CN113749247B

CN113749247B - Method and formulation for regulating dietary restriction and lipid oversynthesis after re-ingestion

Info

Publication number: CN113749247B
Application number: CN202010494332.5A
Authority: CN
Inventors: 翟琦巍; 钟武令
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2020-06-03
Filing date: 2020-06-03
Publication date: 2024-02-02
Anticipated expiration: 2040-06-03
Also published as: CN113749247A

Abstract

The present invention provides a method and formulation for regulating dietary restriction and lipid oversynthesis after re-ingestion. In the present invention, an animal model suitable for lipid metabolism studies or obesity studies was established, revealing the phenomenon that refeeding after dietary restriction would promote the formation of obesity. The present invention also discloses that targeting intestinal lipid absorption by high protein diets is an effective strategy for preventing obesity following dietary restrictions.

Description

Method and formulation for regulating dietary restriction and lipid oversynthesis after re-ingestion

Technical Field

The invention belongs to the field of biological metabolism and food science; more particularly, the present invention relates to a method and formulation for regulating dietary restrictions and lipid oversynthesis following re-ingestion.

Background

Obesity has become increasingly popular worldwide in recent years and is considered one of the biggest public health problems worldwide. Obesity increases the risk of developing a number of diseases, such as type II diabetes and metabolic diseases such as fatty liver; cardiovascular diseases such as hypertension, myocardial infarction, and cerebral apoplexy; musculoskeletal diseases such as osteoarthritis; depression, and the like. In addition, obesity may also be associated with the occurrence of cancer. Obesity shortens the average life of people, reduces the quality of life of people, and increases the medical consumption of people. The world's union of obesity and other organizations announce obesity as a chronic progressive disease, which is a distinct risk factor from other diseases.

For complex body systems, genes interact with environmental factors to regulate energy balance and physiological processes in the body and regulate body weight. Within the hypothalamic arciform nucleus, there are two groups of neurons that are inhibited or excited by circulating neuropeptide hormones, which control energy balance by regulating food intake and energy expenditure. The body's short-term and long-term energy balance controls the central system within the receptor, which can respond to peripheral signals derived from the microorganisms of the flora or adipose tissue, stomach, pancreas and other organs in the body. The brain region outside the hypothalamus may also maintain energy balance through sensory signal input, cognitive, memory, and attention functions. Obesity develops over time when the body is in a positive energy balance for a long period of time. During this process, lipids (mainly triglycerides) accumulate in adipose tissue, while skeletal muscle, liver, and other organs and tissue volumes become large. Lipids are distributed in liposomes in addition to being present in large amounts in adipose tissue. Liposomes are small organelles in the cytoplasm, close to the mitochondria, which are present in many cell types. In the case of obesity, the liposome volume in liver cells increases and large lipid droplets are formed, and a series of pathological characteristics such as non-alcoholic fatty liver, steatohepatitis, cirrhosis and the like are induced. Excessive accumulation of lipid intermediates (e.g., ceramides) in certain non-adipose tissues can lead to lipotoxicity, thereby triggering cellular dysfunction and apoptosis.

The root cause of obesity is an imbalance between intake and consumption of calories. Both genetic and environmental factors can lead to obesity. Epidemiological studies have shown external environmental exposure factors that lead to obesity. Among the environmental factors that lead to obesity, the intake of a large number of high-calorie, savoury and tasty foods and processed foods directly results in excessive energy accumulation. Food companies have adopted powerful marketing strategies to induce people to eat more and more foods available for selection. Meanwhile, the time for watching television or using electronic equipment and other recreational activities is increased, the traffic is convenient, and the physical exercise is weakened, so that the energy consumption in the body is reduced.

While diet and activity are considered to be the primary factors responsible for obesity, experimental evidence from animals and humans suggests that disturbed sleep and rhythm disorders may also cause obesity and metabolic health problems. More time is needed to deal with work, learning and social activities in a busy modern social environment, and the problem of insufficient sleep is prevalent in adults and teenagers. A national statement of health survey shows that about 30% of americans sleep less than 6 hours per night, below the recommended 7 hour sleep time for promoting adult health. Furthermore, the use of electrical lighting delays the timing of the biological clock, allowing work and social activity to take place at times other than during the day. These factors all affect the healthy metabolism of the body and ultimately lead to the development of obesity.

In obese and non-obese mammals, dietary restriction is a very common form of dietary intervention that improves body fat metabolism through dietary control. The current approach to taking more dietary restrictions is daily dietary control, i.e. daily reduction of a certain degree of caloric intake, and in some obese people, while daily dietary restrictions may be an effective means of weight loss, most people have difficulty adhering to it, and others have intermittent dietary restrictions. Weight regain following diet weight loss through dietary restrictions is a great challenge for obesity treatment. Weight maintenance is mediated by a number of processes, including physiological, environmental and behavioral factors. In the regulation of steady state systems, various environmental and behavioral patterns affect ingestion and energy consumption, and the hypothalamus plays a central role in integrating signals related to ingestion, energy balance, weight control, etc. Diet results in a series of adjustments in the steady state balance system, which in turn may cause excessive ingestion and weight regain. Despite the intervention for weight regain after dietary restrictions, the general effect is still relatively limited. In view of the unusually large dieting population, weight rebound after diet restriction is still a major scientific problem to be solved urgently, and more effective and feasible diet intervention measures after diet are still needed to be further studied.

Disclosure of Invention

The invention aims to provide a regulation and preparation for lipid oversynthesis after dietary restriction and re-ingestion.

In a first aspect of the invention, there is provided a method of controlling body weight and lipid biosynthesis comprising: (1) reduced food intake, significant weight and lipid loss; (2) Restoring food intake, intake of a food with a regulated protein content or amino acid content, said food being selected from the group consisting of: high protein content foods, high amino acid content foods, or low protein content foods, thereby controlling body weight and lipid over-synthesis.

In another aspect of the invention, there is provided a method of modulating (inhibiting) dietary restriction and lipid oversynthesis following re-ingestion, comprising: after re-ingestion, ingestion of a food having a protein content or amino acid content regulated, the food selected from the group consisting of: high protein content foods, high amino acid content foods, or low protein content foods, thereby controlling body weight and lipid over-synthesis.

In a preferred embodiment, the high protein content food has a protein content of greater than 400 parts by weight (e.g., 400 to 800 parts by weight); preferably greater than 450 parts by weight; more preferably above 550 parts by weight (e.g., 500, 600, 700 or 800 parts by weight).

In another preferred example, the low protein content food has a protein content of less than 150 parts by weight (e.g., 20 to 150 parts by weight); preferably less than 100 parts by weight; more preferably less than 60 parts by weight (e.g., 120, 100, 80, 60, 50, 40 or 30 parts by weight).

In another preferred embodiment, the protein comprises a protein selected from the group consisting of: casein, whey protein or soy protein.

In another preferred embodiment, the food further comprises cysteine, and the high protein content food comprises cysteine in an amount of more than 5 parts by weight, preferably more than 8 parts by weight (such as 5-20 parts by weight, more particularly such as 6,9, 10, 12 or 15 parts by weight); alternatively, the low protein content food may have a cysteine content of less than 2 parts by weight, preferably less than 1.5 parts by weight (e.g., 0.25 to 2 parts by weight or 0.5 to 1.5 parts by weight; more particularly, 0.4,0.5,0.6,0.8,1,1.2 or 1.6 parts by weight).

In another preferred example, the food also comprises carbohydrate, and the carbohydrate content in the high-protein-content food is 150-350 parts by weight; preferably 180 to 300 parts by weight (e.g., 190, 200, 220, 230, 240 or 260 parts by weight); or, the carbohydrate content in the low protein content food is 650-900 parts by weight; preferably 700 to 850 parts by weight (e.g., 720, 750, 780, 800 or 820 parts by weight).

In another preferred embodiment, the high amino acid content food is a food in which an effective amount of essential amino acids is added to a normal food; preferably, the essential amino acids include: valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine, lysine, histidine.

In another preferred example, among the added amino acids:

the valine content is 16 to 36 parts by weight, preferably 21 to 31 parts by weight;

the isoleucine content is 12 to 32 parts by weight, preferably 17 to 27 parts by weight;

the leucine content is 23 to 43 parts by weight, preferably 28 to 38 parts by weight;

the methionine content is 5 to 15 parts by weight, preferably 8 to 12 parts by weight;

the phenylalanine content is 8 to 28 parts by weight, preferably 13 to 23 parts by weight;

the tryptophan content is 2 to 8 parts by weight, preferably 3 to 6 parts by weight;

the threonine content is 10 to 27 parts by weight, preferably 15 to 23 parts by weight;

the lysine content is 20 to 40 parts by weight, preferably 25 to 35 parts by weight;

the histidine content is 5 to 18 parts by weight, preferably 8 to 15 parts by weight.

In another preferred embodiment, the food further comprises: fat, cellulose, minerals, vitamins;

Preferably, the fat content is 50 to 90 parts by weight, preferably 60 to 80 parts by weight;

preferably, the cellulose content is 30 to 70 parts by weight, preferably 40 to 60 parts by weight;

preferably, the mineral content is 15 to 55 parts by weight, preferably 25 to 45 parts by weight;

preferably, the vitamin content is 8 to 18 parts by weight, preferably 10 to 16 parts by weight.

In another preferred example, the food is preferably a high protein content food or a high amino acid content food.

In another preferred embodiment, the protein content or amino acid content-adjusted food is capable of, after restoration of food intake: decreasing lipid synthesis, increasing lipid oxidation, promoting lipid breakdown, decreasing small intestine lipid absorption, up-regulating lipid oxidation gene expression, up-regulating lipolytic gene Pnpla2, or down-regulating fatty acid and triglyceride synthesis genes (e.g., acly, fasn, etc.).

In another preferred embodiment, the reducing food intake or dietary control includes (but is not limited to): regular diet, intermittent diet, time-limited diet, low-energy diet simulating diet, gradient-increasing or gradient-decreasing diet.

In another preferred embodiment, in step (1), the time taken to reduce food intake is: the time required for significant weight and lipid loss to occur. For example, this time is at least 3 days, at least 6 days, at least 8 days, at least 10 days or more, such as 15, 20, 30, 45, 60, 80, 100 days or more.

In another preferred embodiment, the method of controlling body weight and lipid biosynthesis is a non-diagnostic and therapeutic method.

In another preferred embodiment, the method of modulating (inhibiting) dietary restriction and lipid oversynthesis following re-ingestion is a non-diagnostic and therapeutic method.

In another aspect of the invention, there is provided a composition for modulating dietary restriction and lipid oversynthesis following re-ingestion, which is a food having a modulated protein content or amino acid content, the food selected from the group consisting of: a high protein content food, a high amino acid content food, or a low protein content food; wherein, the protein content in the high protein content food is higher than 400 weight parts (such as 400-800 weight parts); preferably greater than 450 parts by weight; more preferably above 550 parts by weight (e.g., 500, 600, 700 or 800 parts by weight); or, in the low protein content food, the protein content is less than 150 parts by weight (such as 20 to 150 parts by weight); preferably less than 100 parts by weight; more preferably less than 60 parts by weight (e.g., 120, 100, 80, 60, 50, 40 or 30 parts by weight); or, the high amino acid content food is a food in which an effective amount of essential amino acids including: valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine, lysine, histidine.

In a preferred embodiment, the protein comprises a protein selected from the group consisting of: casein, whey protein or soy protein.

In another preferred embodiment, among the essential amino acids,

In another preferred embodiment, the composition for regulating dietary restriction and lipid oversynthesis after re-ingestion is preferably a high protein content food or a high amino acid content food.

In another aspect of the present invention, there is provided a method of preparing a high fat animal model, comprising: (a) Reducing animal food intake, significantly reducing body weight and lipid; (b) And (3) for the animal in the step (a), normal food intake is recovered, and a high-fat animal model is obtained.

In a preferred embodiment, the fatty tissue of the high fat animal model has increased fatty acid uptake, lipid synthesis and storage capacity, decreased lipolysis, and decreased overall lipid oxidation.

In another preferred embodiment, the animal includes (but is not limited to): rodents (including rats, mice, hamsters, etc.), non-human primates (e.g., monkeys, chimpanzees, etc.).

In another preferred embodiment, in step (a), the time taken to reduce food intake in the animal is: the time required for significant weight and lipid loss to occur; preferably 3 to 100 days (more specifically 5,6,8, 10, 15, 20, 30, 50, 70, 80, 90 days).

In another preferred embodiment, in step (b), the time to resume normal food intake is: the time required for the lipid to significantly increase; preferably 3 to 100 days (more specifically 5,6,8, 10, 15, 20, 30, 50, 70, 80, 90 days).

In another preferred embodiment, the reducing food intake or dietary control includes (but is not limited to): regular diet, intermittent diet, time-limited diet, low-energy diet simulating diet, gradient increasing or gradient decreasing diet; preferably a gradient increasing or gradient decreasing diet (e.g., 10%, 25%, 65% food volume for three days, 65%, 25%, 10% food volume for three days, etc.), respectively.

In another aspect of the invention, there is provided the use of the animal model prepared previously for: screening potential substances (foods) for controlling body weight and lipid excessive synthesis; performing obesity study; performing a lipid synthesis or breakdown study; and/or studies of the body's absorption or breakdown of lipids.

In another preferred embodiment, the animal model is used for non-diagnostic and therapeutic applications.

In another aspect of the present invention, there is provided a method of screening for potential substances (foods) that control body weight and lipid oversynthesis, comprising: (1) Reducing food intake in a subject, significantly reducing body weight and lipid; (2) Restoring feeding to normal levels in the subject, the test group subjects administered the test substance, and the control group subjects did not control the type of food;

observing the lipid synthesis of the test group and the control group subjects, and if the lipid synthesis of the test group is obviously lower than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive lipid synthesis;

observing lipid oxidation of the test group and the control group subjects, and if the lipid oxidation of the test group is significantly higher than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive synthesis of lipid;

Observing the lipid decomposition of the test group and the control group subjects, and if the lipid decomposition of the test group is obviously higher than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive synthesis of lipid;

observing the small intestine lipid absorption of the test group and the control group subjects, and if the small intestine lipid absorption of the test group is obviously lower than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive synthesis of lipid;

observing the expression of lipid oxidation genes or lipolytic genes Pnpla2 of the test group and the control group subjects, and if the expression of the lipid oxidation genes or lipolytic genes Pnpla2 of the test group is obviously higher than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and lipid excessive synthesis; or (b)

Observing the expression of the fatty acid and triglyceride synthesis genes of the test group and the control group subjects, and if the expression of the fatty acid and triglyceride synthesis genes of the test group is significantly lower than that of the control group, indicating that the substance to be tested is a potential substance for controlling body weight and lipid excessive synthesis.

In another preferred embodiment, the subject includes, but is not limited to: : rodents (including rats, mice, hamsters, etc.), non-human primates (e.g., monkeys, chimpanzees, etc.).

In another preferred embodiment, the potential substance includes: food, health products, beverage, etc. with different components.

In another preferred embodiment, the screening method is a non-diagnostic and therapeutic method.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

Refeeding after various types of dietary restrictions in fig. 1 induces rapid accumulation of fat. Refeeding after 1 day of food (a-c) or 4 days of food (d) provided to mice within 3 days of a-d significantly increased body fat content. AL, represents any feeding (n=9); DR, representing dietary restrictions. The body fat content of any fed mice or mice with a prescribed diet restriction pattern was measured by nuclear magnetic resonance. DR (10% -25% -65%), representing the amount of food provided 10%, 25%, 65% respectively (n=9) in 1 to 3 days; DR (65% -25% -10%), representing the amount of food provided 65%, 25%, 10% respectively (n=7) in 1 to 3 days; DR (33.3% ×3), which means providing 33.3% food amount per day for 1 to 3 days (n=8); DR (66.7% ×6), which means that 66.7% of the food amount was provided daily for 1 to 6 days (n=9). e, representative pictures of isolated inguinal white adipose tissue collected from DR (10% -25% -65%) group mice. D4d, represents the night on the fourth day. The size of each square was 0.5 cm by 0.5 cm. f, weight of inguinal white adipose tissue in (e) (n=12). g, representative H & E stained sections of inguinal white adipose tissue in panel (E). Refeeding after h-j, 2 days of food (h, i) or 1 day of food (j) provided to mice within 3 days significantly increased body fat content (n=9). Refeeding after 66.7% food volume was provided to mice daily for either k, l,12 days (k) or 24 days (l) significantly increased body fat content (n=9). m, refeeding after 15 cycles of every other day of weaning significantly increased body fat content (n=9). Data are presented as mean ± standard deviation. The significance differences were analyzed by a two-tailed Student's t-test. a, p <0.05; b, p <0.01; c or p <0.001; as compared to either the AL or D0 group.

The re-feeding after various types of diet restrictions of figure 2 significantly increased body fat percentage, reduced lean body mass percentage, up-regulated white adipose tissue content. a,1 to 3 days, mice were provided with a schematic diet restriction with 10%, 25% and 65% food amounts, respectively. Arrows indicate the time points for food supply, weight measurement, body fat measurement, and ingestion measurement. Each mouse was subjected to a single cage feeding adaptation period of 5 days prior to diet restriction, and the average food intake of the mice three days prior to diet restriction was used as a food amount reference standard for diet restriction in the manner of 10% -25% -65% for the next three days. ZT, zeitgeber time. ZT0 represents the on-time and ZT12 represents the off-time. b-e, refeeding after 1 day of food (b-d) or 4 days of food (e) provided to mice within 3 days significantly increased body fat percentage, decreased lean body mass percentage, and/or increased body weight percentage. AL, represents any feeding (n=9); DR, representing dietary restrictions. Body fat and lean body mass levels of either fed mice or mice with prescribed diet restriction patterns were measured by nuclear magnetic resonance apparatus. DR (10% -25% -65%), representing the amount of food provided 10%, 25%, 65% respectively (n=9) in 1 to 3 days; DR (65% -25% -10%), representing the amount of food provided 65%, 25%, 10% respectively (n=7) in 1 to 3 days; DR (33.3% ×3), which means providing 33.3% food amount per day for 1 to 3 days (n=8); DR (66.7% ×6), which means that 66.7% of the food amount was provided daily for 1 to 6 days (n=9). f, schematic diet restrictions of 10%, 25%, 65% food amount were provided to mice over 1 to 3 days, respectively. Arrows indicate the points in time at which food is provided, blood is collected, and tissue samples are collected. g, representative pictures of abdominal fat at ZT10 (D0-D12) and ZT15 (D4D, day 4 night) in mice from DR (10% -25% -65%) group. Yellow arrows indicate inguinal white adipose tissue. h, i, representative pictures of isolated inguinal white adipose tissue (h) and epididymal white adipose tissue (i) collected from mice in (g). The size of each square was 0.5 cm by 0.5 cm. j, weight from epididymal white adipose tissue in (i) (n=12). k, average adipocyte area from inguinal white adipose tissue in (fig. 1 g) (n=6). l, representative H & E stained sections of epididymal white adipose tissue from mice in (g). m, average adipocyte area from epididymal white adipose tissue in (l) (n=6). a, p <0.05; b, p <0.01; c or p <0.001; as compared to either the AL or D0 group.

Re-feeding after 10%, 25%, 65% food was provided to the mice within three days of fig. 3, respectively, promoted hypertrophy of brown adipose tissue at the shoulder blade and induced steatosis of the liver. a, representative pictures of brown adipose tissue at the shoulder blade of mice from DR (10% -25% -65%) group at ZT10 (D0-D12) and ZT15 (D4D, day 4 night). b, weight of brown adipose tissue at the scapula from the mice in (a) (n=12). c, representative H & E stained sections of brown adipose tissue at the shoulder blade from mice in (a). d, representative H & E stained sections of livers from mice in (a). e, triglyceride concentration from the liver of mice in (a) (n=6-11). * P <0.05; * P <0.01; * P <0.001; compared to group D0.

Figure 4, food volume provided for two days in three days, food volume provided for one day in two days, food volume provided for 66.7% per day in 12 or 24 days, and refeeding after 15 cycles of every other day of weaning significantly increased the body fat percentage and decreased the lean body mass percentage of the mice. a-c, refeeding after providing mice with food amounts (a, b) for two days or food amounts (c) for one day for two days significantly increased body fat percentage and decreased lean body mass percentage (n=9). DR (40% -70% -90%), which means that 40%, 70%, 90% of the food amount is provided in 1 to 3 days, respectively; DR (90% -70% -40%), which means that 90%, 70%, 40% of the food amount is provided in 1 to 3 days, respectively; DR (50% ×2) represents the amount of food provided 50% per day for 2 days. Refeeding after providing 66.7% food volume per day or 15 cycles of every other day (f) within days d-f,12 (d) or 24 (e) significantly increased body fat percentage and decreased lean body mass percentage (n=9). DR (66.7% ×12), which means that 66.7% food was provided to mice daily for 1 to 12 days; DR (66.7% ×24), which means that 66.7% food was provided to mice daily for 1 to 24 days; DR (0% -AL) ×15, representing any diet on a fasted day, was performed for 15 cycles. a, <0.05; b, p <0.01; c, p <0.001; as compared to group AL.

Fig. 5, refeeding after dietary restriction up regulates intestinal lipid absorption, enhances fatty acid uptake, lipid synthesis and storage in white adipose tissue, attenuates lipolysis in white adipose tissue and attenuates overall lipid oxidation. a, b, any feeding and average energy expenditure over days 4 and 5 for mice of groups DR (10% -25% -65%) (a) and DR (33.3% ×3) (b). n=6-8 per group. NS, non-salient. c, total feeding of mice of any of the (AL) (n=9), DR (10% -25% -65%) (n=9) and DR (33.3% ×3) (n=8) groups. D, e, refeeding with D0 after providing 10%, 25%, 65% of food amount (D) or 33.3% of food amount per day (e) within 3 days, respectively, significantly increases body fat content (n=9 per group). DR (10% -25% -65%) -100% ×23 and DR (33.3% ×3) -100% ×23, providing 10%, 25%, 65% of food for 1 to three days, respectively, or providing 33.3% of food per day for 3 days, providing 100% of food as much as D0 per day for 23 consecutive days. fecal Triglyceride (TG) concentration (n=8-10) in mice of f, DR (10% -25% -65%) group. g, representative pictures of small intestine villus oil red staining of mice from DR (10% -25% -65%) group at ZT10 (D0-D12) and ZT15 (D4D, day 4 night). h, triglyceride concentration from small intestine tissue in (g) (n=6-8). Serum triglyceride concentration (n=9) after i, any feeding and DR (10% -25% -65%) groups of mice were perfused with olive oil. Mice were gavaged with 15 μl/g body weight of olive oil at ZT12 time of D5D. j, mice in the ad-hoc and DR (10% -25% -65%) group had a mouse fecal BODIPY concentration (n=9) during 10 minutes to 2 hours after D5D gavage containing BODIPY-labeled fatty acids and olive oil. Representative fluorescence pictures of fresh small intestine tissue after 2 hours of D5D lavage with BODIPY-labeled fatty acids and olive oil in mice of the k, ad libitum fed and DR (10% -25% -65%) group. l, representative fluorescence pictures from small intestine villus sections in (k). m is from (k) BODIPY concentration of small intestine tissue (n=9). n, BODIPY concentration from the mouse serum in (j). o, representative fluorescence pictures from the fresh inguinal white adipose tissue and epididymal white adipose tissue of the mice in (j). p, representative fluorescence pictures from inguinal white adipose tissue and epididymal white adipose tissue sections in (o). q, BODIPY concentration from inguinal white adipose tissue and epididymal white adipose tissue in (o) (n=9). Thermogram of differentially expressed genes in inguinal white adipose tissue of mice of the r-t, DR (10% -25% -65%) group, associated with fatty acid uptake (r), fatty acid and triglyceride synthesis process(s), lipolysis and oxidation (t). The heat map represents log of relative mRNA expression levels ₂ Values. Average log of each gene of D0 ₂ The value is set to 0. Three replicates per group were compared to the expression level of D0. Respiratory exchange rate (n=6-8) for u, v, any feeding and DR (10% -25% -65%) and DR (33.3% ×3) group mice. a or p<0.05; b or p<0.01; c or p<0.001; as compared to either the AL or D0 group.

The refeeding after the diet restriction of fig. 6, designated mode, had no significant effect on energy expenditure, physical exercise and body temperature. a-d, energy expenditure (a, c) and physical movement (b, d) of mice fed ad libitum (10% -25% -65%) or DR (33.3% ×3) groups (n=6-8). e-n, body temperature of any fed mice and mice restricted by the prescribed model diet (n=7-9). a, <0.05; b, p <0.01; c, p <0.001; NS, non-salient; as compared to group AL.

The cumulative food intake of the refeeded mice after the diet restriction of fig. 7, designated mode did not significantly increase, and the refeeding according to the food amount of D0 after the diet restriction significantly increased the body fat percentage and reduced the lean body mass percentage. a-h, cumulative food intake of mice on any diet and diet restriction of the specified pattern (n=7-9). Body fat percentage and lean body mass percentage (n=9) of mice of group i, j, any diet and DR (10% -25% -65%) -100% ×23 (i) or DR (33.3% ×3) -100% ×23 (j). DR (10% -25% -65%) -100% ×23 or DR (33.3% ×3) -100% ×23, providing 10%, 25%, 65% food or 33.3% food per day for 3 days respectively, providing the same food as D0, i.e. 100% food per day for 23 consecutive days. k, accumulated feed intake from mice in (i, j). a, p <0.05; b, p <0.01; c, p <0.001; as compared to group AL.

FIG. 8, refeeding after dietary restriction up-regulates the levels of fatty acid intake and gene expression associated with the course of fatty acid and triglyceride biosynthesis, down-regulates the level of expression of lipolytic related genes and reduces overall lipid oxidation. Heat maps of differentially expressed genes associated with fatty acid uptake (a, b), fatty acid and triglyceride synthesis process (c, d), lipolysis and oxidation (e, f) in epididymal white adipose tissue and inter-scapular brown adipose tissue of mice of group a-f, DR (10% -25% -65%). Average Respiratory Exchange Rate (RER) of g, h, any feeding and DR (10% -25% -65%) (g) or DR (33.3% ×3) (h) group mice. * P <0.01; * P <0.001.

Figure 9, refeeding high protein diet effectively maintains the diet-restriction-induced body fat content reducing effect. Serum essential amino acid concentration (n=3-4) of mice of group a, DR (10% -25% -65%). b, high protein diet (HP) maintained body fat content reducing effects induced by the restricted manner of providing 10%, 25%, 65% food amounts, respectively, within 1 to 3 days (n=9-18). NP, normal protein content diet; LP, low protein diet; EAA, essential amino acids. c, representative pictures of white adipose tissue in the groin of mice re-fed normal protein or high protein diet after the diet restriction regimen as in (b). d, weight from the white adipose tissue of the inguinal of the mouse in (c) (n=6-10). E, representative H & E stained sections from inguinal white adipose tissue in (c). f, average adipocyte area from inguinal white adipose tissue in (e) (n=6). g, h, high protein diet (HP) maintained the body fat content reducing effect induced by diet providing 33.3% of food amount (g) per day for 3 days or 66.7% of food amount (h) per day for 12 days (n=9). i, high protein diet (HP) maintained the body fat content reducing effect (n=10) of female mice induced by diets providing 10%, 25%, 65% food amounts, respectively, within 1 to 3 days. a or p <0.05; b or p <0.01; c or p <0.001; the DR-HP group was compared to the AL group or DR-NP group, except as indicated.

Figure 10, refeeding high protein diet maintains the effects of percent body fat reduction and percent lean body mass increase and white fat content reduction induced by diet restriction. Serum semi-essential and non-essential amino acid concentrations (n=3-4) for mice of group a, DR (10% -25% -65%). b, high protein diet (HP) maintained the effects of percent reduction in body fat and percent increase in lean body mass induced by diets providing 10%, 25%, 65% food mass, respectively, over 1 to 3 days (n=9-18). NP, normal protein content diet; LP, low protein diet; EAA, essential amino acids. c, d, representative pictures of the inguinal white adipose tissue (c) and epididymal white adipose tissue (d) of mice re-fed normal protein or high protein diet after the diet restriction regimen as in (b). e, weight of epididymal white adipose tissue from mice in (d) (n=6-10). f, representative H & E stained sections from epididymal white adipose tissue in (d). g, average adipocyte area from epididymal white adipose tissue in (f) (n=6). a or p <0.05; b or p <0.01; c or p <0.001; the DR-HP group was compared to the AL group or DR-NP group, except as indicated.

Fig. 11, high protein diet after dietary restriction did not induce brown adipose tissue hypertrophy and liver steatosis between the shoulder blades. a, representative pictures of brown adipose tissue between shoulder blades of mice fed normal protein or high protein diet after 10%, 25%, 65% food amount, respectively, were provided within 1 to 3 days. b, weight from brown adipose tissue between the shoulder blades in (a) (n=6-10). c, representative pictures from H & E stained sections of brown adipose tissue between the shoulder blades in (a). d, representative pictures of H & E stained sections of liver from mice in (a). e, liver Triglyceride (TG) concentration (n=6-7) from mice in (a). * P <0.05; * P <0.01; * P <0.001.

Figure 12, refeeding high protein diet maintained the effects of percent body fat reduction and percent lean body mass increase induced in male mice by a diet providing 33.3% food amount per day for 3 days or 66.7% food amount per day for 12 days and 10%, 25%, 65% food amount in female mice by 1 to 3 days, respectively. a, b, high protein diets maintained the effects of percent body fat reduction and percent lean body mass increase induced by diets providing either 33.3% food mass per day (a) for 3 days or 66.7% food mass per day (b) for 12 days (n=9). c, the effects of reduced body fat percentage and increased lean body mass percentage induced by diet providing 10%, 25%, 65% of the food amount, respectively, in 1 to 3 days were maintained for a short period of time after refeeding the high protein diet in female mice (n=10). a, p <0.05; b, p <0.01; c, p <0.001; DR-HP group compared with AL group or DR-NP group.

Fig. 13, high protein diet after dietary restriction down-regulates intestinal lipid absorption, reduces anabolism of lipids in white adipose tissue and enhances catabolism of lipids. a, b, energy expenditure in mice fed normal protein (n=6) or high protein feed (n=6) after any diet (n=4) and a diet regimen providing 10%, 25%, 65% of the food amount in 1 to 3 days, respectively. c, d, cumulative ingestion (c) and daily ingestion (d) of mice treated in the same manner as in (a). AL, n=18; DR-NP, n=18; DR-HP, n=9. e, the concentration of Triglyceride (TG) in the feces of mice treated in the same manner as in (a) (n=7-10). f, mice fed ad libitum, DR-NP and DR-HP groups contained BODIPY-labeled fatty acids in the D5 lavage and the concentration of BODIPY in the mouse feces during 10 minutes to 2 hours after olive oil (n=7-9). g, h, representative fluorescence pictures from small intestine tissue (g) of mice in (f) after 2 hours of gavage and their villous section (h). i, j, BODIPY concentration (n=7-9) from small intestine (i) and serum (j) of mice in (g). k, l, representative fluorescence pictures of inguinal white adipose tissue and epididymal white adipose tissue (k) from mice of (f) and their sections (l). m, BODIPY concentration of inguinal white adipose tissue and epididymal white adipose tissue from the mice in (f) (n=9). Heat maps of differentially expressed genes associated with fatty acid uptake (n), fatty acid and triglyceride synthesis process (o), lipolysis and oxidation (p) in white adipose tissue in groin of mice treated in the same manner as in (a). q, r, and the same treatment as in (a) were used for the respiration rate (q) and the average respiration rate (r) (n=4-6). a or p <0.05; b or p <0.01; c or p <0.001; NS, non-salient; the DR-HP group was compared to the AL group or DR-NP group, except as indicated.

Figure 14, high protein diet after diet restriction significantly inhibited mice ingestion. a, b) cumulative food intake (n=9) of male mice re-fed normal or high protein diet after diet restriction in a manner that either an arbitrary diet and a food amount (a) of 33.3% per day or a food amount (b) of 66.7% per day for 3 days were provided. c, cumulative food intake (n=10) of female mice re-fed normal protein or high protein diet after any diet and dietary restriction in a manner to provide 10%, 25%, 65% of the food amount within 1 to 3 days, respectively. d, daily intake of male mice re-fed normal protein or high protein diet (n=9) following any diet and dietary restriction in a manner that provides a food amount of 33.3% per day for 1 to 3 days. a, p <0.05; b, p <0.01; c, p <0.001; DR-HP group compared with AL group or DR-NP group.

Detailed Description

Dietary restrictions are widely used to reduce fat content in obese and non-obese mammals. Nonetheless, weight regain after dieting remains a great challenge, with underlying mechanisms not being well-defined. In view of the current state of the art, the present invention is directed to the research in this respect, and in the course of the intensive research, animal models suitable for the obesity research and the lipid metabolism research are established, and the phenomenon of promoting the formation of obesity by re-feeding after diet restriction is revealed, and thus, important animal models can be provided for the obesity research. The experimental results of the present inventors also demonstrate that the targeting of intestinal lipid absorption by high protein diets is an effective strategy for preventing obesity after dietary restriction.

Terminology

As used herein, the terms "diet (food or food) control", "intake control", "diet (food or food) restriction" are used interchangeably with "diet".

As used herein, a "diet restriction" is a particular stage that differs significantly from a "normal diet"; during this "diet restriction" phase, the subject's food intake is significantly less than for a "normal diet". By "normal diet" is generally meant the daily or natural food intake of the same subject when not or before "diet limiting".

As used herein, the term "restore food intake" is a specific phase, which is different from the "diet restriction" phase; it refers to the return to the subject's natural state of food intake after a "diet restriction" phase.

As used herein, the term "composition of the present invention" includes: foods (compositions), health products (compositions), etc., as long as their protein content or amino acid content is regulated as in the present invention.

As used herein, the term "comprising" or "including" includes "comprising," consisting essentially of … …, "and" consisting of … …. "consisting essentially of … …" means that minor ingredients and/or impurities, in addition to the essential ingredients or components, may be present in the composition in minor amounts without affecting the active ingredient. For example, sweeteners or flavoring agents may be included to improve taste, antioxidants to prevent oxidation, and other additives commonly used in the art.

As used herein, the term "hygienically acceptable" or "pharmaceutically acceptable" ingredients are substances suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

As used herein, the term "effective amount" refers to an amount that is functional or active in and acceptable to a human and/or animal.

As used herein, "parts by weight" or "parts by weight" are used interchangeably and may be any fixed amount expressed in micrograms, milligrams, grams, or kilograms (e.g., 1ug, 1mg, 1g, 2g, 5g, kg, etc.). For example, a composition comprising 1 part by weight of component a and 9 parts by weight of component b may be a composition comprising 1 gram of component a+9 gram of component b, or 10 grams of component a+90 gram of component b, etc. In the composition, the percentage content of a certain component= (the sum of parts by weight of the component/parts by weight of all components) ×100%. Thus, in a composition consisting of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10% and component b is 90%.

As used herein, the terms "unit dosage form" and "unit dosage form" refer to dosage forms that are required to prepare the compositions of the present invention for convenient administration in a single administration, including, but not limited to, various solid (e.g., tablet) and liquid agents. The unit dosage forms contain the compositions of the present invention in amounts suitable for single, single day or unit time administration.

As used herein, the term "lipid oversynthesis" refers to a situation in which the rate of lipid synthesis is significantly increased, for example, in a test subject (e.g., a population that is re-ingested after a pre-diet) when the rate of lipid synthesis is significantly increased with the same amount of energy ingested as a normal subject (e.g., a population that is not pre-diet).

Lipid metabolism after dietary restriction and re-ingestion

The present inventors have conducted intensive studies and found that refeeding after dietary restriction increases intestinal lipid absorption, regulates lipid metabolism of adipose tissue and induces obesity reversible by high protein diet. The inventors have found that refeeding after various types of dietary restrictions causes a rapid accumulation of fat. There were no significant changes in individual energy expenditure, physical movement, and body temperature during refeeding. Experiments to re-provide normal food amounts after dietary restriction indicate that an increase in food intake during re-ingestion is not a major cause of obesity. Further, the inventors found that enhanced intestinal lipid absorption, as well as increased lipid anabolism and decreased lipid catabolism in adipose tissue, are responsible for obesity following dietary restriction. Furthermore, the present inventors have found that a high protein diet can sufficiently suppress the occurrence of obesity caused by dietary restrictions, and even can partially maintain the lipid-lowering effect caused by dietary restrictions. Further studies have found that a high protein diet reduces intestinal lipid absorption and improves lipid metabolism in adipose tissue.

The inventors have found that not only energy expenditure nor increased intake after dietary restriction, but also enhanced intestinal lipid absorption more leads to obesity formation after dietary restriction. Although the small intestine is involved in the processing of fat in the body, its key role is ignored. The results of the present inventors' studies clearly demonstrate that fatty acid uptake, lipid synthesis and storage capacity of adipose tissue increases, lipolysis decreases, and overall lipid oxidation decreases following dietary restriction. In order to find effective nutritional intervention after dietary restriction, the present inventors analyzed the serum amino acid profile of animals before, during and after dietary restriction, and found that many amino acids were up-regulated in expression levels in dietary restriction and after refeeding. The present inventors have thus tried to perform dietary intervention by changing the ratio of proteins or adding amino acids, and as a result have found that a high protein diet, a low protein diet, and a diet supplemented with essential amino acids can suppress an increase in fat content after dietary restriction, as compared with a control group fed with normal protein feed. Particularly remarkable, the high protein dietary intervention after the dietary restriction can effectively prevent the occurrence of obesity, even maintain the effect of reducing the fat content induced by the dietary restriction, and further provide a potential method for preventing the occurrence of obesity after the dietary restriction. The high protein diet improves weight maintenance in overweight or obese patients following a low calorie diet, indicating that the high protein diet can prevent obesity following a dietary restriction and can even maintain the fat-reducing effects of the dietary restriction. The experimental results of the present inventors can provide valuable references for those who want to lose weight and worry about weight regain, i.e., eat high protein ratio foods after weight loss by diet restriction to prevent weight regain caused by binge eating. In addition, high protein diets are a relatively simple intervention to prevent weight regain after dietary restrictions, and have great popularity.

The inventors have found that a high protein diet can effectively suppress ingestion. Through experimental analysis, the inventors have clarified that feeding factors and energy consumption factors are not the main causes of preventing obesity from occurring after dietary restriction, triglyceride concentration in the feces of animals with high protein diet intervention after dietary restriction is significantly increased, and small intestine lipid absorption is an important factor. Gastric lavage fluorescence labeled fatty acid experiments demonstrated that high protein diets after dietary restriction reduced small intestine lipid absorption. Further, the present inventors found that high protein diets intervened in the reduced lipid anabolism and increased catabolism of the group white fat. Furthermore, the respiratory exchange rate of the intervention group fed the high protein diet after dietary restriction was significantly reduced, indicating that high protein diet intervention significantly increased overall lipid oxidation.

Thus, the present inventors have found that refeeding after dietary restriction induces obesity occurrence, thereby providing a new view of the obesity occurrence mechanism under such conditions, i.e., increased intestinal lipid absorption and increased anabolic catabolism of adipose tissue lipids leading to obesity occurrence associated with dietary restriction, and that obesity induced in this way can provide a new animal model for the study of obesity mechanism and prevention. Further, the present inventors have found that high protein dietary intervention can prevent the occurrence of obesity induced by dietary restrictions and can even maintain the lipid-lowering effect of dietary restrictions, mainly because the high protein diet after dietary restrictions can attenuate lipid absorption in the intestinal tract and improve lipid metabolism in adipose tissue. Experiments by the inventors suggest that obesity occurrence after dietary restriction can be prevented and controlled by high protein dietary intervention or by some strategy targeting intestinal lipid absorption.

Based on the new findings of the present inventors, a method for controlling body weight and lipid biosynthesis is provided, comprising: (1) reduced food intake, significant weight and lipid loss; (2) Restoring food intake, intake of a food with a regulated protein content or amino acid content, said food being selected from the group consisting of: high protein content foods, high amino acid content foods, or low protein content foods, thereby controlling body weight and lipid over-synthesis; preferably, the high amino acid content food is a high essential amino acid content food, i.e., a food in which an effective amount of essential amino acids is added to a normal food.

As another alternative, there is provided a method of modulating (inhibiting) dietary restriction and lipid oversynthesis following re-ingestion, comprising: after re-ingestion, ingestion of a food having a protein content or amino acid content regulated, the food selected from the group consisting of: high protein content foods, high amino acid content foods, or low protein content foods, thereby controlling body weight and lipid over-synthesis.

According to the results of the present inventors, the foods having the protein content or amino acid content regulated after the early diet and the restoration of food intake achieve the control of body weight and the inhibition of lipid oversynthesis by means of weakening lipid synthesis, increasing lipid oxidation, promoting lipid decomposition, weakening small intestine lipid absorption, up-regulating lipid oxidation gene expression, up-regulating lipolytic gene pnpla2, or down-regulating fatty acid and triglyceride synthesis genes (such as Acly, fasn, etc.).

In the methods of the invention, the reduction of food intake or diet control is performed in a preliminary stage, and this process may be performed in a variety of ways, including, but not limited to: regular diet, intermittent diet, time-limited diet, low-energy diet simulating diet, gradient-increasing or gradient-decreasing diet. Food intake or diet control is performed according to the needs or plan of the subject; for example, for a human, the planning may be performed over a longer (e.g., 3-6 months or longer) or medium length such as (1-3 months) or shorter (e.g., 3-30 days). This process is typically measured as a significant reduction in weight as a test criterion, also measured according to the needs or plan of the subject, e.g., a significant reduction of 2-40% in weight; more specifically, for example, 3%, 5%, 8%, 10%, 15%, 20%, 30%, 35, and the like.

In the present invention, the type of food to be ingested in the "diet restriction" process is not particularly limited, and may be a conventional food, but the amount of food to be ingested may be controlled (the amount of food to be ingested is significantly reduced), or a low-energy food may be ingested. However, as a preferred mode of the invention, the "diet restriction" is a gradient increasing or gradient decreasing restriction scheme, such as programming daily intake, increasing or decreasing regularly, rhythmically or wavelike according to the programmed diet restriction time. Preferably, even if there is a period of time to increment to a higher point, the higher point is below the level of "normal diet".

In said "restore food intake" phase, preferably a food with a regulated protein content or amino acid content is taken, said food being selected from the group consisting of: high protein content foods, high amino acid content foods, or low protein content foods, thereby controlling body weight and lipid over-synthesis; preferably, the high amino acid content food is a high essential amino acid content food; more preferably, the essential amino acids include: valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine, lysine, histidine.

Composition and method for producing the same

Based on the new findings of the present inventors, there is also provided a composition for regulating dietary restriction and lipid oversynthesis after re-ingestion, which is a food with a regulated protein content or amino acid content, selected from the group consisting of: high protein content food, high amino acid content food, or low protein content food.

As a preferred mode of the present invention, the food is a high protein content food, wherein the protein content is more than 400 parts by weight; preferably greater than 450 parts by weight; more preferably above 550 parts by weight.

As another preferred mode of the present invention, the high amino acid content food is a high essential amino acid content food, and the essential amino acids include: valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, threonine, lysine, histidine.

As another alternative of the present invention, the food is a low protein content food, wherein the protein content is less than 150 parts by weight; preferably less than 100 parts by weight; more preferably less than 60 parts by weight. In some embodiments, the inventors have unexpectedly found that low protein content foods are also beneficial in regulating dietary restrictions and lipid oversynthesis after re-ingestion.

As a preferred mode of the present invention, the protein includes a protein selected from the group consisting of: casein, whey protein or soy protein.

As a preferred mode of the invention, cysteine is also included in the food. In the high protein content foods, the cysteine content is significantly higher than in the normal protein content foods.

As a preferred mode of the invention, carbohydrates are also included in the food. In the high protein content foods, the carbohydrate content is significantly lower than in normal protein content foods.

As a preferred mode of the present invention, in the high amino acid content food, the content of the essential amino acid is optimally designed according to the results of the study by the present inventors. Preferably, the valine content is 16-36 parts by weight, the isoleucine content is 12-32 parts by weight, the leucine content is 23-43 parts by weight, the methionine content is 5-15 parts by weight, the phenylalanine content is 8-28 parts by weight, the tryptophan content is 2-8 parts by weight, the threonine content is 10-27 parts by weight, the lysine content is 20-40 parts by weight, and the histidine content is 5-18 parts by weight.

As a preferred embodiment of the present invention, the food further comprises: fat, cellulose, minerals, vitamins. More preferably, the fat content is 50 to 90 parts by weight, the cellulose content is 30 to 70 parts by weight, the mineral content is 15 to 55 parts by weight, and the vitamin content is 8 to 18 parts by weight.

The composition may be a food composition, and in some embodiments, the composition may further comprise a pharmaceutically or nutraceutically acceptable carrier.

The formula ranges shown in the present invention can be used as reference guidelines. It will be appreciated that the effective dosage of each component may also vary with the actual application when used to develop a prepared food or composition. Such as being in a concentrated form or in a diluted form, etc., which are also intended to be encompassed by the present invention.

In some preferred embodiments of the invention, the composition is in unit dosage form. When the composition is prepared into a unit dosage form, 2 to 6 doses of the composition in the unit dosage form are taken every day, for example, 2,3 and 4 doses are taken according to the diet rule.

Animal model

Based on the new findings of the present inventors, the present invention provides an economically convenient preparation of high fat animal models, which can provide other important and convenient animal models for obesity research, while providing a new strategy for re-feeding after dietary restrictions to prevent obesity.

In the present inventors' embodiments, up to 10 types of diet restriction experiments were performed, systematically studying the effect of re-feeding after diet restriction on obesity, all data demonstrating that re-feeding after diet restriction resulted in rapid accumulation of fat content. In view of the popularity of active and passive dietary restrictions in today's society, not only overweight and obese people will choose to diet, but also normal weight people will diet, and the concern about becoming fat will also place a lean person on dietary restrictions. There is an urgent need to fully understand the deleterious effects of re-feeding after dietary restrictions and to communicate this information to the public. For individuals of normal weight, while dietary restrictions may bring short-term benefits, controlling body weight by diet may increase the risk of overweight thereafter. In addition, small rodents, including rats, and in particular mice, are very close in physiological characteristics to humans, and are the most widely studied preclinical animal models of obesity. Common mouse obesity models include single factor models of the leptin pathway such as ob/ob mice and db/db mice; multifactorial diet-induced models such as high fat diet models; and surgically and chemically induced models such as the mouse obesity model for intra-abdominal hypothalamic lesions. Experiments by the present inventors demonstrate that refeeding after dietary restriction can induce obesity in mice with concomitant hypertrophy of brown fat and liver steatosis. High fat diet induced obese mice are often used to study the mechanisms of obesity and strategies to combat obesity, however this model is time consuming and expensive. The technical scheme of the invention can effectively change the situation.

The invention provides a method for preparing the high-fat animal model, which comprises the following steps: (a) Reducing animal food intake, significantly reducing body weight and lipid; (b) And (3) for the animal in the step (a), normal food intake is recovered, and a high-fat animal model is obtained.

In another preferred example, the normal food means food whose protein content or amino acid content is not regulated. That is, in order to obtain a high-fat animal model, some of the preferable embodiments of the present invention are not applied to foods with high protein content, foods with high amino acid content or foods with low protein content, which are advantageous in maintaining or reducing body weight, reducing body fat after re-intake.

In a preferred mode of the invention, the time taken to reduce food intake in an animal is: the time required for significant weight and lipid loss to occur; preferably 3 to 100 days; or, the time to resume normal food intake is: the time required for the lipid to significantly increase; preferably 3 to 100 days. The time may be further varied according to the animal to which the method of the present invention is directed.

In a preferred mode of the invention, the reduced food intake or diet control includes, but is not limited to: regular diet, intermittent diet, time-limited diet, low-energy diet simulating diet, gradient increasing or gradient decreasing diet; preferably a gradient increasing or decreasing diet; such as 10%, 25%, 65% food in three days, and 65%, 25%, 10% food in three days, respectively. With reference to the method of the embodiments of the present invention, the diet programming may be further varied depending on the animal to which it is directed.

The high-fat animal model prepared by the method has the advantages of increased fatty acid uptake, lipid synthesis and storage capacity of adipose tissues, reduced lipolysis and reduced overall lipid oxidation.

Although the examples of the present invention provide a high fat animal model in murine models, it will be appreciated that this embodiment of the invention is also applicable to other mammals, including for example (but not limited to): rodents (including rats, mice, hamsters, etc.), non-human primates (e.g., monkeys, chimpanzees, etc.), and the like.

The animal models of the present invention find utility in a variety of applications, such as, but not limited to, for: screening potential substances (foods) for controlling body weight and lipid excessive synthesis; performing obesity study; performing a lipid synthesis or breakdown study; and/or studies of the body's absorption or breakdown of lipids.

Screening method

According to the present invention, after a series of characteristics such as dietary restriction and lipid oversynthesis after re-ingestion are known, substances (e.g., foods or health products) effective for controlling body weight, alleviating or avoiding such lipid oversynthesis can be screened based on these characteristics. Although the inventors' studies have given some effective substances, further studies and screening are still necessary.

In a preferred embodiment of the present invention, the screening method may be performed using the high-fat animal model of the present invention as a subject.

As a preferred mode of the present invention, the method for screening potential substances for controlling body weight and lipid biosynthesis comprises: (1) Reducing food intake in a subject, significantly reducing body weight and lipid; and, (2) restoring food intake to normal levels in the subject, the test group subjects administered the test substance, and the control group subjects did not control food type. Further, observing the lipid synthesis of the test group and the control group subjects, and if the lipid synthesis of the test group is significantly lower than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive lipid synthesis; or observing lipid oxidation of the test group and the control group subjects, and if the lipid oxidation of the test group is significantly higher than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive synthesis of lipid; or, observing the lipid decomposition of the test group and the control group subjects, if the lipid decomposition of the test group is significantly higher than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive synthesis of lipid; or observing the small intestine lipid absorption of the test group and the control group subjects, and if the small intestine lipid absorption of the test group is obviously lower than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and excessive synthesis of lipid; or observing the expression of the lipid oxidation gene or the lipolytic gene Pnpla2 of the test group and the control group subjects, and if the expression of the lipid oxidation gene or the lipolytic gene Pnpla2 of the test group is obviously higher than that of the control group, indicating that the substance to be tested is a potential substance for controlling weight and lipid oversynthesis; or observing the expression of the fatty acid and triglyceride synthesis genes of the test group and the control group subjects, and if the expression of the fatty acid and triglyceride synthesis genes of the test group is significantly lower than that of the control group, indicating that the substance to be tested is a potential substance for controlling body weight and lipid excessive synthesis.

In the screening method, the subjects include but are not limited to: rodents (including rats, mice, hamsters, etc.), non-human primates (e.g., monkeys, chimpanzees, etc.), and the like. The potential materials may include, but are not limited to: food, health products, drinks, etc. having a difference in components; can be natural substances or processed products.

As a preferred mode of the present invention, the method further comprises: further cellular and/or animal experiments are performed on the potential substances obtained to further select and identify substances that are truly useful for inhibition of dietary restriction, lipid oversynthesis after re-ingestion, and the like.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

In the present invention, abbreviations and their full names are shown in table 1.

TABLE 1

Mice and feeds

All mice used in the experiments were C57BL/6J strain mice purchased from Shanghai Laike laboratory animal Limited. Normal standard feeds for mice were supplied from animal houses, purchased from Shanghai Laek laboratory animal Limited, and other feeds were purchased from Shanghai Saibo biotechnology Co. Wherein 20% of the normal protein feed was made according to AIN-93G rodent diet formulation (D10012G, research Diets inc.) except that the antioxidant tBHQ was not contained and that the sucrose was replaced with corn starch comprising 20% casein, 0.3% cysteine and 49.7% corn starch.

High protein feed: is prepared on the basis of 20% of normal protein feed, and comprises 60% of casein, 0.9% of cysteine and 9.1% of corn starch.

Low protein feed: made on a 20% normal protein feed base comprising 5% casein, 0.075% cysteine and 64.925% corn starch.

Feed supplemented with essential amino acids: is prepared on the basis of 20% of normal protein feed, and comprises 32.5% of corn starch, 2.6% of valine, 2.2% of isoleucine, 3.32% of leucine, 1% of methionine, 1.8% of phenylalanine, 0.44% of tryptophan, 1.76% of threonine, 2.96% of lysine and 1.12% of histidine; the amount of each essential amino acid to be supplemented is calculated from the portion of the high protein feed that is more than the normal protein feed.

Reagent and kit

BODIPY 500/510 C1, C12 fatty acids, available from Molecular Probes; OCT compound (Optimal cutting temperature compound), available from Sakura corporation; nonidet P-40, available from Sangon Biotech; chloral hydrate, methanol, chloroform, absolute ethanol, purchased from Shanghai, shibasi technologies development Co., ltd; paraformaldehyde, purchased from national pharmaceutical groups chemical reagent limited; hematoxylin and eosin stain from wuhansai wil biotechnology limited; valine, histidine, available from beijing enokio technologies limited; isoleucine, available from the company of Biotechnology, inc.; leucine, purchased from Shao Yuan technology Co., ltd; methionine available from Shanghai Taitan technologiesCompany limited; phenylalanine, available from psa prandial; tryptophan, available from Bio-engineering Co., ltd; threonine, lysine, purchased from calico biotechnology limited. aTRAQ ^TM Kit, purchased from abscix corporation; triglyceride detection kit, available from Shanghai Shen Suoyou Fu medical diagnostic articles Inc.

Diet restriction experiment

All mouse experiments were performed according to the guidelines of the animal administration and use committee of Shanghai institute of nutrition and health. Mice of the 8 week old C57BL6/J strain were acclimatized in the animal house for 3-5 days after purchase. Mice were then fed in single cages for a period of 5 days, followed by diet restriction and refeeding the experimental mice were also fed in single cages. In the single cage feeding stage prior to diet restriction, the mice were fed with feed for diet restriction. The average food intake of mice three days before dietary restriction was used as a dietary reference for the following experiments. Diet restriction was performed by adding specified amounts of food to mice at ZT12 (19:00). Unless otherwise specified, all animal experiments were performed on male mice.

Body weight and body composition measurements

Mouse body weight and body composition were measured at ZT10 (17:00). Fat and lean body mass content were determined using an echo mri-100H body composition analyzer (echo mri).

Body temperature measurement

The body temperature of the mice was measured by using a RET-3 rectal probe (Physiomp) attached to a BAT-12 thermometer (Physiomp) for a period of time at ZT3 (10:00).

Metabolic rate and physical Activity measurements

Metabolic rate and physical activity of the designated mice were measured by a 16-chamber environmental control integrated laboratory animal monitoring system (Columbus Instruments). Mice were acclimatized for at least 24 hours in cages prior to formal recording of the data. The average Respiratory Exchange Rate (RER) refers to the amount of carbon dioxide produced (VCO 2) divided by the amount of oxygen consumed (VO 2) (both units are liters/hour). Energy expenditure (in kilocalories/hour) is calculated by the following formula: 3.815 XVO ₂ +1.232×VCO ₂ (Singh et al.，2019)。

Blood and tissue collection

Mice were anesthetized with 6% chloral hydrate at the indicated time points, then the tissues of interest were isolated, weighed, photographed, then fixed with 4% paraformaldehyde or snap frozen with liquid nitrogen and stored in a-80 ℃ freezer. Blood was collected at the apex needle using a 1ml syringe, and the collected blood was centrifuged at 1000g for 30 minutes at 4℃and then serum was collected and stored in a-80℃refrigerator.

Hematoxylin eosin staining and adipocyte size quantification

The tissue was fixed with 4% paraformaldehyde at 4℃for 24-48 hours, followed by dehydration, paraffin embedding, sectioning (4 μm thick) and finally staining with hematoxylin and eosin. The adipocyte size was quantitatively calculated by ImageJ software (version 1.51).

Oil red dyeing

The tissue was fixed with 4% paraformaldehyde at 4 ℃ for 24-48 hours, followed by dehydration, embedding with OCT compound, sectioning (10 μm thick), and finally staining with oil red solution for 10 minutes and counterstaining with hematoxylin.

Measurement of triglyceride concentration

The extraction and measurement of triglycerides from the liver, small intestine and faeces was carried out using the method described previously, with minor modifications on this basis. Specifically, after a liver or small intestine sample is collected, it is sheared with scissors. The collected feces were freeze-dried and ground with a mortar and pestle. 70-90mg of liver fragments or 40-60mg of jejunal fragments, or about 100mg of fecal powder, were placed in 1.2ml of a methanol/chloroform (volume: 1:2) mixture and 2mm diameter porcelain beads 5 were added and homogenized for 10 minutes at 30 Hz using a tissue Lyser II (Qiagen) instrument. The sample was then centrifuged at 1000g for 10 minutes, 700-800ul of the supernatant was transferred to a fresh EP tube, then 150ul of 0.9% saline was added, and the sample was vortexed and centrifuged at 1000g for 10 minutes. Transfer 300-400. Mu.l of the organic phase to a fresh 1.5ml centrifuge tube and then air dry in a fume hood at room temperature. The completely air-dried sample was dissolved in 200-300. Mu.l of absolute ethanol. These samples dissolved in absolute ethanol were assayed for triglyceride concentration using an enzyme detection kit. Collected serum samples were also assayed for triglyceride concentration using an enzyme detection kit.

Lipid and fatty acid absorption experiments

Mice were perfused with either gastric olive oil (15 μl/g body weight) or a mixture of BODIPY 500/510 C1, C12 fatty acid (1 μl/g body weight) and olive oil (10 μl/g body weight) at ZT12 (19:00). The mice were provided with sufficient water but no food after gavage. For the olive oil gastric lavage experiment, tail end blood collection is carried out on the mice before gastric lavage and 1, 2, 4 and 6 hours after gastric lavage respectively, and then serum is obtained through centrifugation, so that the concentration of triglyceride is measured. For the BODIPY gastric lavage experiments, the mouse feces were first collected in the interval from 10 minutes to 2 hours after gastric lavage, then freeze-dried and ground with a mortar and pestle, and then stored in a-20℃refrigerator. Mice were anesthetized with 6% chloral hydrate 2 hours after gastric lavage and then tissue and blood samples of interest were collected. The isolated proximal jejunum, inguinal white adipose tissue or epididymal white adipose tissue was directly observed under a fluorescence microscope or embedded in OCT compound and then sectioned. For the concentration detection of BODIPY. Specifically, the proximal jejunum, inguinal white adipose tissue or epididymal white adipose tissue was first homogenized in RIPA lysis buffer (50 mM Tris-HCl pH7.5, 150mM NaCl,1% Nonidet P-40,1% sodium deoxycholate, 0.1% SDS) and the supernatant was centrifuged to read the fluorescent signal. The fluorescence signal of the extracted tissue sample or serum sample was measured by an enzyme-labeled instrument, the corresponding excitation wavelength was 492nm, and the emission wavelength was 520nm. The dried and ground stool samples were treated with a mixture of water and chloroform at night (volume ratio: 1:2) and the organic phase was centrifuged to measure the fluorescent signal.

Serum amino acid concentration measurement

Serum amino acids were first treated with sulfosalicylic acid and then treated with the aTRAQTM kit (ABSciex)Labeling, then adding a reagent containing Norvaline and NorleuThe internal standards of the cine were mixed. Finally, serum amino acid concentrations were determined using a liquid chromatograph-tandem mass spectrometer (LC-MS/MS) equipped with an Agilent 1200 series HPCL system and a 4000Q-Trap tandem mass spectrometer of electrospray ionization source.

High throughput RNA sequencing and analysis

Designated tissues were collected at ZT10 (17:00) followed by extraction of RNA with TRIzol reagent (Thermo). Libraries for sequencing were generated using a TruSeq RNA sample preparation kit (Illumina) followed by sequencing by a HiSeq X TEN system (Illumina) resulting in 2X 150 base double-ended Reads (Reads), the sequencing depth of each library being 30-60 million Reads. High quality readings were obtained by a Cutadapt (version 1.15) software screen. The reference genome index was established by Bowtie2 (version 2.2.6) software and high quality reads were aligned to the reference genome (mm 10) using Tophat2 (version 2.0.14) software. The P-value of the genetic statistical difference was calculated by using DESeq (version 1.30.0) software. Genes with P <0.01 and average RPM (reads for a gene per million reads) of greater than 2 before and after dietary restriction were considered significantly differentially expressed. Fatty acid uptake-related genes (including Cd36, slc27a1-7, fabp1-9, acsl1, acsl 3-6), fatty acid and triglyceride synthesis process-related genes (GO: 0006633 and GO:0019432, designated by MGI), lipolysis-related genes (including Pnpla2, lip, mgll), fatty acid oxidation-related genes (GO: 0019395, designated by GO Central) were analyzed and heat maps were generated using GraphPad Prism software.

Data analysis and statistics

The statistical analysis of the data adopts Excel software, and all numerical calculation results are displayed in the form of mean value +/-standard deviation. All figures were drawn using GraphPad Prism software. Significant differences between the different groups were analyzed by two-tailed Student's t-test. p values less than 0.05 were considered statistically significant differences. The experiments in FIGS. 1a-d, 1h-j, 1k-m, 2b-e, 4a-c, 4d-f, 5c and 7a-b, 5d-e, 6e-h, 6i-k, 6l-n, 7c-e, 7f-h, and 7i-j were each performed simultaneously. To clearly show these data, control mouse data for the same arbitrary diet group are shown in different figures.

Example 1 refeeding after dietary restriction resulted in obesity in mice

In this and subsequent examples, the diet given to the mice was normal standard feed unless otherwise indicated.

To investigate the effect of re-feeding after dietary restriction on body fat in mice, the inventors devised different modes of re-feeding experiments after dietary restriction. First, the mice were provided with food for only 1 day in the first 3 days, or for only 4 days in the first 6 days; the mice were then provided with sufficient food for refeeding. The inventors found that the body fat content of mice was significantly reduced after feeding 10%, 25%, 65% of food to the mice at the indicated time points from day 1 to day 3, which was consistent with expectations (fig. 1a; fig. 2 a). Unexpectedly, however, refeeding after 10%, 25%, 65% food was fed, inducing rapid accumulation of fat in the mice (fig. 1 a). At the same time, the percentage of body fat in mice increased significantly and the percentage of lean body mass decreased significantly (fig. 2 b). Similarly, following diet restriction in a manner that provided 65%, 25%, 10% of the diet to the mice, or 33.3% of the diet per day for 3 days, and 66.7% of the diet per day for 6 days, respectively, the subsequent re-normal feeding resulted in a rapid accumulation of fat content in the mice (figures 1 b-d), with a significant increase in body fat percentage and a significant decrease in lean body mass percentage in the corresponding mice (figures 2 c-e).

To further demonstrate the effect of refeeding after dietary restriction on mouse fat, mice that were dietary restricted by 10% -25% -65% were sacrificed and dissected at designated time points (fig. 2 f), blood and tissues of interest were collected. The inventors found that during the period of feeding the mice with 10% -25% -65% of the food amount, the fat content of the abdomen of the mice was significantly reduced, while the fat content of the abdomen after refeeding was significantly increased (fig. 2 g). By analysing the isolated tissue, it was found that the morphology of the Inguinal White Adipose Tissue (iWAT) of the mice became smaller and its weight significantly reduced at the time of diet restriction, whereas the morphology of the inguinal white adipose tissue became significantly larger and exceeded the initial level after refeeding, and the corresponding weight was also significantly higher than the initial level (fig. 1e and f; fig. 2 h). Likewise, the size and weight of epididymal white adipose tissue (eWAT) in mice after refeeding also increased significantly and above the original level (FIGS. 2i and j). The results of hematoxylin and eosin (H & E) staining showed that refeeding after dietary restriction significantly increased the adipocyte size of inguinal white adipose tissue and epididymal white adipose tissue (FIG. 1g; FIGS. 2 k-m).

Furthermore, the inventors found that the bright morphology of brown adipose tissue between the shoulder blades of mice became significantly larger after refeeding (fig. 3 a), the corresponding weight was significantly higher than the initial level (fig. 3 b), and the results of hematoxylin and eosin staining showed a significant increase in large lipid droplet accumulation in adipocytes after refeeding (fig. 3 c). Re-feeding after dietary restriction thus induced hypertrophy of brown tissue between the mice shoulder blades. The liver of mice at the diet restriction and refeeding stage was sectioned and stained with hematoxylin and eosin, and large lipid droplet accumulation was found in the liver cells after refeeding (fig. 3 d). Further, the concentration of liver Triglycerides (TG) was examined and found to increase significantly on the first day when mice were fed with 10% food, i.e. acute fasting resulted in accumulation of liver triglycerides. The triglyceride levels in the liver were again reduced to pre-dietary levels during subsequent dietary restrictions and increased significantly after refeeding (fig. 3 e), so that refeeding after dietary restrictions induced liver steatosis in mice.

To fully investigate the effect of re-feeding after dietary restriction on fat content, the inventors performed different modes of re-feeding experiments after dietary restriction. First, the mice were provided with a food amount of 2 days in three days or a food amount of one day in 2 days, specifically, the following were included: 40%, 70%, 90% of the diet was provided to mice from day one to day three, respectively; 90%, 70%, 40% of the diet was provided to mice from day one to day three, respectively; mice were provided with 50% food daily from day one to day two. These three modes of dietary restriction allowed the mice to eat less food for one day. As a result, it was found that refeeding after these three modes of diet restriction also induced rapid accumulation of fat in mice (fig. 1 h-j), and a significant increase in body fat percentage and a significant decrease in lean body mass percentage (fig. 4 a-c). Subsequently, a comparison was made with the conventional diet restriction mode, i.e. the mice were reduced in food intake by a certain amount daily over a longer period of time. Specifically, the method comprises the following two steps: mice were provided with 66.7% food per day for 12 consecutive days; mice were provided with 66.7% food per day for 24 consecutive days. It was also surprising that once the mice were given sufficient food, the fat content of the mice increased rapidly over a short period of time (fig. 1k and l), with the corresponding mice having a significantly higher percentage of body fat than any fed mice and a significantly lower percentage of lean body mass than any fed mice (fig. 4d and e). Finally, the inventors performed diet restriction experiments on alternate days. Specifically, the first day of complete fasting did not provide the mice with food, and the second day provided enough food for the mice to eat ad libitum, for 15 such cycles in succession. As a result, it was found (fig. 1 m) that the body fat content of mice in those days who were completely fasted was significantly lower than that of the control group in the first two weeks of the alternate-day diet restriction, suggesting that such alternate-day diet restriction had a certain body fat content-reducing effect in a short period of time. However, on those days fed ad libitum from day 10 to day 30, mice had significantly higher body fat levels than the control group, suggesting that this alternate dietary restriction did not contribute to a decrease in body fat levels over a longer period of time. After the end of the daily diet restriction, the present inventors provided the mice with enough food to eat at will, and can see that the body fat content of the mice rapidly accumulated, while the corresponding body fat percentage significantly increased and the lean body mass percentage significantly decreased (fig. 4 f). Taken together, these results demonstrate that refeeding after dietary restriction can induce rapid accumulation of body fat in mice.

Example 2 enhancement of intestinal lipid absorption and reduction of anabolism enhancement and catabolism of adipose tissue lipids after dietary restriction

To reveal the obesity mechanism caused by refeeding after dietary restriction, the present inventors first analyzed the energy expenditure of mice. The recorded results of the mouse metabolic monitoring system showed that the diet-restricted mice were not significantly changed in energy consumption during refeeding (fig. 5a; fig. 6 a) in a 10% -25% -65% manner compared to any fed mice, while the recorded results of the movement profile of the mice showed no significant change in physical movement of the mice during refeeding (fig. 6 b). To further explore the energy expenditure profile of mice, the metabolic profile of mice fed with a diet restriction followed by refeeding in such a way that a food amount of 33.3% was provided per day over 3 days was monitored, as was the finding that there were no significant changes in energy expenditure and physical movement of mice during refeeding (fig. 5b; fig. 6c and d). In addition, the present inventors examined the body temperature of mice during diet restriction and refeeding, and found that the body temperature of mice was significantly lowered at the time of diet restriction, whereas the body temperature of mice was quickly restored to normal level after refeeding, and the body temperature of mice did not significantly change after refeeding as a whole although there were fluctuations in the body temperature of mice at several time points (fig. 6 e-n). These data demonstrate that obesity following dietary restriction is not caused by reduced energy expenditure.

Next the inventors studied whether or not ingestion of mice involved obesity after dietary restriction. As shown in fig. 1a-d and h-m, the mice were found to have significantly increased food intake for the first few days of refeeding after dietary restriction. To determine whether increased food intake is the primary cause of rapid accumulation of fat content following dietary restriction, the inventors analyzed the cumulative food intake of mice. Interestingly, the cumulative food intake of all mice with various modes of diet restriction measured was either similar to or significantly lower than that of any fed mice (FIG. 5c; FIGS. 7 a-h). Notably, the total feed to mice in either of these modes was significantly lower than any fed mice throughout the refeeding process, with a diet restriction of 66.7% food for either 12 days or 24 days. To explore the effect of increased food intake on body fat in mice for the first few days of the refeeding period after dietary restriction, mice were provided with 10%, 25%, 65% food amount or 33.3% food amount per day for three days, respectively, after which the mice were not given any diet, but were provided with 100% food per day, which is equivalent to the food intake of the mice before dietary restriction. Similarly, providing mice with 100% food following dietary restriction also induced rapid accumulation of fat content (fig. 5d and e), while mice had significantly increased body fat percentage and significantly decreased lean body mass percentage (fig. 7i and j). As expected results, the cumulative food intake of both diet-restricted mice was significantly lower than that of any diet mice (fig. 7 k). These data demonstrate that the accumulated food intake does not significantly contribute positively to obesity after dietary restriction, and that increased food intake for the first few days of the refeeding period is not the main cause of obesity after dietary restriction.

To further investigate the mechanism of obesity after dietary restriction, the present inventors analyzed whether increased intestinal lipid absorption was involved. Mice fed 10%, 25%, 65% of their food respectively on consecutive 3 days were found to have significantly reduced concentration of fecal Triglycerides (TG) during the diet restriction and refeeding phases (fig. 5 f). Furthermore, the results of red staining of the sections of small intestine tissue showed a significant increase in Triglyceride (TG) content of the small intestine after refeeding (fig. 5 g), while the direct measurement of small intestine tissue triglyceride content also showed that refeeding after dietary restriction increased triglyceride accumulation in the small intestine of the mice (fig. 5 h). Further, mice were gavaged with olive oil during the refeeding phase, and a significant increase in serum triglyceride levels was found in mice following diet restriction compared to the control group (fig. 5 i). These all demonstrate an enhanced lipid absorption capacity in the small intestine of the re-fed mice following dietary restriction. To more clearly examine the lipid absorption of the small intestine, mice were subjected to experiments of lavage with a mixture of BODIPY fluorescent-labeled fatty acid and olive oil, and the feces produced by the mice during the period of 10 minutes to 2 hours after the lavage were collected and the BODIPY concentration was examined, and it was found that the BODIPY concentration of the feces of the mice re-fed after the diet restriction was significantly reduced (FIG. 5 j). Meanwhile, blood and proximal jejunal tissue of the mice were collected 2 hours after gastric lavage. Fresh proximal jejunal tissue was taken and directly observed under a fluorescence microscope and the fluorescence intensity of the tissue corresponding to the diet-restricted mice was found to be significantly enhanced (fig. 5 k). The inventors subsequently performed frozen sections of proximal jejunal tissue and could see a significant increase in fluorescence intensity in proximal jejunum villus in diet-restricted mice (fig. 5 l). In addition, treatment of proximal jejunal tissue with RIPA lysate followed by centrifugation to clarify the fluorescence intensity was examined and as a result, a significant increase in BODIPY concentration was found in the proximal jejunal tissue corresponding to diet-restricted mice (fig. 5, m). Meanwhile, the inventors also examined the fluorescence intensity in the serum of mice after gastric lavage, and found that the serum BODIPY concentration of mice after diet restriction was significantly increased (fig. 5 n). The results of the detection of BODIPY concentrations in the intestinal tract and serum further demonstrate the enhancement of the intestinal lipid absorption capacity of mice. Taken together, these data demonstrate that enhanced intestinal lipid absorption results in obesity after dietary restriction.

To further understand how increased intestinal lipid absorption ultimately leads to rapid accumulation of fat following dietary restrictions, the inventors analyzed synthesis and catabolism in adipose tissue. The white adipose tissues of the inguinal and epididymis of the mice were collected 2 hours after the gastric lavage BODIPY fluorescence-labeled fatty acid and olive oil mixture. Fresh inguinal and epididymal white adipose tissues were taken and directly observed under a fluorescence microscope, and the fluorescence intensity of the tissues corresponding to the mice after diet restriction was found to be significantly enhanced (fig. 5 o). Subsequently, inguinal and epididymal white adipose tissues were frozen and sectioned, and a significant increase in fluorescence intensity was also seen in the white adipose tissue sections after diet restriction (fig. 5 p). Furthermore, the inventors treated inguinal and epididymal white adipose tissues with RIPA lysate, followed by centrifugation to clarify and examined fluorescence intensity, and as a result found that the BODIPY concentration of the corresponding tissues of diet-restricted mice was significantly increased (fig. 5 q). The results from BODIPY-labeled fatty acid lavage experiments demonstrate that refeeding after dietary restriction induces an increase in lipid uptake capacity of white adipose tissue. Next, the present inventors performed high-throughput RNA sequencing analysis on adipose tissue before and after dietary restriction, and as a result, found that the expression levels of Fabp5, acsl3, acsl1 and the like genes involved in fatty acid uptake in inguinal white adipose tissue were significantly increased after dietary restriction (fig. 5 r), and the differentially expressed genes related to the progress of fatty acid and triglyceride synthesis were almost all up-regulated (fig. 5 s), indicating that the anabolism of lipids was significantly increased in inguinal white adipose tissue. Meanwhile, the present inventors also found that the differentially expressed genes involved in fatty acid uptake were mostly up-regulated in epididymal white fat (fig. 8 a), and that the differentially expressed genes associated with the progress of fatty acid and triglyceride synthesis were mostly up-regulated (fig. 8 c), indicating that anabolism of lipids was also significantly increased in epididymal white adipose tissue. In the brown adipose tissue between the shoulder blades, the differentially expressed genes involved in fatty acid uptake were up-regulated and down-regulated (fig. 8 b), the differentially expressed genes involved in the synthesis process of fatty acids and triglycerides were up-regulated and down-regulated (fig. 8 d), but the gene expression of Acly, fasn, etc. directly involved in the synthesis process was significantly up-regulated. Analysis of genes involved in catabolism in inguinal white adipose tissue revealed a significant decrease in expression of pn pla2 among three genes of pn pla2, cope, mgll, which directly participated in lipolysis (fig. 5 t). The ATGL encoded by the Pnpla2 gene is the first step in catalyzing lipolysis, and is critical to lipolysis. A significant decrease in expression of Pnpla2 was also seen in epididymal white adipose tissue and brown adipose tissue between the shoulder blades (fig. 8e and f). The differentially expressed genes associated with fatty acid oxidation were up-regulated in inguinal white adipose tissue (fig. 5 t), and also were down-regulated in epididymal white adipose tissue (fig. 8 e), and were down-regulated in brown adipose tissue between shoulder blades (fig. 8 f). Brown adipose tissue is one of the sites of highest oxidation rate of body fatty acids. Thus, fatty acid oxidation of brown adipose tissue is more representative for adipose tissue. Furthermore, by monitoring the metabolism of mice, the inventors found that the Respiratory Exchange Rate (RER) levels of mice after dietary restriction were significantly increased (FIGS. 5u and v; FIGS. 8g and h), with high RER values representing the body predominantly consumed carbohydrates and low RER values representing the body predominantly consumed fat, thus indicating a significant reduction in lipid oxidation in mice after dietary restriction. Taken together, these data demonstrate that increased lipid anabolism and decreased lipid catabolism in adipose tissue results in rapid accumulation of fat content following dietary restriction.

Example 3 re-feeding a high protein diet can prevent diet-restriction induced obesity

In order to find potential dietary intervention measures to prevent obesity occurrence after dietary restriction, the present inventors analyzed serum amino acid expression profiles before, during and after dietary restriction, and found that the concentration of essential amino acids (Essential amino acids, EAAs) other than methionine (also called methionine) was significantly increased during and after dietary restriction (fig. 9 a). In addition, among other semi-essential and non-essential amino acids, the concentrations of other amino acids besides glutamic acid and aspartic acid increased significantly after dietary restriction (fig. 10 a).

To further explore the potential effect of amino acids on obesity after dietary restriction, after dietary restriction was performed in such a way that mice were provided with 10%, 25%, 65% of food amounts for 3 consecutive days, respectively, the present inventors provided mice with a high protein diet (HP), a low protein diet (LP), and a normal protein diet (np+eaa) supplemented with essential amino acids, with the normal protein diet (NP) as a control; the method comprises the following steps: NP,20% normal protein diet; HP,60% high protein diet; LP,5% low protein diet; np+eaa,20% normal protein+essential amino acid diet. More specific formulations are shown in Table 2.

TABLE 2 dietary composition

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As shown in fig. 9b, both of these three modes of dietary intervention prevented obesity from occurring after dietary restriction, and particularly high protein diets could even maintain the diet-induced fat-content-reducing effects. At the same time, these three modes of dietary intervention significantly inhibited the increase in body fat percentage and decrease in lean body mass percentage compared to the normal protein diet following dietary restriction (fig. 10 b), wherein the high protein diet maintained the effects of diet-restricted induced decrease in body fat percentage and increase in lean body mass percentage.

To further demonstrate the effect of high protein dietary intervention following dietary restriction on fat content, mice following normal protein or high protein diet following dietary restriction in a manner of 10% -25% -65% were sacrificed and dissected at specific time points and tissues of interest were collected. The inventors found that the size and weight of inguinal white adipose tissue of mice fed high protein diet after dietary restriction was significantly lower than that of mice fed normal protein diet (FIGS. 9c and d; FIG. 10 c). Likewise, mice on a high protein diet following dietary restriction also had significantly lower epididymal white adipose tissue size and weight than mice fed normal protein feed (fig. 10d and e). The results of hematoxylin eosin (H & E) staining showed that feeding high protein feed significantly inhibited the increase in adipocyte size in the inguinal and epididymal white adipose tissue of mice compared to mice fed normal protein feed following dietary restriction (FIGS. 9E and f; FIGS. 10f and g).

In addition, the inventors found that the enlargement of the morphology of the brown adipose tissue between the shoulder blades of the mice after refeeding was significantly inhibited by the high protein diet (fig. 11 a), the corresponding increase in tissue weight was also significantly inhibited (fig. 11 b), and the result of hematoxylin eosin staining showed that the high protein diet interfered with no large lipid accumulation in the adipocytes of the brown adipose tissue between the shoulder blades of the mice (fig. 11 c), and thus the high protein diet after diet restriction did not induce hypertrophy of the brown adipose tissue between the shoulder blades. Slicing and hematoxylin eosin staining of the liver of mice fed normal protein and high protein feed following dietary restriction, the inventors found that the liver cells of mice fed high protein feed did not show large lipid droplet accumulation (fig. 11 d). Further, the present inventors examined the concentration of liver Triglyceride (TG), and found that the high protein diet after diet restriction significantly suppressed the increase in the concentration of liver triglyceride (fig. 11 e), and thus the high protein diet after diet restriction did not induce the occurrence of liver steatosis in mice.

To further confirm the inhibitory effect of the high protein diet on body fat rebound after dietary restriction, the present inventors provided the high protein diet to mice after dietary restriction in such a manner that the mice were provided with 33.3% of food amount per day for 3 days or 66.7% of food amount per day for 12 days. As expected by the present inventors, the high protein diet after both of these dietary restrictions prevented the occurrence of obesity and maintained the effect of reduced fat content induced by the dietary restrictions (fig. 9g and h). The high protein diet after both dietary restrictions also maintained the effects of percent body fat reduction and percent lean body mass elevation (figures 12a and b). Furthermore, to explore the effects of refeeding after dietary restriction on female mice and the intervention of high protein diet, diet restriction experiments were performed with female mice provided with 10%, 25%, 65% diet, respectively, within three days, after which the female mice were provided with normal protein or high protein diet, and it was found that refeeding after dietary restriction induced rapid accumulation of fat in female mice, whereas high protein diet could prevent occurrence of female mice obesity (fig. 9 i). Also, by analysis, the inventors found that refeeding after diet restriction significantly increased body fat percentage, decreased lean body mass percentage of female mice, whereas high protein diets could partially maintain the diet-induced body fat percentage decrease and lean body mass percentage increase effects of female mice (fig. 12 c).

Example 4 high protein diet after dietary restriction reduced intestinal lipid absorption and improved adipose tissue lipid metabolism

To investigate how a diet-restricted high protein diet prevents the development of obesity, the inventors first analyzed the energy expenditure of mice. As shown in fig. 13a and b, after diet restriction in such a way that 10%, 25%, 65% of the food amount was provided within three days, respectively, mice with high protein diet intervention had significantly lower energy consumption than mice with normal protein diet. This suggests that high protein diets do not act by increasing energy expenditure on obesity inhibition following dietary restriction.

Next, the present inventors investigated whether ingestion of mice was involved in inhibition of obesity after dietary restriction by high protein diets. As shown in fig. 13c, the cumulative food intake was significantly reduced in mice on high protein diets compared to mice on normal protein diets following any feeding and diet restriction in the 10% -25% -65% regimen. Likewise, providing a high protein diet significantly reduced the cumulative food intake of mice after dietary restriction in a manner that provided 33.3% food amount per day for 3 days and 66.7% food amount per day for 12 days (fig. 14a and b). In addition, the inventors found that feeding high protein feed after dietary restriction could also significantly reduce the cumulative intake of female mice (fig. 14 c). Still further, the daily intake of mice on the first few days after diet restriction was significantly reduced compared to the daily intake of mice on normal protein diet after diet restriction, and the daily intake of mice on any diet was close (FIG. 13d; FIG. 14 d). As shown in fig. 5d and e and fig. 7i and j, feeding 100% of the diet to mice after diet restriction also induced rapid accumulation of fat. Thus, the relative decrease in the first few days of feeding with high protein feed following a dietary restriction is not a major cause of obesity following a dietary restriction.

To further investigate how high protein dietary intervention prevented obesity from occurring after dietary restriction, the present inventors analyzed whether lipid absorption in the small intestine was involved. The inventors found that the concentration of fecal triglycerides in mice fed high protein diet after dietary restriction was significantly higher than in mice fed normal protein diet after dietary restriction (fig. 13 e), suggesting that high protein dietary intervention would result in reduced intestinal lipid absorption. To more clearly demonstrate the lipid absorption of the small intestine, the inventors perfused mice with a mixture of BODIPY fluorescent-labeled fatty acids and olive oil, and found that the levels of BODIPY in the feces of mice on a high protein diet following dietary restriction were significantly higher than those of mice on a normal protein diet following dietary restriction (fig. 13 f). Fresh proximal jejunal tissue after 2 hours of gastric lavage was directly observed under a fluorescence microscope and found to have significantly reduced fluorescence intensity in tissues corresponding to high protein diet intervention group mice (fig. 13 g). The fluorescence intensity of frozen sections of proximal jejunum villus from high protein diet-interfered group mice was also significantly reduced (fig. 13 h). In addition, the quantitative detection result of the fluorescence intensity of the tissues also showed that the proximal jejunal tissue BODIPY level of the mice of the high protein diet intervention group was significantly reduced (fig. 13 i), while the inventors found that the serum BODIPY level of the mice of the high protein diet intervention group was also significantly reduced (fig. 13 j). These results demonstrate that reduced intestinal lipid absorption is responsible for the high protein dietary intervention preventing obesity from occurring after dietary restriction.

To further understand how the reduced intestinal lipid absorption ultimately inhibits fat accumulation following dietary restrictions, the inventors analyzed lipid anabolism and catabolism in adipose tissue. The white adipose tissue in the groin and epididymis of mice after 2 hours of the gastric lavage BODIPY fluorescent-labeled fatty acid and olive oil mixture was collected and observed under a fluorescence microscope, and the tissue fluorescence intensity of the high protein diet intervention group after diet restriction was found to be significantly lower than that of the normal protein diet group after diet restriction (fig. 13 k). Fluorescence intensity of frozen sections of inguinal and epididymal white adipose tissue of high protein diet intervention group was also significantly reduced (fig. 13 l). Furthermore, by quantifying the fluorescence intensity of the tissues, the inguinal and epididymal white adipose tissue BODIPY levels of the high protein diet intervention group after diet restriction were found to be significantly lower than that of the normal protein diet group after diet restriction (fig. 13 m). The results from BODIPY-labeled fatty acid lavage experiments demonstrate that high protein dietary intervention following dietary restriction inhibits the lipid uptake capacity of white adipose tissue. Further, the present inventors performed high-throughput RNA sequencing analysis of adipose tissues of mice on normal protein or high protein diets before and after dietary restriction, and as a result, found that in the tissues of mice on normal protein diets after dietary restriction, there were down-regulated and up-regulated by high protein diet intervention among those differentially expressed genes involved in fatty acid intake (FIG. 13 n). Differentially expressed genes associated with fatty acid and triglyceride synthesis processes were mostly down-regulated by high protein dietary intervention, such as Acly, fasn, etc. directly involved in the synthesis process (fig. 13 o). High protein dietary intervention significantly upregulated the expression level of the key gene Pnpla2 for lipolysis and of the partial gene for fatty acid oxidation (fig. 13 p). Furthermore, by monitoring the metabolic status of mice, the inventors found that the Respiratory Exchange Rate (RER) level of mice in the high protein diet intervention group was significantly reduced, indicating that the administration of high protein diet after diet restriction significantly increased lipid oxidation (fig. 13q and r). These data demonstrate that high protein dietary intervention reduces lipid anabolism, increases lipid catabolism and thus prevents obesity from occurring after dietary restrictions.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A method of controlling body weight and lipid biosynthesis comprising:

(1) Reducing food intake, significantly reducing body weight and lipid;

(2) Restoring food intake, which is a high protein content food, to ingest a food having a regulated protein content, thereby controlling body weight and lipid over-synthesis; the protein is casein; the high protein content food also comprises cysteine, carbohydrate and fat;

in the high-protein-content food, the protein content is 400-800 parts by weight, the cysteine content is 5-20 parts by weight, the carbohydrate content is 150-350 parts by weight, and the fat content is 50-90 parts by weight;

the method of controlling body weight and lipid biosynthesis is a non-therapeutic method.

2. The method of claim 1, wherein the high protein content food comprises 450 to 700 parts by weight protein.

3. The method according to claim 1, wherein the high protein content food has a cysteine content of 8 to 20 parts by weight.

4. The method of claim 1, wherein the high protein content food has a carbohydrate content of 180 to 300 parts by weight.

5. The method according to claim 1, wherein the high protein content food has a fat content of 60 to 80 parts by weight.

6. The method of claim 1, wherein the high protein content food further comprises: cellulose, minerals, vitamins; the cellulose content is 30-70 parts by weight; the mineral content is 15-55 weight parts; the vitamin content is 8-18 parts by weight.

7. The method according to claim 6, wherein the high protein content food has a cellulose content of 40 to 60 parts by weight; the mineral content is 25-45 weight parts; the vitamin content is 10-16 parts by weight.

8. The method of claim 1, wherein said reducing food intake comprises: regular diet, intermittent diet, time-limited diet, low-energy diet simulating diet, gradient-increasing or gradient-decreasing diet.

9. A method of controlling weight, comprising:

(1) Reducing food intake, significantly reducing body weight and lipid;

(2) Restoring food intake, which is a high amino acid content food, to intake a food with a regulated amino acid content, thereby controlling body weight; the high amino acid content food also comprises cysteine, carbohydrate and fat;

the high amino acid content food is a food in which an effective amount of amino acids is added to a normal food; the amino acid is: 16-36 parts of valine, 12-32 parts of isoleucine, 23-43 parts of leucine, 5-15 parts of methionine, 8-28 parts of phenylalanine, 2-8 parts of tryptophan, 10-27 parts of threonine, 20-40 parts of lysine and 5-18 parts of histidine;

in the high amino acid content food, the carbohydrate content is 150-350 parts by weight, and the fat content is 50-90 parts by weight;

the method of controlling body weight is a non-therapeutic method.

10. The method of claim 9, wherein, among the added amino acids:

the valine content is 21-31 parts by weight;

the isoleucine content is 17-27 parts by weight;

The leucine content is 28-38 parts by weight;

the methionine content is 8-12 parts by weight;

13-23 parts by weight of phenylalanine;

the tryptophan content is 3-6 parts by weight;

the threonine content is 15-23 parts by weight;

the lysine content is 25-35 parts by weight;

the histidine content is 5-18 parts by weight.

11. The method of claim 9, wherein said reducing food intake comprises: regular diet, intermittent diet, time-limited diet, low-energy diet simulating diet, gradient-increasing or gradient-decreasing diet.

12. Use of a composition for the preparation of a food for controlling body weight and lipid excess synthesis, said composition being a high protein content food; the protein is casein; the high protein content food also comprises cysteine, carbohydrate and fat;

in the high-protein-content food, the protein content is 400-800 parts by weight, the cysteine content is 5-20 parts by weight, the carbohydrate content is 150-350 parts by weight, and the fat content is 50-90 parts by weight.

13. The use according to claim 12, wherein the high protein content food comprises 450 to 700 parts by weight of protein.

14. The use according to claim 12, wherein the high protein content food has a cysteine content of 8 to 20 parts by weight.

15. The use according to claim 12, wherein the carbohydrate content of the high protein content food is 180 to 300 parts by weight.

16. The use according to claim 12, wherein in the high protein content food, further comprising: cellulose, minerals, vitamins;

the cellulose content is 30-70 parts by weight;

the mineral content is 15-55 weight parts;

the vitamin content is 8-18 parts by weight.

17. Use of a composition for the preparation of a food for controlling body weight and lipid excess synthesis, said composition being a high amino acid content food; the high amino acid content food also comprises cysteine, carbohydrate and fat;

the high amino acid content food is a food in which an effective amount of amino acids is added to a normal food; the amino acids include: 16-36 parts of valine, 12-32 parts of isoleucine, 23-43 parts of leucine, 5-15 parts of methionine, 8-28 parts of phenylalanine, 2-8 parts of tryptophan, 10-27 parts of threonine, 20-40 parts of lysine and 5-18 parts of histidine;

In the high amino acid content food, the carbohydrate content is 150-350 parts by weight, and the fat content is 50-90 parts by weight.

18. The use according to claim 17, wherein, among the amino acids,