Abstract

   The adverse metabolic impacts of branched-chain amino acids (BCAA) have
   been elucidated are mediated by isoleucine and valine. Dietary
   restriction of isoleucine promotes metabolic health and increases
   lifespan. However, a high protein diet enriched in BCAA is presently
   the most useful therapeutic strategy for nonalcoholic fatty liver
   disease (NAFLD), yet, its underlying mechanism remains largely unknown.
   Fatty liver hemorrhagic syndrome (FLHS), a specialized laying hen NAFLD
   model, can spontaneously develop fatty liver and hepatic steatosis
   under a high-energy and high-protein dietary background that the
   pathogenesis of FLHS is similar to human NAFLD. The mechanism
   underlying dietary BCAA control of NAFLD development in laying hens
   remains unclear. Herein, we demonstrate that dietary supplementation
   with 67 % High BCAA has unique mitigative impacts on NAFLD in laying
   hens. A High BCAA diet alleviates NAFLD, by inhibiting the
   tryptophan-ILA-AHR axis and MAPK9-mediated de novo lipogenesis (DNL),
   promoting ketogenesis and energy metabolism, and activating PPAR-RXR
   and pexophagy to promote fatty acid β-oxidation. Furthermore, we
   uncover that High BCAA strongly activates ubiquitin-proteasome
   autophagy via downregulating UFMylation to trigger MAPK9-mediated DNL,
   fatty acid elongation and lipid droplet formation-related proteins
   ubiquitination degradation, activating PPAR-RXR and pexophagy mediated
   fatty acid β-oxidation and lipolysis. Together, our data highlight
   moderating intake of high BCAA by inhibiting the AHR/MAPK9 are
   promising new strategies in NAFLD and FLHS treatment.

   Keywords: Branched-chain amino acids, Nonalcoholic fatty liver disease,
   Tryptophan-ILA-AHR, MAPK9, Ubiquitination

1. Introduction

   Nonalcoholic fatty liver disease (NAFLD), also referred to as metabolic
   -associated fatty liver disease (MAFLD), has become a global epidemic
   with no approved therapeutic drugs by FDA [[41]1]. It is characterized
   by the accumulation of lipid droplets (LD) in the liver and accompanied
   by elevated nonesterified fatty acids (NEFA) and triglycerides (TG),
   resulting in steatosis and insulin resistance (IR) [[42]2]. Excessive
   lipid accumulation in intracellular triggers a series of events within
   hepatocytes, including oxidative stress, autophagy inhibition,
   endoplasmic reticulum (ER) stress, and chronic inflammation, ultimately
   leading to hepatocyte death and fibrosis [[43]3]. NAFLD stands out as
   one of the most prevalent metabolic chronic liver conditions globally,
   affecting up to 25 % of the world's population and exhibiting a rising
   incidence [[44]4]. Approximately a quarter of patients with NAFLD
   develop from simple steatosis to nonalcoholic steatohepatitis (NASH),
   which over time advance to cirrhosis and hepatocellular carcinoma
   (HCC), statistically [[45]2,[46]5]. Obesity-related NAFLD serves as a
   significant risk factor for its prevalence, and patients with NAFLD
   face heightened risks of developing various systemic and metabolic
   complications [[47]6]. For example, IR, type-2 diabetes (T2D),
   cardiovascular diseases, and extrahepatic cancers [[48]6]. The burden
   of NAFLD is increasing because of its complex pathogenesis, yet there
   is currently no FDA-approved agent available for NAFLD [[49]5].

   Indeed, recent reports have pointed out that amino acid metabolic
   imbalance has been related to a raised risk and disease severity of
   obesity and NAFLD [[50][7], [51][8], [52][9], [53][10]]. For instance,
   increased circulating levels of branched-chain amino acids (BCAA)
   including leucine, isoleucine, and valine, in particular, and aromatic
   amino acids (AAA, i.e., tyrosine, phenylalanine, and tryptophan), have
   been associated with a raised risk of metabolic diseases, including
   NAFLD [[54][11], [55][12], [56][13], [57][14]]. BCAA have garnered
   significant attention, both because the three BCAA are almost always
   eaten and combusted together and their impact on the mammalian target
   of rapamycin (mTOR) pathways, which play a crucial role in linking
   nutrition to metabolic health and disease [[58]11]. Additionally,
   circulating levels of BCAA have been positively correlated with
   obesity, IR, and metabolic disorders in both rodents and humans and are
   a possible biomarker of NAFLD [[59][15], [60][16], [61][17]]. Long-term
   intake of high BCAA diets results in hyperphagia, reduced lifespan and
   obesity-associated NAFLD via promoting de novo lipogenesis (DNL) and
   hepatic steatosis [[62]9]. These adverse impacts are not solely
   attributed to elevated BCAA levels or the activation of hepatic mTOR
   but instead contribute to a change in the proportion of dietary BCAA
   compared to other amino acids, particularly tryptophan and threonine
   [[63]9]. However, lifelong restriction of BCAA leads to a 30 % increase
   in lifespan and a decrease in frailty among males, but not include
   female [[64]18]. The adverse metabolic impacts of BCAA have been
   confirmed to be induced by isoleucine and valine and isoleucine has the
   strongest role [[65]19]. Restriction of isoleucine promotes metabolic
   health by remodeling liver and adipose metabolism, increasing hepatic
   insulin sensitivity and ketogenesis and promoting energy expenditure,
   activating the fibroblast growth factor 21 (FGF21)-uncoupling protein 1
   (UCP1) axis [[66]19]. Restriction of dietary isoleucine is enough to
   attenuate western diet-induced (WD) obese-associated hepatic steatosis
   by reducing hepatic lipid deposition and smaller LD [[67]19]. In
   addition, reduction of dietary isoleucine is enough to promote the
   metabolic health of young and old HET3 mice, improving leanness and
   glycemic control in both sexes, reducing frailty, extending the
   lifespan, and remodeling hepatic metabolism in a sex-dependent manner
   [[68]20]. However, a high-protein diet that is enriched in BCAA, is
   presently the most useful therapeutic for NAFLD which leads to a
   reduction of liver fat by 36%–48 % in NAFLD patients within 6 weeks
   [[69][21], [70][22], [71][23]]. In particular, high protein diet
   enriched in BCAA has been widely used to treat hepatic
   steatosis-related liver disease in both preclinical and clinical trials
   [[72][24], [73][25], [74][26]]. Recent research also found that dietary
   leucine and isoleucine ameliorate steatosis by promoting
   polyubiquitination of PLIN2 via powerfully binding to and activating
   UBR1, targeting the degradation of PLIN2 [[75]27]. Leucine and
   isoleucine-induced activation of UBR1/2 ubiquitination is necessary to
   ameliorate hepatic steatosis and obesity induced by high-fat diet (HFD)
   [[76]27]. Nevertheless, these contradictory findings uncover the
   complex role of BCAA in the pathogenesis and progress of NAFLD.

   Fatty liver hemorrhagic syndrome (FLHS) induced by NAFLD in poultry,
   notably laying hens, has been a giant cause of death in commercial
   caged laying hens [[77]28]. FLHS is characterized by the accumulation
   of large amounts of hepatic lipid and abdominal fat and accompanied by
   hepatic hemorrhage [[78]29]. The pathogenesis of FLHS is similar to
   NAFLD in humans, resulting in considerable mortality of laying hens
   during and after the laying peak period due to liver rupture leading to
   internal bleeding [[79]29]. It is worth noting that, until now, our
   knowledge about the impacts of BCAA on the pathogenesis of NAFLD was
   primarily based on either a single dietary BCAA restriction or intake
   of all three BCAA at the same time. However, the metabolic impacts of
   dietary BCAA supplementation on laying hens under the pathological
   circumstances of FLHS remains unknown. Therefore, an overall analysis
   of the functions of dietary supplementation with BCAA on NAFLD and
   FLHS, applying to laying hen models that partly imitate the human NAFLD
   and appropriate dosing, is urgent to be solved. Given the load of NAFLD
   on humans and FLHS on laying hens, lack of effective therapies, and
   contradictory reports relating dietary BCAA intake to NAFLD progress,
   there is a strong reason to better comprehend the impacts of dietary
   BCAA supplementation on NAFLD, which can find effective therapeutics.
   Here, we attempt to detect whether and how high dietary BCAA
   supplementation contributes to NAFLD development by performing
   transcriptome, proteome, and metabolome in the serum and liver of
   middle-aged laying hens with NAFLD. We suggest that supplementation of
   67 % three High BCAA (a maximum tolerance dose), but not leucine,
   isoleucine, and leucine plus isoleucine, is both required and enough to
   ameliorate NAFLD by reprogramming liver metabolism. These data indicate
   that dietary High BCAA is a useful treatment for FLHS in laying hens or
   potentially human NAFLD and recommend that a modest increase of dietary
   BCAA may be a convertible way to ameliorate and treat NAFLD and FLHS.

2. Materials and methods

2.1. Diets, birds, and experimental management

   Corn and soybean meals are major ingredients of a corn-soybean-type
   basal diet and are made according to NRC (1994) [[80]30] and China
   National Feeding Standard of Chicken (NY/T33-2004) [[81]31]. To
   determine the requirement of dietary BCAA in laying hens during the
   laying peak period, we collected the published research references