Growth hormone resistance induced by amino acid deprivation in fao cells is independent of FGF21.

Adequate dietary intake of amino acids is imperative for normal animal growth. Our previous work using rat hepatocarcinoma Fao cells demonstrated that growth hormone (GH) resistance, coupled with a concurrent reduction in insulin-like growth factor 1 (Igf1) mRNA levels, may underlie the growth retardation associated with a low-protein diet (LPD). In this study, we investigated whether FGF21 contributes to liver GH resistance in Fao rat hepatoma cells under amino acid deprivation conditions. Mice subjected to an LPD exhibited growth retardation, compromised GH signaling in the liver, and decreased blood IGF-1 levels compared with those on a control diet. To assess the potential involvement of fibroblast growth factor (FGF) 21, produced in response to amino acid deficiency, in the development of GH resistance, we examined GH signaling and Igf1 mRNA levels in Fao cells cultured in amino acid-deprived medium. Despite the inhibition of Fgf21 expression by the integrated stress response inhibitor, an inhibitor of the eukaryotic initiation factor 2-activating transcription factor 4 pathway, GH resistance persisted in response to amino acid deprivation. Additionally, the introduction of FGF21 into the control medium did not impair either GH signaling or GH-induced Igf1 transcription. These data suggest that, in Fao cells, amino acid deprivation induces GH resistance independently of FGF21 activity. By shedding light on the mechanisms behind growth retardation-associated GH resistance linked to amino acid deficiencies, our findings provide valuable insights for clinicians in formulating effective treatment strategies for individuals facing these challenges.

Moderate protein intake percentage in mice for maintaining metabolic health during approach to old age.

Nutritional requirements for maintaining metabolic health may vary with each life stage, such as young, middle, and old age. To investigate the appropriate ratio of nutrients, particularly proteins, for maintaining metabolic health while approaching old age, young (6-month-old) and middle-aged (16-month-old) mice were fed isocaloric diets with varying protein percentages (5%, 15%, 25%, 35%, and 45% by calorie ratio) for two months. The low-protein diet developed mild fatty liver, with middle-aged mice showing more lipids than young mice, whereas the moderate-protein diet suppressed lipid contents and lowered the levels of blood glucose and lipids. Self-organizing map (SOM) analysis revealed that plasma amino acid profiles differed depending on age and difference in protein diet and were associated with hepatic triglyceride and cholesterol levels. Results indicate that the moderate protein intake percentages (25% and 35%) are required for maintaining metabolic health in middle-aged mice, which is similar to that in young mice.

Macroglossia and less advanced dystrophic change in the tongue muscle of the Duchenne muscular dystrophy rat.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease caused by a complete lack of dystrophin, which stabilizes the plasma membrane of myofibers. The orofacial function is affected in an advanced stage of DMD and this often leads to an eating disorder such as dysphagia. Dysphagia is caused by multiple etiologies including decreased mastication and swallowing. Therefore, preventing the functional declines of mastication and swallowing in DMD is important to improve the patient's quality of life. In the present study, using a rat model of DMD we generated previously, we performed analyses on the masseter and tongue muscles, both are required for proper eating function. METHODS: Age-related changes of the masseter and tongue muscle of DMD rats were analyzed morphometrically, histologically, and immunohistochemically. Also, transcription of cellular senescent markers, and utrophin (Utrn), a functional analog of dystrophin, was examined. RESULTS: The masseter muscle of DMD rats showed progressive dystrophic changes as observed in their hindlimb muscle, accompanied by increased transcription of p16 and p19. On the other hand, the tongue of DMD rats showed macroglossia due to hypertrophy of myofibers with less dystrophic changes. Proliferative activity was preserved in the satellite cells from the tongue muscle but was perturbed severely in those from the masseter muscle. While Utrn transcription was increased in the masseter muscle of DMD rats compared to WT rats, probably due to a compensatory mechanism, its level in the tongue muscle was comparable between WT and DMD rats and was similar to that in the masseter muscle of DMD rats. CONCLUSIONS: Muscular dystrophy is less advanced in the tongue muscle compared to the masseter muscle in the DMD rat.

Essential Amino Acid Intake Is Required for Sustaining Serum Insulin-like Growth Factor-I Levels but Is Not Necessarily Needed for Body Growth.

Essential amino acids (EAAs) are those that cannot be synthesized enough to meet organismal demand; therefore, it is believed that they must be taken from the diet for optimal growth. The growth hormone (GH)/insulin-like growth factor-I (IGF-I) system is also considered significant for growth regulation in mammals. This study aimed to evaluate the relative contributions of protein nutrition and the GH/IGF-I system to body growth regulation. Experiments using rodents and hepatocyte-derived cell lines subjected to EAA deficiency showed that a reduction in the serum EAA concentration hinders Igf1 transcription in the liver in a cell-autonomous manner, thereby decreasing serum IGF-I levels. Remarkably, when the serum IGF-I level of mice on a low-protein diet was restored by the recombinant IGF-I infusion, the body growth was mostly rescued, although the mice were still deficient in EAA intake. Meanwhile, the GH signal activation and subsequent Igf1 transcription were also dramatically diminished by EAA deprivation in the cell culture model. Altogether, we demonstrate that EAAs are not strictly necessary for animal growth as building blocks but are required as IGF-I-tropic cues. The results will bring a paradigm shift regarding the definition of "essential" amino acids.

Dietary lysine restriction induces lipid accumulation in skeletal muscle through an increase in serum threonine levels in rats.

We previously reported that dietary amino acid restriction induces the accumulation of triglycerides (TAG) in the liver of growing rats. However, differences in TAG accumulation in individual cell types or other tissues were not examined. In this study, we show that TAG also accumulates in the muscle and adipose tissues of rats fed a low amino acid (low-AA) diet. In addition, dietary lysine restriction (low-Lys) induces lipid accumulation in muscle and adipose tissues. In adjusting the nitrogen content to that of the control diet, we found that glutamic acid supplementation to the low-AA diet blocked lipid accumulation, but supplementation with the low-Lys diet did not, suggesting that a shortage of nitrogen caused lipids to accumulate in the skeletal muscle in the rats fed a low-AA diet. Serum amino acid measurement revealed that, in rats fed a low-Lys diet, serum lysine levels were decreased, while serum threonine levels were significantly increased compared with the control rats. When the threonine content was restricted in the low-Lys diet, TAG accumulation induced by the low-Lys diet was completely abolished in skeletal muscle. Moreover, in L6 myotubes cultured in medium containing high threonine and low lysine, fatty acid uptake was enhanced compared with that in cells cultured in control medium. These findings suggest that the increased serum threonine in rats fed a low-Lys diet resulted in lipid incorporation into skeletal muscle, leading to the formation of fatty muscle tissue. Collectively, we propose conceptual hypothesis that "amino-acid signal" based on lysine and threonine regulates lipid metabolism.

A novel amino acid signaling process governs glucose-6-phosphatase transcription.

Emerging evidence has shown that amino acids act as metabolic regulatory signals. Here, we showed that glucose-6-phosphatase (G6Pase) mRNA levels in cultured hepatocyte models were downregulated in an amino-acid-depleted medium. Inversely, stimulation with amino acids increased G6Pase mRNA levels, demonstrating that G6Pase mRNA level is directly controlled by amino acids in a reversible manner. Promoter assay revealed that these amino-acid-mediated changes in G6Pase mRNA levels were attributable to transcriptional regulation, independent of canonical hormone signaling pathways. Metabolomic analysis revealed that amino acid starvation induces a defect in the urea cycle, decreasing ornithine, a major intermediate, and supplementation of ornithine in an amino-acid-depleted medium fully rescued G6Pase mRNA transcription, similar to the effects of amino acid stimulation. This pathway was also independent of established mammalian target of rapamycin complex 1 pathway. Collectively, we present a hypothetical concept of "metabolic regulatory amino acid signal," possibly mediated by ornithine.

Alteration of serum amino acid profiles by dietary adenine supplementation inhibits fatty liver development in rats.

Studies on animal models have demonstrated that feeding a low-arginine diet inhibits triacylglycerol (TAG) secretion from the liver, resulting in marked fatty liver development in rats. Here, we first showed that culturing hepatocytes in the medium mimicking the serum amino acid profile of low-arginine diet-fed rats induced TAG accumulation in the cells, indicating that the specific amino acid profile caused TAG accumulation in hepatocytes. Dietary adenine supplementation completely recovered hepatic TAG secretion and abolished hepatic TAG accumulation in rats. A comprehensive non-linear analysis revealed that inhibition of hepatic TAG accumulation by dietary adenine supplementation could be predicted using only serum amino acid concentration data. Comparison of serum amino acid concentrations indicated that histidine, methionine, and branched-chain amino acid (BCAA) concentrations were altered by adenine supplementation. Furthermore, when the serum amino acid profiles of low-arginine diet-fed rats were altered by modifying methionine or BCAA concentrations in their diets, their hepatic TAG accumulation was abolished. Altogether, these results suggest that an increase in methionine and BCAA levels in the serum in response to dietary arginine deficiency is a key causative factor for hepatic TAG accumulation, and dietary adenine supplementation could disrupt this phenomenon by altering serum amino acid profiles.

Low-arginine and low-protein diets induce hepatic lipid accumulation through different mechanisms in growing rats.

BACKGROUND: Dietary protein deficiency and amino acid imbalance cause hepatic fat accumulation. We previously demonstrated that only arginine deficiency or total amino acid deficiency in a diet caused significant hepatic triglyceride (TG) accumulation in young Wistar rats. In this study, we explored the mechanisms of fatty liver formation in these models. METHODS: We fed 6-week-old male Wistar rats a control diet (containing an amino acid mixture equivalent to 15% protein), a low-total-amino acid diet (equivalent to 5% protein; 5PAA), and a low-arginine diet (only the arginine content is as low as that of the 5PAA diet) for 2 weeks. RESULTS: Much greater hepatic TG accumulation was observed in the low-arginine group than in the low-total-amino acid group. The lipid consumption rate and fatty acid uptake in the liver did not significantly differ between the groups. In contrast, the low-total-amino acid diet potentiated insulin sensitivity and related signaling in the liver and enhanced de novo lipogenesis. The low-arginine diet also inhibited hepatic very-low-density lipoprotein secretion without affecting hepatic insulin signaling and lipogenesis. CONCLUSIONS: Although the arginine content of the low-arginine diet was as low as that of the low-total-amino acid diet, the two diets caused fatty liver via completely different mechanisms. Enhanced lipogenesis was the primary cause of a low-protein diet-induced fatty liver, whereas lower very-low-density lipoprotein secretion caused low-arginine diet-induced fatty liver.

Importance of Serum Amino Acid Profile for Induction of Hepatic Steatosis under Protein Malnutrition.

We previously reported that a low-protein diet caused animals to develop fatty liver containing a high level of triglycerides (TG), similar to the human nutritional disorder "kwashiorkor". To investigate the underlying mechanisms, we cultured hepatocytes in amino acid-sufficient or deficient medium. Surprisingly, the intracellular TG level was increased by amino acid deficiency without addition of any lipids or hormones, accompanied by enhanced lipid synthesis, indicating that hepatocytes themselves monitored the extracellular amino acid concentrations to induce lipid accumulation in a cell-autonomous manner. We then confirmed that a low-amino acid diet also resulted in the development of fatty liver, and supplementation of the low-amino acid diet with glutamic acid to compensate the loss of nitrogen source did not completely suppress the hepatic TG accumulation. Only a dietary arginine or threonine deficiency was sufficient to induce hepatic TG accumulation. However, supplementation of a low-amino acid diet with arginine or threonine failed to reverse it. In silico analysis succeeded in predicting liver TG level from the serum amino acid profile. Based on these results, we conclude that dietary amino acid composition dynamically affects the serum amino acid profile, which is sensed by hepatocytes and lipid synthesis was activated cell-autonomously, leading to hepatic steatosis.