown in aged mouse patellar tendon impacts fibril morphology, scar area, and mechanical properties.

Small leucine-rich proteoglycans, such as decorin and biglycan, play pivotal roles in collagen fibrillogenesis during development, healing, and aging in tendon. Previous work has shown that the absence of decorin and biglycan affects fibril shape and mechanical properties during tendon healing. However, the roles of decorin and biglycan in the healing process of aged tendons are unclear. Therefore the objective of this study was to evaluate the differential roles of decorin and biglycan during healing of patellar tendon injury in aged mice. Aged (300 days old) female Dcn+/+/Bgn+/+ control (WT, n = 52), Dcnflox/flox (I-Dcn-/-, n = 36), Bgnflox/flox (I-Bgn-/-, n = 36), and compound Dcnflox/flox/Bgnflox/flox (I-Dcn-/-/Bgn-/-, n = 36) mice with a tamoxifen-inducible Cre were utilized. Targeted gene expression, collagen fibril diameter distributions, mechanical properties, and histological assays were employed to assess the effects of knockdown of decorin and/or biglycan at the time of injury. Knockdown resulted in alterations in fibril diameter distribution and scar area, but surprisingly did not lead to many differences in mechanical properties. Biglycan played a larger role in early healing stages, while decorin is more significant in later stages, particularly in scar remodeling. This study highlights some of the differential roles of biglycan and decorin in the regulation of fibril structure and scar area, as well as influencing gene expression during healing in aged mice.

A Study on the Effect of Various Media and the Supplementation of Organic Compounds on the Enhanced Production of Astaxanthin from Haematococcus lacustris (Girod-Chantrans) Rostafinski (Chlorophyta).

Natural astaxanthin is in high demand due to its multiple health benefits. The microalga Haematococcus lacustris has been used for the commercial production of astaxanthin. In this study, we investigated the effects of six different media with and without a nitrogen source and supplementation with nine organic compounds on the growth and astaxanthin accumulation of H. lacustris. The highest astaxanthin contents were observed in cultures of H. lacustris in Jaworski's medium (JM), with a level of 9.099 mg/L in JM with a nitrogen source supplemented with leucine (0.65 g/L) and of 20.484 mg/L in JM without a nitrogen source supplemented with sodium glutamate (0.325 g/L). Six of the nine organic compounds examined (leucine, lysine, alanine, sodium glutamate, glutamine, and cellulose) enhanced the production of astaxanthin in H. lacustris, while malic acid, benzoic acid, and maltose showed no beneficial effects.

Protein Requirements for Maximal Muscle Mass and Athletic Performance Are Achieved with Completely Plant-Based Diets Scaled to Meet Energy Needs: A Modeling Study in Professional American Football Players.

American football players consume large quantities of animal-sourced protein in adherence with traditional recommendations to maximize muscle development and athletic performance. This contrasts with dietary guidelines, which recommend reducing meat intake and increasing consumption of plant-based foods to promote health and reduce the risk of chronic disease. The capacity of completely plant-based diets to meet the nutritional needs of American football players has not been studied. This modeling study scaled dietary data from a large cohort following completely plant-based diets to meet the energy requirements of professional American football players to determine whether protein, leucine, and micronutrient needs for physical performance and health were met. The Cunningham equation was used to estimate calorie requirements. Nutrient intakes from the Adventist Health Study 2 were then scaled to this calorie level. Protein values ranged from 1.6-2.2 g/kg/day and leucine values ranged from 3.8-4.1 g/meal at each of four daily meals, therefore meeting and exceeding levels theorized to maximize muscle mass, muscle strength, and muscle protein synthesis, respectively. Plant-based diets scaled to meet the energy needs of professional American football players satisfied protein, leucine, and micronutrient requirements for muscle development and athletic performance. These findings suggest that completely plant-based diets could bridge the gap between dietary recommendations for chronic disease prevention and athletic performance in American football players.

Fructose Reduces Mitochondrial Metabolism and Increases Extracellular BCAA during Insulin Resistance in C2C12 Myotubes.

Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been correlated with the severity of insulin resistance. Most recently, the effect of fructose consumption on the downregulation of BCAA catabolic enzymes was observed. Thus, this mechanistic study investigated the effects of physiologically attainable levels of fructose, both with and without concurrent insulin resistance, in a myotube model of skeletal muscle. METHODS: C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. RESULTS: Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. DISCUSSION: Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.

NRC immune receptor networks show diversified hierarchical genetic architecture across plant lineages.

Plants' complex immune systems include nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, which help recognize invading pathogens. In solanaceous plants, the NRC (NLR required for cell death) family includes helper NLRs that form a complex genetic network with multiple sensor NLRs to provide resistance against pathogens. However, the evolution and function of NRC networks outside solanaceous plants are currently unclear. Here, we conducted phylogenomic and macroevolutionary analyses comparing NLRs identified from different asterid lineages and found that NRC networks expanded significantly in most lamiids but not in Ericales and campanulids. Using transient expression assays in Nicotiana benthamiana, we showed that NRC networks are simple in Ericales and campanulids, but have high complexity in lamiids. Phylogenetic analyses grouped the NRC helper NLRs into three NRC0 subclades that are conserved, and several family-specific NRC subclades of lamiids that show signatures of diversifying selection. Functional analyses revealed that members of the NRC0 subclades are partially interchangeable, whereas family-specific NRC members in lamiids lack interchangeability. Our findings highlight the distinctive evolutionary patterns of the NRC networks in asterids and provide potential insights into transferring disease resistance across plant lineages.

LUZP1 regulates the maturation of contractile actomyosin bundles.

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

Variations in the LINGO2 and GLIS3 Genes and Gene-Environment Interactions Increase Gestational Diabetes Mellitus Risk in Chinese Women.

Gestational diabetes mellitus (GDM) has been found to be a common complication in pregnant women, known to escalate the risk of negative obstetric outcomes. In our study, we genotyped 1,566 Chinese pregnant women for two single nucleotide polymorphisms (SNPs) in the LINGO2 gene and one SNP in the GLIS3 gene, utilizing targeted next-generation sequencing. The impact of two interacting genes, and the interaction of genes with the environment─including exposure to particulate matter (PM2.5), ozone (O3), and variations in prepregnancy body mass index (BMI)─on the incidence of GDM were analyzed using logistic regression. Our findings identify the variants LINGO2 rs10968576 (P = 0.022, OR = 1.224) and rs1412239 (P = 0.018, OR = 1.231), as well as GLIS3 rs10814916 (P = 0.028, OR = 1.172), as risk mutations significantly linked to increased susceptibility to GDM. Further analysis underscores the crucial role of gene-gene and gene-environment interactions in the development of GDM among Chinese women (P < 0.05). Particularly, the individuals carrying the rs10968576 G-rs1412239 G-rs10814916 C haplotype exhibit increased susceptibility to GDM during the prepregnancy period when interacting with PM2.5, O3, and BMI (P = 8.004 × 10-7, OR = 1.206; P = 6.3264 × 10-11, OR = 1.280; P = 9.928 × 10-7, OR = 1.334, respectively). In conclusion, our research emphasizes the importance of the interaction between specific gene variations─LINGO2 and GLIS3─and environmental factors in influencing GDM risk. Notably, we found significant associations between these gene variations and GDM risk across various environmental exposure periods.

Effects of bound nucleotides on the secondary structure, thermal stability, and phosphorylation of Rab3A.

Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2.

Trophoblast-specific overexpression of the LAT1 increases transplacental transport of essential amino acids and fetal growth in mice.

Placental System L amino acid transporter activity is decreased in pregnancies complicated by intrauterine growth restriction (IUGR) and increased in fetal overgrowth. However, it is unknown if changes in the expression/activity of placental Large Neutral Amino Acid Transporter Small Subunit 1 (Slc7a5/LAT1) are mechanistically linked to placental function and fetal growth. We hypothesized that trophoblast-specific Slc7a5 overexpression increases placental transport of essential amino acids, activates the placental mechanistic target of rapamycin (mTOR) signaling, and promotes fetal growth in mice. Using lentiviral transduction of blastocysts with a Slc7a5 transgene, we achieved trophoblast-specific overexpression of Slc7a5 (Slc7a5 OX) with increased fetal (+27%) and placental weights (+10%). Trophoblast-specific Slc7a5 overexpression increased trophoblast plasma membrane (TPM) LAT1 protein abundance and TPM System L transporter (+53%) and System A transporter activity (+ 21%). Slc7a5 overexpression also increased transplacental transport of leucine (+ 85%) but not of the System A tracer, 14C-methylamino isobutyric acid, in vivo. Trophoblast-specific overexpression of Slc7a5 activated placental mTORC1, as assessed by increased (+44%) phosphorylation of S6 ribosomal protein (Ser 235/236), and mTORC2 as indicated by phosphorylation of PKCα-Tyr-657 (+47%) and Akt-Ser 473 (+96%). This is the first demonstration that placental transport of essential amino acids is mechanistically linked to fetal growth. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter in some cases of fetal overgrowth may directly contribute to the development of these pregnancy complications.

Effectiveness of Nutritional Guidance Focusing on Leucine Intake During Cardiac Rehabilitation Maintenance.

Objective: Due to the lack of information on the effects of nutritional guidance focused on leucine intake in patients undergoing maintenance cardiac rehabilitation, this study investigated on plasma leucine concentrations, lean body mass, and muscle strength. Methods: Nutritional guidance, focused on leucine (intervention group) or general nutritional guidance (control group), was provided for six months to patients participating in cardiac rehabilitation. Body composition, grip strength, hematological test results, and diet of both groups were compared before and after the intervention. Results: Seven patients in the intervention group (53.2 ±â€…18.2 years) and 7 patients in the control group (58.6 ±â€…15.3 years) were included. Dietary survey results showed that the six-month intervention significantly (p < 0.05) increased protein intake and estimated leucine intake only in the intervention group. There was no significant difference in the rate of change in plasma leucine concentration between the two groups. The rate of change in lean body mass was significantly higher in the intervention group compared to the control group (p = 0.035). The rate of change in plasma leucine concentration and that in lean body mass was positively correlated only in the intervention group (r = 0.777, p = 0.040), and the rate of change in plasma leucine concentration was also positively correlated with the rate of change in grip strength (ρ = 0.857, p = 0.014). Conclusions: In the patients undergoing maintenance cardiac rehabilitation, increased plasma leucine concentration by nutritional guidance focused on leucine increased lean body mass without any increasing the training load.

Leucine Supplementation Modulates Lipid Metabolism and Inflammation in Early Weaning Piglets.

Early weaning can induce the programmed dysregulation of glycolipid metabolism and inflammation in adult animals. The primary objective of this study was to evaluate the efficacy of leucine supplementation administered promptly after early weaning in mitigating these adverse effects in piglets. At day 21, 24 piglets were randomly selected and divided into 3 groups: EW group where the piglets were weaned at day 21 and fed basal diet, EWL group where the piglets were weaned at day 21 and fed the basal diet with supplementation of 1% leucine, and C group where the piglets were fed basal diet and weaned at 28 days. Each group contained eight replicates, with one piglet per replicate. The results indicated that early weaning had an impact on gut health and could activate the inhibitor of the kappa B kinase gamma/inhibitor kappa B alpha/NF-kappa-B (IKKγ/IκBα/NF-κB) signaling pathway to ameliorate pro-inflammatory factor and apoptosis levels. Furthermore, early weaning reduced the activity of fatty acid ß oxidation (FAßO) and affected genes linked with lipid metabolism. Supplementing with leucine can improve the effects of these factors. In summary, leucine may alleviate the influences of early weaning on the lipid metabolism and inflammation in piglets.

The NRC0 gene cluster of sensor and helper NLR immune receptors is functionally conserved across asterid plants.

Nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) proteins can form complex receptor networks to confer innate immunity. NLR-REQUIRED FOR CELL DEATH (NRCs) are phylogenetically related nodes that function downstream of a massively expanded network of disease resistance proteins that protect against multiple plant pathogens. Here, we used phylogenomic methods to reconstruct the macroevolution of the NRC family. One of the NRCs, termed NRC0, is the only family member shared across asterid plants, leading us to investigate its evolutionary history and genetic organization. In several asterid species, NRC0 is genetically clustered with other NLRs that are phylogenetically related to NRC-dependent disease resistance genes. This prompted us to hypothesize that the ancestral state of the NRC network is an NLR helper-sensor gene cluster that was present early during asterid evolution. We provide support for this hypothesis by demonstrating that NRC0 is essential for the hypersensitive cell death that is induced by its genetically linked sensor NLR partners in four divergent asterid species: tomato (Solanum lycopersicum), wild sweet potato (Ipomoea trifida), coffee (Coffea canephora), and carrot (Daucus carota). In addition, activation of a sensor NLR leads to higher-order complex formation of its genetically linked NRC0, similar to other NRCs. Our findings map out contrasting evolutionary dynamics in the macroevolution of the NRC network over the last 125 million years, from a functionally conserved NLR gene cluster to a massive genetically dispersed network.

Early change in serum leucine-rich α-2-glycoprotein predicts clinical and endoscopic response in ulcerative colitis.

Background/Aims: Leucine-rich α-2-glycoprotein (LRG) is a new serum biomarker reflecting the disease activity of ulcerative colitis (UC), but its change during the acute phase has not been enough investigated. Methods: Patients with UC who initiated the induction therapy with steroid or advanced therapy (biologics or Janus kinase inhibitors) were prospectively enrolled. Associations of LRG, C-reactive protein (CRP) and fecal calprotectin (FC) at baseline, week 1, and week 8 with clinical remission at week 8 and subsequent endoscopic improvement within 1 year (Mayo endoscopic subscore of 0 or 1) were assessed. Results: A total of 143 patients with UC were included. LRG and CRP at week 1 were significantly lower in the clinical remission group than in the non-remission group (LRG, 20.6 µg/mL vs. 28.4 µg/mL, P< 0.001; CRP, 0.9 mg/dL vs. 2.3 mg/dL, P< 0.001) while FC demonstrated the difference between groups only at week 8. The area under the curves of week 1 LRG, CRP, and FC for week 8 clinical remission using the receiver operating characteristic curves analysis were 0.68, 0.71, and 0.57, respectively. Furthermore, LRG and CRP predicted subsequent endoscopic improvement as early as week 1, while FC was predictive only at week 8. Conclusions: LRG can be an early-phase biomarker predicting subsequent clinical and endoscopic response to induction therapy.

Combining Flexible Region Design and Automatic Design to Enhance the Thermal Stability and Catalytic Efficiency of Leucine Dehydrogenase.

Leucine dehydrogenase (LeuDH, EC 1.4.1.9) can reversibly catalyze the oxidative deamination of l-leucine and some other specific α-amino acids to form the corresponding α-ketoacids. This reaction has great significance in the field of food additives and the pharmaceutical industry. The LeuDH from Exiguobacterium sibiricum (EsLeuDH) has high catalytic efficiency but limited thermal stability, hindering its widespread industrial application. In this study, a mutant N5F/I12L/A352Y of EsLeuDH (referred to as M2) was developed with enhanced thermal stability and catalytic activity through rational modification. The M2 mutant exhibits a half-life at 60 °C (t1/2(60 °C)) of 975.7 min and a specific activity of 69.6 U mg-1, which is 5.4 and 2.1 times higher than those of EsLeuDH, respectively. This research may facilitate the utilization of EsLeuDH at elevated temperatures, enhancing its potential for industrial applications. The findings offer a practical and efficient approach for optimizing LeuDH and other industrial enzymes.

How Parkinson's Disease-Linked LRRK2 Mutations Affect Different CNS Cell Types.

LRRK2 is a relatively common genetic risk factor for Parkinson's disease (PD), with six coding variants known to cause familial PD. Non-coding variation at the same locus is also associated with sporadic PD. LRRK2 plays a role in many different intracellular signaling cascades including those involved in endolysosomal function, cytoskeletal dynamics, and Ca2+ homeostasis. PD-causing LRRK2 mutations cause hyperactive LRRK2 kinase activity, resulting in altered cellular signaling. Importantly, LRRK2 is lowly expressed in neurons and prominently expressed in non-neuronal cells in the brain. In this review, we will summarize recent and novel findings on the effects of PD-causing LRRK2 mutations in different nervous system cell types. This review will also provide novel insight into future areas of research at the intersection of LRRK2 cell biology, cell type specificity, and PD.

Local complement activation and modulation in mucosal immunity.

The complement system is an evolutionarily conserved arm of innate immunity, which forms one of the first lines of host response to pathogens and assists in the clearance of debris. A deficiency in key activators/amplifiers of the cascade results in recurrent infection, whereas a deficiency in regulating the cascade predisposes to accelerated organ failure, as observed in colitis and transplant rejection. Given that there are over 60 proteins in this system, it has become an attractive target for immunotherapeutics, many of which are United States Food and Drug Administration-approved or in multiple phase 2/3 clinical trials. Moreover, there have been key advances in the last few years in the understanding of how the complement system operates locally in tissues, independent of its activities in circulation. In this review, we will put into perspective the abovementioned discoveries to optimally modulate the spatiotemporal nature of complement activation and regulation at mucosal surfaces.

Identification of inhibitors for neurodegenerative diseases targeting dual leucine zipper kinase through virtual screening and molecular dynamics simulations.

Neurodegenerative diseases lead to a gradual decline in cognitive and motor functions due to the progressive loss of neurons in the central nervous system. The role of dual leucine zipper kinase (DLK) in regulating stress responses and neuronal death pathways highlights its significance as a target against neurodegenerative diseases. The non-availability of FDA-approved drugs emphasizes a need to identify novel DLK-inhibitors. We screened NPAtlas (Natural products) and MedChemExpress (FDA-approved) libraries to identify potent ATP-competitive DLK inhibitors. ADMET analyses identified four compounds (two natural products and two FDA-approved) with favourable features. Subsequently, we performed molecular dynamics simulations to examine the binding-stability and ligand-induced conformational dynamics. Molecular mechanics Poisson Boltzmann surface area (MM-PBSA) calculations demonstrated CID139591660, dithranol, and danthron having greater affinity, while CID156581477 showed lower affinity than control sunitinib. PCA and network analysis results indicated structural and network alteration post-ligand binding. Furthermore, we identified an analogue of CID156581477 using the deep learning-based web server DeLA Drug which demonstrated a higher affinity than its parent compound and the control and identified several crucial interacting residues. Overall, our study provides significant theoretical guidance for designing potent novel DLK inhibitors and compounds that could emerge as promising drug candidates against DLK following laboratory validation.

New insights into evolution and functional diversification of Camellia sinensis LRR-RLKs.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subgroup of receptor-like kinases (RLKs) in plants. While some LRR-RLK members play a role in regulating various plant growth processes related to morphogenesis, disease resistance, and stress response, the functions of most LRR-RLK genes remain unclear. In this study, we identified 397 LRR-RLK genes from the genome of Camellia sinensis and categorized them into 16 subfamilies. Approximately 62% of CsLRR-RLK genes are situated in regions resulting from segmental duplications, suggesting that the expansion of CsLRR-RLK genes is due to segmental duplications. Analysis of gene expression patterns revealed differential expression of CsLRR-RLK genes across different tissues and in response to stress. Furthermore, we demonstrated that CssEMS1 localizes to the cell membrane and can complement Arabidopsis ems1 mutant. This study is the initial in-depth evolutionary examination of LRR-RLKs in tea and provides a basis for future investigations into their functionality. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01458-1.

Exploring the novel benefits of leucine: Protecting nitrite-induced liver damage in sub-adult grass carp (Ctenopharyngodon idella) through regulating mitochondria quality control.

Leucine is an essential amino acid for fish. The ability of leucine to resist stress in fish has not been reported. Nitrite is a common pollutant in the aquatic environment. Therefore, we investigated the effects of dietary leucine on growth performance and nitrite-induced liver damage, mitochondrial dysfunction, autophagy, and apoptosis for sub-adult grass carp. A total of 450 grass carp (615.91 ± 1.15 g) were selected and randomly placed into 18 net cages. The leucine contents of the six diets were 2.91, 5.90, 8.92, 11.91, 14.93, and 17.92 g/kg, respectively. After a 9-week feeding trial, the nitrite exposure experiment was set up for 96 h. These results indicated that dietary leucine significantly promoted FW, WG, PWG, and SGR of sub-adult grass carp (P < 0.05). Appropriate levels of dietary leucine (11.91-17.92 g/kg) decreased the activities of serum parameters (glucose, cortisol, and methemoglobin contents, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactate dehydrogenase), the contents of reactive oxygen species (ROS), nitric oxide (NO) and peroxynitrite (ONOO-). In addition, appropriate levels of dietary leucine (11.91-17.92 g/kg) increased the mRNA levels of mitochondrial biogenesis genes (PGC-1α, Nrf1/2, TFAM), fusion-related genes (Opa1, Mfn1/2) (P < 0.05), and decreased the mRNA levels of caspase 3, caspase 8, caspase 9, fission-related gene (Drp1), mitophagy-related genes (Pink1, Parkin) and autophagy-related genes (Beclin1, Ulk1, Atg5, Atg7, Atg12) (P < 0.05). Appropriate levels of dietary leucine (8.92-17.92 g/kg) also increased the protein levels of AMP-activated protein kinase (AMPK), prostacyclin (p62) and decreased the protein levels of protein light chain 3 (LC3), E3 ubiquitin ligase (Parkin), and Cytochrome c (Cytc). Appropriate levels of leucine (8.92-17.92 g/kg) could promote growth performance and alleviate nitrite-induced mitochondrial dysfunction, autophagy, apoptosis for sub-adult grass carp. Based on quadratic regression analysis of PWG and serum GPT activity, dietary leucine requirements of sub-adult grass carp were recommended to be 12.47 g/kg diet and 12.55 g/kg diet, respectively.