Methods, compositions and applications for modulating salt taste receptors and salt taste perception

Abstract

This invention belongs to the field of biotechnology and relates to methods, compositions, and applications for regulating salty taste receptors and salty taste perception. Specifically, the activity level of GCN2 on the tongue can serve as a target for assessing and / or regulating salty taste perception ability. Activating GCN2 can enhance the subject's ability to perceive salty taste, while inhibiting GCN2 can suppress the subject's ability to perceive salty taste.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to methods, compositions, and applications for regulating salty taste receptors and salty taste perception. Background Technology

[0002] Saltiness in food is a key sensory characteristic that enhances appetite.

[0003] For example, high blood pressure is the leading cause of morbidity and death from cardiovascular diseases such as stroke, heart failure, myocardial infarction, and chronic kidney disease. Individuals with hypertension-related conditions or at risk of developing hypertension require a low-sodium diet.

[0004] For example, there are some core diseases that clinically require a high-salt diet, such as adrenocortical insufficiency, extensive burns, cystic fibrosis, and vasovagal syncope. For these diseases, a high-salt diet should be followed under clinical guidance.

[0005] Therefore, determining a person's sensitivity to saltiness is of great value in nutrition and health, food research and development, and clinical medicine.

[0006] From a health management perspective, an individual's sensitivity to saltiness directly affects salt intake. Those with high sensitivity can often satisfy their taste buds with less salt, thereby reducing the risk of hypertension, cardiovascular disease, and stomach cancer. Conversely, those with low sensitivity are prone to unintentionally consuming excessive salt. Therefore, sensitivity testing can be used to develop personalized salt reduction intervention programs. In the food industry, by assessing consumers' saltiness sensitivity thresholds, companies can optimize low-sodium product formulations (such as using potassium salt substitutes or flavor enhancers) without sacrificing saltiness perception, thus meeting both food safety standards and market demands. Furthermore, in clinical practice, abnormal saltiness sensitivity can serve as an auxiliary diagnostic indicator for certain endocrine disorders (such as adrenal insufficiency), neurological lesions, or nutritional deficiencies. For example, zinc deficiency often leads to decreased taste sensitivity, and taste testing can screen for potential pathological conditions early. Additionally, for the common salt preference problem among the elderly due to taste degeneration, medical institutions can design dietary interventions to improve their cardiovascular prognosis.

[0007] Therefore, it is of great significance to provide biomarkers for the diagnosis and / or treatment of salty taste perception sensitivity. Summary of the Invention

[0008] The purpose of this invention is to provide a method, composition and application of modulating salty taste receptors.

[0009] This invention provides the application of GCN2 as a target in detecting and / or modulating the sensitivity of test subjects to salty taste.

[0010] In a first aspect of the invention, the use of GCN2 as a biomarker in the preparation of products for assessing and / or modulating the sensitivity of test subjects to salty tastes is provided.

[0011] In a second aspect of the invention, the use of a GCN2 agonist is provided for improving the sensitivity of a test subject to salty taste.

[0012] In another preferred embodiment, the use is for non-diagnostic and non-therapeutic purposes.

[0013] In another preferred embodiment, it is used to prepare a formulation or composition for improving the sensitivity of a test subject to salt.

[0014] In another preferred embodiment, the GCN2 agonist is the sole active ingredient in the formulation or composition.

[0015] In another preferred embodiment, the test subjects include: humans or non-human mammals.

[0016] In another preferred embodiment, the non-human mammal includes: mouse, rat, pig, cow, sheep, or rabbit.

[0017] In a specific implementation, it is used to improve the subject's sensitivity to salty tastes while maintaining the subject's sensitivity to sweet, bitter, sour, and / or umami tastes.

[0018] In another preferred embodiment, increasing the subject's sensitivity to saltiness includes: (Z1) significantly increased the expression level of salty taste receptors in the subjects; (Z2) Improve subjects' rating of the saltiness of the same food; and / or (Z3) Reduce the salt intake level of the subjects.

[0019] In another preferred embodiment, the salty taste receptor comprises: SCNN1α, SCNN1β, SCNN1γ, or a combination thereof.

[0020] In another preferred embodiment, "significantly improved" means that, before administration of the GCN2 agonist or a formulation or composition containing the GCN2 agonist, the expression level Y1 of the salty taste receptor in the test subject is Y2, and after administration of the GCN2 agonist or a formulation or composition containing the GCN2 agonist, the expression level Y2 of the salty taste receptor in the test subject is Y2 / Y1 ≥ 1.5, preferably ≥ 2, more preferably ≥ 3, and most preferably ≥ 4.

[0021] In another preferred embodiment, the reduction in salt intake of the test subject means that after administration of a GCN2 agonist or a formulation or composition containing a GCN2 agonist, the salt intake of the test subject is reduced by ≥10%, more preferably by ≥30%, more preferably by ≥50%, even more preferably by ≥70%, and most preferably by ≥90%.

[0022] In another preferred embodiment, the GCN2 agonist significantly increases GCN2 levels on the tongue.

[0023] In another preferred embodiment, the tongue comprises lingual epithelial cells.

[0024] In another preferred embodiment, the GCN2 level includes: the activity level of GCN2.

[0025] In another preferred embodiment, the GCN2 level includes: the gene level and / or protein level of GCN2.

[0026] In another preferred embodiment, "significantly improved" means that the ratio of the subject's GCN2 level X1 before administration of the GCN2 agonist to the subject's GCN2 level X2 after administration of the GCN2 agonist is ≥1.3, preferably ≥1.5, more preferably ≥1.8, and most preferably ≥2.

[0027] In another preferred embodiment, the application comprises: oral administration, wet compress, or a combination thereof.

[0028] In another preferred embodiment, the activity level is characterized by a parameter selected from the group consisting of: the expression level of phosphorylated GCN2 or the expression level of p-eIF2a.

[0029] In another preferred embodiment, the GCN2 agonist comprises: a dietary composition for a specific amino acid deficiency. The specific amino acids include: leucine, tryptophan, valine, lysine, or combinations thereof.

[0030] In another preferred embodiment, the deficiency refers to a condition in which the level L1 of a specific amino acid in the dietary composition is preferably ≤0.3 and most preferably ≤0.1 compared to the recommended intake level L0 of the specific amino acid.

[0031] In another preferred embodiment, the dietary composition comprises the following components: protein, carbohydrates, and fat.

[0032] In another preferred embodiment, the carbohydrate comprises: corn syrup, modified corn starch, or a combination thereof.

[0033] In another preferred embodiment, the fat comprises: high-oleic safflower oil, coconut oil, soybean oil, or a combination thereof.

[0034] In another preferred embodiment, the carbohydrates in the dietary composition comprise 45-70 parts by weight.

[0035] In another preferred embodiment, the fat content in the dietary composition is 15-25 parts by weight.

[0036] In another preferred embodiment, the protein in the dietary composition comprises 10-25 parts by weight.

[0037] In another preferred embodiment, the protein is a mixture of amino acids that does not contain a specific amino acid.

[0038] In another preferred embodiment, the specific amino acid comprises: leucine, tryptophan, valine, lysine, or a combination thereof.

[0039] In another preferred embodiment, the protein is composed of L-arginine, L-hydrated histidine, L-isoleucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, L-alanine, L-hydrated asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-proline, L-serine, and L-tyrosine.

[0040] In another preferred embodiment, the protein is composed of L-arginine, L-hydrated histidine, L-isoleucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-leucine, L-valine, L-alanine, L-hydrated asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-proline, L-serine, and L-tyrosine.

[0041] In another preferred embodiment, the protein is composed of L-arginine, L-hydrated histidine, L-isoleucine, L-tryptophan, L-methionine, L-phenylalanine, L-threonine, L-leucine, L-valine, L-alanine, L-hydrated asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-proline, L-serine, and L-tyrosine.

[0042] In another preferred embodiment, the protein is composed of L-arginine, L-hydrated histidine, L-isoleucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-leucine, L-tryptophan, L-alanine, L-hydrated asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-proline, L-serine, and L-tyrosine.

[0043] In another preferred embodiment, the GCN2 agonist comprises: an agent that specifically inhibits the digestion, absorption, and / or metabolism of a particular amino acid. The specific amino acids include: leucine, tryptophan, valine, lysine, or combinations thereof.

[0044] In a third aspect of the invention, a dietary combination is provided for activating GCN2, thereby increasing the sensitivity of a subject to salty taste, the dietary combination comprising carbohydrates, protein, and fat; The protein is deficient in a specific amino acid, which includes leucine, tryptophan, valine, lysine, or a combination thereof.

[0045] In another preferred embodiment, the combination further includes a reagent for detecting the expression level of salty taste receptors.

[0046] In another preferred embodiment, the dietary combination is an artificially formulated oral preparation, injectable preparation, and / or tongue application preparation.

[0047] In another preferred embodiment, the dietary combination can replace the subject's normal diet.

[0048] In another preferred embodiment, the substitution is a short-term substitution, where short-term means ≤2 days, preferably ≤3 days, and most preferably ≤5 days.

[0049] In a fourth aspect of the invention, the use of a GCN2 inhibitor is provided for reducing the sensitivity of a subject to salty taste.

[0050] In another preferred embodiment, it is used to prepare a formulation or composition for reducing the tolerance of a test subject to salt.

[0051] In another preferred embodiment, it is used to reduce the subject's sensitivity to salty tastes while maintaining the subject's sensitivity to sweet, bitter, sour, and / or umami tastes.

[0052] In another preferred embodiment, reducing the sensitivity of the test subject includes: (Z1) significantly reduced the expression level of salty taste receptors; (Z2) Reduce the subject's rating of the saltiness of the same food; and / or (Z3) Increase the salt intake level of the subjects.

[0053] In another preferred embodiment, "significantly reduced" means that, before the administration of the GCN2 inhibitor or a formulation or composition containing the GCN2 inhibitor, the expression level Y3 of the salty taste receptor in the test subject is Y4, and after the administration of the GCN2 inhibitor or a formulation or composition containing the GCN2 inhibitor, the expression level Y4 of the salty taste receptor in the test subject is Y4 / Y3 ≤ 0.9, preferably ≤ 0.85, and most preferably ≤ 0.8.

[0054] In another preferred embodiment, the increase in salt intake level of the test subject means that after administration of a GCN2 inhibitor or a formulation or composition containing a GCN2 inhibitor, the salt intake level of the test subject is increased by ≥10%, more preferably by ≥30%, more preferably by ≥50%, even more preferably by ≥70%, and most preferably by ≥90%.

[0055] In another preferred embodiment, the GCN2 inhibitor significantly inhibits the level of GCN2.

[0056] In another preferred embodiment, the significant inhibition means that the ratio of the level of GCN2 in the subject before administration of the GCN2 inhibitor (X3) to the level of GCN2 in the subject after administration of the GCN2 inhibitor (X4) is ≤50%, preferably ≤30%, and most preferably ≤20%.

[0057] In another preferred embodiment, the level includes gene level, protein level, and activity level.

[0058] In another preferred embodiment, the GCN2 inhibitor comprises: GCN2iB and a GCN2 knockout agent.

[0059] In a fifth aspect of the invention, the use of a GCN2 detection reagent is provided for assessing the sensitivity of a test subject to salty taste.

[0060] In another preferred embodiment, a formulation or composition is prepared for evaluating the sensitivity of a test subject to saltiness.

[0061] In another preferred embodiment, the GCN2 detection reagent detects the GCN2 level of the test subject.

[0062] In another preferred embodiment, the GCN2 level is the tongue GCN2 level.

[0063] In a sixth aspect of the invention, a method is provided for increasing the sensitivity of a subject to salty taste for non-diagnostic and non-therapeutic purposes by administering a GCN2 agonist or the dietary combination described in the third aspect of the invention to the subject in need.

[0064] In another preferred embodiment, the application includes oral administration or topical application.

[0065] In another preferred embodiment, the application is made to the tongue.

[0066] In another preferred embodiment, the application is performed for ≥1 day, preferably ≥2 days, and most preferably ≥3 days.

[0067] In a seventh aspect of the invention, a method for reducing the sensitivity of a subject to salty taste for non-diagnostic and non-therapeutic purposes is provided by administering a GCN2 inhibitor to the subject in need.

[0068] In an eighth aspect of the invention, a method for assessing a subject's sensitivity to saltiness for non-diagnostic and non-therapeutic purposes is provided, comprising the following steps: (S1) Provide the subject's tongue epithelial cells; (S2) Detect the GCN2 level of the tongue epithelial cells to assess the subject’s sensitivity to salty taste.

[0069] In a ninth aspect of the invention, a formulation combination is provided, the formulation combination comprising: GCN2 agonists or GCN2 inhibitors; and Reagents for detecting the expression level of salt taste receptors.

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0071] Figure 1 The study showed that a dietary intervention for leucine deficiency affected the perception of salty taste in leucine-containing foods. Figure 1 A shows the timeline and pattern diagram of the population intervention; Figure 1 B shows the population's rating of the saltiness of the control formula; Figure 1 C shows the population's ratings of sweetness, sourness, bitterness, and umami for the control formula; Figure 1 D shows the liquid food formula for the population; Figure 1 E shows the population's taste questionnaire.

[0072] Figure 2 The study showed that leucine deficiency treatment in mice could increase the expression of salty taste receptors on the tongue. Figure 2 A shows the mRNA expression level of salty taste receptors in mouse tongue epithelial cells after 24 h of treatment with either a control or leucine-deficient culture medium. Figure 2 Figure B shows the calcium signal response of mouse tongue epithelial cells after treatment with control or leucine-deficient culture medium for 24 h, followed by the addition of a calcium signal indicator (green) and a GCN2 inhibitor, and then the detection of calcium signal response before and after the addition of 10 μM Leu. Left panel: Representative calcium signal fluorescence image; Right panel: Statistical graph of calcium signal response (cells with a calcium signal change greater than 10% are considered responsive). Figure 2 C shows a schematic diagram of mice subjected to specific amino acid (leucine, tryptophan, lysine, valine) deficiency or control food treatment; Figure 2 D、 Figure 2 E, Figure 2 F shows the mRNA expression level of the salty taste receptor on the tongue of mice with specific amino acid deficiencies.

[0073] Figure 3 The study showed that leucine deficiency treatment can increase the activity of GCN2 in the tongue of mice and humans; Figure 3 A shows the expression level of p-eIF2α protein (a signal reflecting GCN2 activity) in the tongue of mice after 3 days of leucine deficiency; Cont: control diet; (-) Leu: leucine-deficient diet; Figure 3 B shows the expression level of p-eIF2α protein in lingual epithelial cells after 2 days of intervention with a leucine-deficient formula; Cont: control formula drink; (-) Leu: leucine-deficient formula drink.

[0074] Figure 4 The study showed that knocking down GCN2 on the mouse tongue can inhibit the expression of salty taste receptors on the tongue and alter food choices. Figure 4 A shows a diagram illustrating the GCN2 knockdown virus injection and food treatment pattern in mice; Figure 4 B shows the GCN2 mRNA expression level in the tongue of mice after GCN2 knockdown; Figure 4 C shows the expression level of p-eIF2α protein in the tongue of mice after GCN2 knockdown; Figure 4 D shows the mRNA expression level of the salty taste receptor on the tongue after GCN2 knockdown in mice; Figure 4 E shows the percentage of times mice touched the control food with their mouths after being given a leucine-deficient diet; Figure 4 F shows the preference index of mice after being given a control diet and a leucine-deficient diet, which was used to detect their intake of different foods.

[0075] Figure 5This study showed that knocking down salt taste receptors on the tongue of mice altered their food preferences. Figure 5 A shows a diagram of the salt taste receptor knockdown virus injected into the tongue of mice and the food treatment pattern; Figure 5 B shows the mRNA expression level of the salt taste receptor on the tongue after knockdown of the salt taste receptor in mice. Figure 5 C shows the percentage of times mice touched the control food with their mouths after being given a leucine-deficient diet; Figure 5 D shows the preference index of mice after being given a control diet and a leucine-deficient diet, which was used to detect their intake of different foods.

[0076] All experimental results are expressed as mean ± standard error. Detailed Implementation

[0077] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that GCN2 can serve as a target for assessing and / or regulating salty taste perception. Activating GCN2 can enhance the subject's ability to perceive salty taste, while inhibiting GCN2 can suppress the subject's ability to perceive salty taste. Based on this, the present invention was completed.

[0078] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0079] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0080] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0081] As used in this article, the term "or a combination thereof" means "or any combination thereof".

[0082] As used in this article, terms such as "enhanced perception of saltiness" and "increased sensitivity to saltiness" can be used interchangeably. They refer to the increased perceived saltiness of the test subject for the same formulation; or they can refer to the test subject's perceived saltiness for a formulation with lower salt content that perfectly matches their individual needs.

[0083] As used herein, the recommended intake level for a specific amino acid refers to the recommended intake level for that specific amino acid derived from the dietary guidelines issued by the National Health Commission. In a specific implementation, the dietary guidelines include the "Chinese Dietary Guidelines (2022)". In a specific implementation, the dietary guidelines include the "Core Information on 'Healthy Eating and Reasonable Diet'".

[0084] As used herein, maintaining the subject's sensitivity to sweet, bitter, sour, and / or umami tastes means that the subject's ability to perceive sweet, bitter, sour, and / or umami tastes does not increase or decrease significantly, but remains at the level before the intervention.

[0085] Salty taste perception When food enters the mouth, it activates different taste receptors, allowing the organism to perceive the specific taste of the food. Taste perception is crucial for making informed food choices.

[0086] Among the many compounds recognized by the human taste system, sodium ions (Na+) are particularly noteworthy. + Sodium ions play a particularly important role in maintaining key physiological functions such as fluid balance and nerve conduction. They trigger a specific sensation, a salty taste; low to moderate concentrations of table salt (NaCl, a common sodium-containing chemical we use to flavor food) are perceived as pleasant and appealing.

[0087] The perception of saltiness begins the moment a taste activator dissolves in saliva and interacts with specific receptors within the taste buds. Epithelial sodium channels (ENaCs, salt taste receptors) play a crucial role in maintaining fluid volume and sodium ion balance, a function fundamental to the pathogenesis of salt-sensitive hypertension. ENaCs have been identified as receptors that recognize sodium... + And key proteins that mediate preference for low to moderate salt concentrations. Salty diets activate the salty taste receptor ENaC, which in turn activates the downstream cascade of taste reactions. Through transmission in multiple brain regions in the central nervous system, organisms can perceive saltiness.

[0088] Increase saltiness sensitivity The balance between salt and body fluids is essential for regulating blood pressure. Reducing sodium ions (Na+) in food is crucial. + Salt intake can reduce high blood pressure and cardiovascular-related morbidity and mortality. Therefore, finding new salty alternatives to reduce dietary salt intake has become a research hotspot.

[0089] The core principle of salt substitutes is that they are substances capable of partially or completely replacing traditional table salt. Their aim is to effectively reduce the sodium content of food by providing a similar salty taste or enhancing the overall flavor of the food. A wide variety of commercially available ionic and specialty salt products have emerged on the market. These products not only have lower sodium content but also effectively meet consumers' urgent need for healthy eating while maintaining the delicious taste of food. These salt substitutes can be broadly categorized into four types: ionic and specialty salts, extracted / synthetic salts, and salts with a salty taste. However, common salt substitutes, such as potassium salts, pose a risk of hyperkalemia and their taste differs significantly from natural table salt, sodium chloride.

[0090] In food science, adding umami (glutamate), sour (citric acid), sweet, or spicy flavors can reduce the perceived weight of saltiness at the brain level. Even if the sodium content of a food is low, the perceived "insufficient saltiness" is significantly reduced due to the "competition" from other flavors, making the overall eating experience less bland. However, to mask "insufficient saltiness" or "the bitterness caused by insufficient saltiness," this method often requires the addition of other ingredients, which may introduce new health risks.

[0091] Therefore, there is an urgent need to provide a method to improve the level of saltiness perception, so as to fundamentally increase people's sensitivity to salt, thereby achieving the effect of eating less salt while experiencing normal saltiness.

[0092] Reduce saltiness sensitivity For those working in high-temperature environments, excessive sweating leads to significant sodium loss. If they are overly sensitive to salty tastes, they may instinctively resist replenishing electrolytes through hydration, instead tending to drink large amounts of plain water, resulting in hyponatremia (low sodium levels). Appropriately reducing their sensitivity can help them more naturally accept salty drinks.

[0093] For astronauts, the altered distribution of bodily fluids in microgravity often leads to sensations similar to "high sodium," making food taste bland. While this usually necessitates additional seasoning, research into the regulation of salt sensitivity can help optimize their diet.

[0094] In addition, there are some core diseases that clinically require a high-salt diet, such as adrenocortical insufficiency, extensive burns, cystic fibrosis, and vasovagal syncope. For these diseases, a high-salt diet should be followed under clinical guidance.

[0095] Saltiness sensitivity test Early detection of salt sensitivity can help identify individuals with high salt intake. Approximately 25% to 40% of hypertensive patients suffer from "salt-sensitive hypertension," meaning their blood pressure rises significantly after salt intake. This group is typically also more sensitive to salty tastes. Detecting salt sensitivity can help identify salt-sensitive individuals, thus guiding more stringent salt restriction interventions and controls.

[0096] Furthermore, testing salt sensitivity can guide individual dietary levels. Individual salt sensitivity varies greatly. For those with high sensitivity, even a slight reduction in salt can be perceived as "bland," requiring flavor enhancement (such as spices or acidity) to facilitate a transition. For those with low sensitivity, more systematic behavioral interventions are needed to help them gradually adapt to a low-salt environment. The test results allow nutritionists to provide more targeted advice. Children, in particular, are still developing their taste sensitivity; early testing helps identify "stronger flavor" tendencies, allowing for timely adjustments to family diets and preventing the development of high-salt habits in adulthood.

[0097] Salt sensitivity testing can also be used for consumer sensory testing and the screening of professional sensory evaluators. When developing products such as low-sodium salt, reduced-sodium soy sauce, and low-sodium snacks, food companies recruit participants to grade their salt sensitivity to assess the product's acceptance by groups with different sensitivity levels. This helps determine the optimal salt reduction ratio and taste masking strategies. Sensory evaluation teams (evaluators) in food companies typically undergo salt sensitivity screening, requiring members to have normal and stable taste sensitivity to ensure the consistency and reliability of evaluation results.

[0098] GCN2 GCN2 (General Control Nondepressible kinase 2), also known as EIF2AK4, is an evolutionarily conserved serine / threonine protein kinase. GCN2 is a powerful cellular "stress control center" whose core function is to help cells cope with challenges and maintain homeostasis by finely regulating protein synthesis when cells face stress.

[0099] GCN2 can be activated by various intracellular and extracellular stress signals. Specifically, when a certain amino acid is deficient, unloaded tRNA corresponding to that amino acid accumulates intracellularly (i.e., "empty tRNA"), thereby activating GCN2 in tumor cells, intestinal cells, and tissues such as the liver and brain. Furthermore, ultraviolet radiation, oxidative stress, and hypoxia can also activate GCN2. Once activated, GCN2 specifically phosphorylates a protein called eIF2α. Phosphorylation of eIF2α leads to two seemingly contradictory results: on the one hand, the synthesis of most common proteins is temporarily inhibited, thus conserving energy and resources for the cell; on the other hand, a small subset of messenger RNAs that help the cell cope with stress (such as ATF4) are preferentially translated, further activating a series of downstream genes involved in amino acid synthesis and transport, antioxidation, autophagy, and other processes, helping the cell restore homeostasis.

[0100] Abnormalities in GCN2 are associated with neurodegenerative diseases, cancer, inflammatory diseases, and pulmonary hypertension. For example, in diabetic neuropathy, GCN2 activation amplifies pain signals. Regulating GCN2 activity has become a potential therapeutic strategy. On the one hand, in cancer, inhibiting GCN2 activity may block tumor cells' ability to adapt to nutrient-deficient environments, thereby inhibiting tumor growth. On the other hand, in neurodegenerative diseases, moderate GCN2 activation may help clear misfolded proteins. Several inhibitors and activators targeting GCN2 are currently available and may be applied to the treatment of related diseases in the future.

[0101] Numerous compounds have been reported as both activators and inhibitors of GCN2. Activators include HC-7366, GCN2activator-1 (C20), and Halofuginone (Halo), while inhibitors include GCN2iB (the most commonly used), GCN2-IN-1, and GCN2-IN-6. Some of these can be taken orally, while others are undergoing clinical application research.

[0102] However, there are currently no reports on the relationship between GCN2 on the tongue and taste, or whether it can regulate taste receptors.

[0103] Methods and uses of the present invention This invention provides a method to directly alter an organism's perception of saltiness in food and change salty taste receptors by activating / inhibiting GCN2, offering a new approach to reducing salt intake; it will provide a scientific basis for the food industry to develop low-salt products and for the development of drugs to prevent and treat hypertension.

[0104] In a first aspect of the invention, there is a method for preparing a formulation to enhance the perception of saltiness, wherein the formulation is used for intervention for 1-3 days. The method comprises: mixing protein, carbohydrates, and fat to obtain a dietary composition; The protein is obtained by mixing the amino acids required by the body. The carbohydrates mentioned are corn syrup and modified corn starch; The fat is a mixture of high-oleic safflower seed oil, coconut oil, and soybean oil; Based on a total weight of 100 parts by weight of the dietary composition, the protein content accounts for 10-25 parts by weight of the total dietary composition, the carbohydrate content accounts for 45-70 parts by weight of the total dietary composition, and the fat content accounts for 15-25 parts by weight of the total dietary composition; and The protein is formulated according to the recommended intake levels of each amino acid, wherein the weight fraction of a specific amino acid is adjusted to 0, and the specific amino acid includes: leucine, tryptophan, lysine, valine, or a combination thereof.

[0105] In another preferred embodiment, when the protein is formulated according to the recommended intake levels of each amino acid, the weight parts of each amino acid are as follows: Preferably, the improvement of salty taste perception is achieved by detecting a person's taste score for food.

[0106] Preferably, the present invention does not alter the taste ratings for sweetness, sourness, bitterness, and umami.

[0107] In another aspect of the invention, there is a method for altering the expression of salty taste receptors, including a formulation method for improving the expression of salty taste receptors and taste response, and a molecular target for altering the expression of salty taste receptors.

[0108] In a preferred embodiment, the composition for enhancing the salty taste receptor comprises the combination formulation of the present invention, treated for 1-3 days; wherein, in the combination formulation, leucine, tryptophan, lysine, or valine is adjusted to 0; the molecular target of the salty taste receptor ENaC is modified to be general control non-derepressible protein kinase 2 (GCN2), and activation of GCN2 can enhance the salty taste receptor (e.g., GCN2 activator), while decreases of GCN2 expression or activity (decreases of GCN2 expression can be achieved by interfering with adeno-associated virus, and decreases of GCN2 activity can be achieved by the GCN2 inhibitor GCN2iB) can inhibit the expression of the salty taste receptor. The salty taste receptor ENaC comprises three ligands: amiloride-sensitive sodium channel protein 1α (SCNN1α), amiloride-sensitive sodium channel protein 1β (SCNN1β), and amiloride-sensitive sodium channel protein 1γ (SCNN1γ).

[0109] Furthermore, the GCN2 target knockdown can alter dietary preferences. These dietary preferences are determined by administering a leucine-free diet for 3 days prior to treatment, followed by simultaneous administration of both a leucine-free and leucine-containing diets. Animal food intake is then measured, and dietary preferences are statistically analyzed. Specifically, under normal circumstances, animals consume roughly the same amount of both diets (leucine-containing and leucine-free) over a period of time, with no significant difference. However, after 3 days of treatment with a leucine-free diet, animals will actively choose the leucine-containing diet (i.e., consuming significantly more leucine-containing food than leucine-free food, exhibiting an amino acid preference). Knocking down GCN2 on the tongue at this point will change their preference (at which point the intake of the two diets will again be indistinguishable).

[0110] In another aspect of the invention, an application of reducing salt taste receptors is provided: reducing salt taste receptors on the tongue can alter dietary preferences.

[0111] In a preferred embodiment, the salty taste receptors include SCNN1α, SCNN1β, and SCNN1γ. The dietary preference was determined by administering a leucine-free diet for 3 days prior to pretreatment, followed by simultaneous administration of both a leucine-free and leucine-containing diets. Animal food intake was then measured, and dietary preferences were statistically analyzed.

[0112] The dietary compositions provided by this invention may also incorporate one or more pharmaceutically acceptable carriers. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field.

[0113] The dietary compositions provided by this invention can be in various forms, such as poultices, tablets, injections, capsules, powders, syrups, solutions, suspensions, and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterilization devices for injection or infusion.

[0114] The dietary compositions of the present invention can be formulated into ordinary preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems.

[0115] In order to formulate the dietary composition of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0116] Many patients experience taste impairment (diminished taste) due to illness or medication, leading to anorexia. This controlled amino acid deficiency stimulation may temporarily awaken their basal perception of saltiness, helping to restore appetite. Therefore, the dietary composition of this invention has practical value.

[0117] The main advantages of this invention include: (a) The present invention provides an objective and quantitative method for assessing saltiness sensitivity. By detecting the activity level of GCN2, it replaces the traditional sensory test that relies on subjective feedback, thereby significantly improving the accuracy and repeatability of the assessment results.

[0118] (b) This invention achieves precise bidirectional regulation of salty taste perception. It can both activate GCN2 to provide a solution for people on low-salt diets to reduce sodium intake without sacrificing the salty taste experience, and inhibit GCN2 to increase salt intake (e.g. for outdoor people or specific groups such as those with vasovagal syncope), demonstrating broad application prospects in the food industry and clinical nutrition.

[0119] (c) This invention directly links molecular targets with sensory physiology, opening up new avenues for personalized dietary intervention and health management. By using the activity of this target as a biomarker, customized dietary recommendations or adjunctive treatment plans can be developed for different individuals’ varying sensitivities to saltiness, thereby effectively preventing or adjunctive treating hypertension and other related diseases.

[0120] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0121] Example 1: Experimental method.

[0122] (a) Population recruitment and intervention The population trial was a randomized, double-blind, placebo-controlled, parallel-group study. Eligible participants were aged 20 to 40 years, in good health, and had a normal body mass index (BMI) (between 18.5 and 24 kg / m²). 2 (between). A total of 32 participants were recruited through advertising, but 4 were excluded during the screening process. A total of 28 healthy and eligible participants were randomly assigned to two groups: (1) intervention group: n=14, receiving a 2-day liquid food formula lacking leucine (containing all nutrients but leucine); (2) control group: n=14, receiving a 2-day control liquid food formula (leucine was added to the formula lacking leucine). All participants completed the intervention; Dietary Intervention: To ensure dietary standardization, all participants underwent a two-day adaptation period before the intervention, during which they consumed a standardized diet. During the intervention, participants consumed a liquid food formula (leucine-deficient formula or control formula) three times daily, accompanied by an apple (320 kcal daily, containing very low leucine), for two consecutive days. Formula intake varied by gender: 400 g (1920 kcal) / day for men and 300 g (1440 kcal) / day for women. All participants consumed the entire liquid food formula at each meal, with no waste. The formulas for the intervention and control groups were identical in appearance, taste, and quality, differing only in leucine content. Both the leucine-deficient formula (I-VALEX-1, Abbott Laboratories) and the leucine-fortified formula (Wanbang Chemical Technology Co., Ltd., Henan) were commercially available; see formula details below. Figure 1 D; On the mornings of Day 0 and Day 2 of the intervention, tongue epithelial cell samples were collected by smearing the front, back, left, and right sides of the participants' tongues five times each using cotton swabs. Participants were then given two cups of control formula beverage and one cup of leucine-deficient formula beverage (double-blind). After tasting each beverage, participants completed a taste questionnaire, and different taste scores were calculated based on the questionnaire results. The questionnaire content is available in [link to questionnaire]. Figure 1 E.

[0123] (b) Handling of laboratory animals All mice, possessing the C57BL / 6J gene background, were purchased from the Shanghai Slack Laboratory Animal Center. Prior to the experiment, mice were housed in groups under constant temperature (23-25 ​​degrees Celsius), humidity (50%-60%), and a 12-hour light / dark cycle (lights on at 8:00 AM / lights off at 8:00 PM). Mice were kept individually in each cage for at least 3 days before the start of the experiment.

[0124] (c) Animal diet and food selection The experimental control group (nutritionalally complete amino acid) diet, and diets deficient in leucine [(-)Leu], tryptophan [(-)Trp], lysine [(-)Lys], and valine [(-)Val] were purchased from Nutrition Animal Feed Technology Co., Ltd. (Nantong, China). All diets were isocaloric and identical except for the experimental components; the caloric deficit due to amino acid deficiency was compensated for by carbohydrates. At the start of the feeding experiment, mice were acclimatized to the control diet for 3 days, followed by the control diet and the diet deficient in specific amino acids for 3 days (or a specified time period). Mice could choose to consume either the control diet or the leucine-deficient diet during the selection process. In the first 10 minutes, the number of times the mice touched or bit either food was counted, and the percentage of total touches was calculated. Food intake for both diets was measured at 10 min, 30 min, 1 h, and 2 h. The preference index (PI) was calculated as follows: where f c f is the weight of the control diet ingested by the mice. d This represents the weight of amino acid-deficient food consumed by mice. A preference coefficient greater than 0 and the higher it is, indicates a preference for food containing specific amino acids (such as leucine), i.e., an amino acid preference. A preference coefficient near 0 indicates no significant preference. A preference coefficient less than 0 and the lower it is, indicates a preference for food lacking amino acids.

[0125] (d) Mouse tongue epithelial cell culture, amino acid and drug treatment Mouse tongue epithelial cells were cultured in culture dishes until they reached 70%-80% confluence. Nutrient-complete and leucine-deficient culture media were prepared, and the cells were treated with both media for 24 hours. Afterward, the tongue epithelial cells were collected, proteins were extracted, and the level of p-eIF2α was detected. Another group of mouse tongue epithelial cells were treated with complete and leucine-deficient culture media for 24 hours, followed by the addition of a calcium signaling indicator and the GCN2 inhibitor GCN2iB (MCE, HY-112654). Fluorescence images were captured under a fluorescence microscope before and after the addition of 10 μM leucine, and the fluorescence intensity was statistically analyzed to indicate the intensity of the taste signal (calcium signal) response of the tongue epithelial cells.

[0126] (e) Injection of adeno-associated virus (AAV) into the tongue of mice To knock out the GCN2 gene in the tongue of wild-type mice, AAV2 / DJ-shGCN2-EGFP (titer 2 × 10⁻⁶) was injected into the tongue of the mice. 11 Pfu / mL (Heyuan Biotechnology Co., Ltd.) or AAV2 / DJ-EGFP (titer 2×10⁻⁶) 11 Pfu / mL (and Yuan Biotechnology Co., Ltd.) was used as a control group. The target sequence of GCN2 was 5'-TCTGGATGGATTAGCTTATA-3'. Specifically, the mouse tongue was punctured with an insulin needle parallel to the lingual epithelium, from the tip to the middle and back of the tongue, creating a channel between the epithelial and muscle layers. Using a 25 μL microsyringe, the virus was injected into the middle and back of the tongue along this channel. The syringe was slowly withdrawn while injecting the virus, changing the direction of the needle tip to ensure uniform infection of different parts of the tongue. 2-3 μL of virus was injected into each site each time, for a total injection volume of 25 μL. Finally, the wound was sealed with tissue glue to prevent virus leakage. Three weeks later, 2 μL of virus was dripped onto the tongue surface each time, repeating this procedure until the virus was absorbed, reaching a total injection volume of 30 μL. The experiment was conducted one week later.

[0127] The salt taste receptors on the tongue were knocked down using the same method described above. The injected virus was AAV2 / DJ-shSCNN1α-EGFP (titer 2 × 10⁻⁶). 11 Pfu / mL, Heyuan Biotechnology Co., Ltd.), AAV2 / DJ-shSCNN1β-EGFP (titer of 2×10⁻⁶) 11 Pfu / mL (Heyuan Biotechnology Co., Ltd.) and AAV2 / DJ-shSCNN1γ-EGFP (titer of 2×10⁻⁶) 11 A mixture of three viruses (Pfu / mL, and Yuan Biotechnology Co., Ltd.), or a control virus AAV2 / DJ-EGFP (titer 2×10⁻⁶). 11 Pfu / mL, and Yuan Biotech Co., Ltd. The target sequences of the three viruses are: shScnn1α: GCAGTGTGACCAACTACAA, shScnn1β: CAAGAGTTCAACTACCGTA, shScnn1γ: GGAACTGCTACACGTTCAA.

[0128] (f) RNA extraction and RT-PCR RNA was extracted using TRIzol reagent (Invitrogen). mRNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific) and quantitative real-time PCR analysis was performed using SYBR Green I major mixture on an ABI 7900 instrument (Applied Biosystems).

[0129] (g) Data statistics Statistical analysis was performed using GraphPad Prism software (version 8.0; GraphPad Software, San Diego, California, USA). All data are presented as mean ± standard error (SEM). Two-tailed unpaired Student's t-tests were used to compare two groups. For experiments involving multiple comparisons, two-way ANOVA followed by Tukey's multiple comparison test was used for analysis. Individual data points on each histogram reflect individual differences in measurements and sample size. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0130] Example 2: Dietary intervention for leucine deficiency improves human perception of saltiness.

[0131] The recruited population was divided into a control group and a leucine deficiency intervention group. After two days of standardized diet, the control group was given a placebo-formulated liquid food, while the leucine-deficient group was given a leucine-deficient liquid food (e.g., [formula missing]). Figure 1 (As shown in D) Over 2 days, participants were given 3 small cups of [something] on days 0 and 2. Figure 1 The participants were given the liquid beverage shown in E and asked to fill out a corresponding taste questionnaire. The participants' perception scores for different tastes were collected. Figure 1 A).

[0132] The inventors found that, compared with the control group, participants in the leucine-deficient group (experimental group) significantly increased their saltiness ratings when consuming the control formula food on the second day, while the saltiness ratings of the food in the control group did not change significantly. Figure 1 B).

[0133] There were no significant changes in the sweetness, sourness, bitterness, and umami taste of the food in either the experimental or control groups. Figure 1 C).

[0134] The above results indicate that dietary intervention for leucine deficiency significantly improved the subjects' perception of saltiness in the control diet, but had no significant effect on other tastes.

[0135] Example 3: Treatment with specific amino acid deficiency can increase the expression of salty taste receptors on the tongue.

[0136] Mouse tongue epithelial cells collected on day 0 of intervention were treated with complete culture medium and leucine-deficient culture medium for 24 h, respectively. After cell collection, mRNA was extracted from primary tongue epithelial cells, and the expression level of salt taste receptors (ENaC, including Scnn1α, Scnn1β and Scnn1γ) was detected. The results showed that, compared with the control group, the expression levels of salt taste receptors Scnn1α, Scnn1β, and Scnn1γ in lingual epithelial cells treated with leucine-deficient culture medium were significantly increased. Figure 2 A).

[0137] Mouse tongue epithelial cells collected on day 0 of intervention were treated with complete culture medium and leucine-deficient culture medium for 24 h. Then, calcium signaling indicator and GCN2 inhibitor GCN2iB (MCE, HY-112654) were added. Fluorescence images were taken before and after the addition of 10 μM leucine under a fluorescence microscope, and the fluorescence intensity was counted to indicate the intensity of taste signal (calcium signal) response of tongue epithelial cells. The results showed that in the control group, there were no significant changes in taste signals before and after leucine treatment; in the leucine-deficient group, taste signals (calcium signals) were significantly activated after the addition of leucine; and after GCN2 inhibition, even in the leucine-deficient group, taste signals were no longer activated. Figure 2 B). This indicates that the target site for leucine deficiency to induce taste response is GCN2.

[0138] Wild-type mice were acclimatized to a control diet for 3 days and then randomly divided into two groups, one group receiving the control diet and the other receiving a specific amino acid-deficient diet for 3 days. Figure 2 C), collect its tongue tissue, extract mRNA, and detect the expression level of salt taste receptors; The results showed that, in vivo, the expression levels of Scnn1α and Scnn1β in the tongue of mice fed a leucine-deficient diet were significantly increased. Figure 2 D); In mice fed a tryptophan-deficient diet, the levels of Scnn1α and Scnn1γ in the tongue were significantly increased (D); Figure 2 E); The expression level of Scnn1β in the tongue of mice fed a lysine-deficient diet was significantly increased ( Figure 2 F); The expression levels of Scnn1α, Scnn1β and Scnn1γ in the tongue of mice fed a valine-deficient diet were significantly increased (F). Figure 2 F).

[0139] Example 4. Activation of GCN2 signaling in the tongue after leucine deficiency treatment in animals and humans.

[0140] After wild-type mice were acclimatized to the control diet for 3 days, they were randomly divided into two groups, one group was given the control diet and the other group was given the leucine-deficient diet for 3 days. Their tongue tissues were collected, proteins were extracted, and the GCN2 signal in the mouse tongue was detected (the activity was reflected by its downstream p-Eif2a). The results showed that, compared with the control group, leucine-deficient mice exhibited activated GCN2 signaling in the tongue (with increased downstream p-Eif2a). Figure 3 (A) This is the first time that an amino acid deficiency has been found to activate GCN2 on the tongue, thereby increasing sensitivity to salty tastes.

[0141] After two days of intervention with a control formula drink and a leucine-deficient formula drink, tongue epithelial cells were collected, proteins were extracted, and GCN2 signaling in human tongue epithelial cells was detected. The results showed that intervention with human leucine-deficient formulations significantly activated GCN2 signaling in the lingual epithelium. Figure 3 B).

[0142] Example 5: Knockdown of GCN2 on the tongue of mice inhibits the expression of salty taste receptors on the tongue and alters food preferences.

[0143] Wild-type mice were injected lingually with GCN2 knockdown adeno-associated virus or control virus. After viral expression, they were fed either a control diet or a leucine-deficient diet for 3 days. Then, they were simultaneously fed control food and leucine-deficient food, and the mice were subjected to a food choice experiment to detect their preferences. Figure 4 A), and at the same time, tongue tissue was collected, mRNA was extracted, and the expression level of salty taste receptors was detected.

[0144] The results showed that knocking down GCN2 in the mouse tongue reduced both GCN2 levels in the tongue and p-Eif2a, which reflects GCN2 activity. Figure 4 B and Figure 4 C) indicates successful knockout; the expression levels of Scnn1α and Scnn1γ in the tongue are significantly reduced ( Figure 4 D). Dietary preferences changed in GCN2 knockdown mice: In mice without GCN2 knockdown, after leucine deficiency treatment, the percentage of mice biting or touching the control diet increased significantly, and a food preference was developed (a preference for the control diet was observed, with a food preference index (PI) significantly greater than 0). In contrast, in GCN2 knockdown mice, after leucine deficiency treatment, the percentage of mice biting or touching the control diet for a short period did not increase, and no food preference was developed (preference index was near 0), and the levels were significantly lower than in the non-GCN2 knockdown group mice treated with amino acid deficiency. Figure 4 E and Figure 4 F).

[0145] The results of this embodiment indicate that knocking down GCN2 on the tongue can inhibit the expression of salty taste receptors and alter food preferences.

[0146] Activating GCN2 can increase the level of salty taste receptors, causing mice to have a high sensitivity to salty tastes in food. At this time, without amino acid deficiency pretreatment, mice can develop a preference for foods containing amino acids and actively choose diets containing specific amino acids.

[0147] Example 6. Knockdown of salty taste receptors on the tongue of mice alters their food preferences.

[0148] Wild-type mice were injected into the tongue with salt taste receptors (Scnn1α, Scnn1β, and Scnn1γ) to knock down adeno-associated virus or control virus. After viral expression, the mice were fed a leucine-deficient diet for 3 days, followed by simultaneous administration of control and leucine-deficient diets. The mice were then subjected to a food choice experiment to determine their preferences. Figure 5 A), and at the same time, tongue tissue was collected, mRNA was extracted, and the expression level of salty taste receptors was detected.

[0149] The results showed that knocking down salt taste receptors on the tongue of mice significantly reduced the expression levels of Scnn1α, Scnn1β, and Scnn1γ on the tongue. Figure 5 B). Analysis of their food preferences revealed that mice with knocked-down salt taste receptors exhibited a lower percentage of biting or touching control food, and a significantly lower food preference index. Figure 5 C and Figure 5 D). This indicates that knocking down salt taste receptors on the tongue can alter food preferences.

[0150] discuss This invention has a wide range of applications. For example, it can alter the expression of salty taste receptors by activating or inhibiting GCN2; it can change the dietary preferences of people with abnormal eating habits through GCN2 regulation; it can increase or decrease the level of salty taste perception in a population; or it can evaluate salty taste sensitivity or dietary preferences through GCN2 detection.

[0151] GCN2 activity in the tongue can serve as a biomarker for detecting and / or treating salty taste preferences and dietary preferences. Tongue epithelial cells can be collected by smearing them with a cotton swab; after processing, gene and protein levels can be detected. This detection method is non-invasive and may have wider applications in the future.

[0152] In individuals with taste disorders (such as an inability to perceive saltiness or excessive sensitivity to saltiness), an amino acid-deficient diet or GCN2 activators can be used to increase the expression of salt taste receptors, while GCN2 inhibitors can be used to decrease their expression, thereby reducing salt taste sensitivity. The diet consists of common nutrients (rather than medications), making its application potentially safer and more widespread. GCN2 activators or inhibitors can be administered via the tongue (orally or as a wet compress), causing no invasive damage.

[0153] In individuals with picky eating habits or abnormal dietary preferences, such as a particular fondness or aversion to protein-rich foods, the underlying cause may be alterations in salt taste receptors. If such alterations are detected, the nutritional combination or GCN2 activator / inhibitor of this invention can be used to regulate these preferences, thereby changing dietary habits. Specifically, for individuals who are particularly accustomed to protein-rich diets, administering a GCN2 inhibitor can reduce their preference for protein and balance their intake of other foods; for individuals who particularly dislike protein-rich diets, administering a GCN2 activator can promote their preference for amino acid-rich foods and increase their intake of corresponding high-protein foods.

[0154] Salt substitutes, especially potassium-rich, low-sodium salt, have been proven to be an effective strategy for assisting in the control of hypertension. The latest high-quality studies and clinical guidelines indicate that for most hypertensive patients, replacing regular table salt with low-sodium salt can not only lower blood pressure but also reduce the risk of cardiovascular events and death. However, this method is not suitable for everyone, with the greatest risk being hyperkalemia. For individuals with renal insufficiency (such as chronic kidney disease) or those taking potassium-sparing diuretics, angiotensin-converting enzyme inhibitors / angiotensin II receptor antagonists, or other medications that affect potassium excretion, using low-sodium salt may lead to dangerously high blood potassium levels. Our nutritional combination addresses amino acid deficiencies, increasing the saltiness score of a person's diet (rather than directly adding salt substitutes without increasing the amount of other substances). This allows for the use of less salt in food to meet the individual's salty taste buds, reducing overall salt intake and thus helping to prevent or reduce hypertension.

[0155] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of GCN2 as a biomarker in the preparation of products for assessing and / or modulating the sensitivity of test subjects to salty taste.

2. The use of a GCN2 agonist, characterized in that, It is used to increase the subject's sensitivity to salty taste.

3. The use as described in claim 2, characterized in that, The method of increasing the subject's sensitivity to salty taste includes: (Z1) significantly increased the expression level of salty taste receptors in the subjects; (Z2) Improve subjects' rating of the saltiness of the same food; and / or (Z3) Reduce the salt intake level of the subjects.

4. The use as described in claim 2, characterized in that, The GCN2 agonist comprises: a dietary composition for a specific amino acid deficiency. The specific amino acids include: leucine, tryptophan, valine, lysine, or combinations thereof.

5. A dietary combination for activating GCN2, thereby increasing the sensitivity of subjects to salty taste, characterized in that, The dietary combination includes carbohydrates, protein, and fat; The protein is deficient in a specific amino acid, which includes leucine, tryptophan, valine, lysine, or a combination thereof.

6. The use of a GCN2 inhibitor, characterized in that, It is used to reduce the subject's sensitivity to salty taste.

7. The use of a GCN2 detection reagent, characterized in that, Used to assess the subject's sensitivity to salty taste.

8. A method for improving the sensitivity of a subject to salty taste for non-diagnostic and non-therapeutic purposes, characterized in that, Administer the GCN2 agonist or the dietary combination of claim 5 to the subjects in need.

9. A method for reducing the sensitivity of a subject to salty taste for non-diagnostic and non-therapeutic purposes, characterized in that, GCN2 inhibitors were administered to subjects who needed them.

10. A method for assessing a subject's sensitivity to salty taste for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: (S1) Provide the subject's tongue epithelial cells; (S2) Detect the GCN2 level of the tongue epithelial cells to assess the subject’s sensitivity to salty taste.

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