Enteric capsuled protein reduced food intake and inhibited high-fat diet-induced weight gain in mice

Abstract

To understand the mechanisms of food intake reduction after metabolic bariatric surgery, we investigated the potential antiobesity effects of undigested proteins delivered to the small intestine using enteric capsules. We utilized EUDRAGIT-coated capsules (enteric capsules) to deliver contents not into the stomach but into the small intestine. Wild-type mice were administered various proteins (soy, pea, chicken, or whey) in the enteric capsules, and the amount of food intake and weight gain by the high-fat diet were evaluated. Protein aggregation by heat treatment and vagal nerve ablation by capsaicin treatment were conducted to determine whether they affect food intake. We found that: (1) Single administration of less than 4 milligrams of soy protein in enteric capsules significantly reduced food intake. Similar effects were observed with other proteins. (2) Heat treatment increased the food intake reduction effect of whey protein with increasing levels of the enteric hormone PYY. Vagal nerve ablation by capsaicin abolished the effects of such food intake reduction. (3) Multiple administrations of soy protein in enteric capsules reduced body weight gain and liver triglyceride accumulation under high-fat diet conditions. We concluded that proteins delivered to the small intestine via enteric capsules reduced food intake and inhibited high-fat diet-induced weight gain in mice. The aggregation of protein and the capsaicin-sensitive vagal afferent nerve might play a role in this effect.

1. Introduction

Obesity is associated with several complications, such as type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, non-alcoholic fatty liver disease, and obstructive sleep apnea, and has become a major contributor to the global burden of chronic disease [1]. One of the most effective treatments for obesity is bariatric metabolic surgery, such as sleeve gastrectomy and Roux-en-Y gastric bypass. Several factors, including neurotransmitters such as Glucagon-like peptide-1 (GLP-1) and Peptide YY (PYY) [2], the vagal afferent nerve [3], and mechanical stress in the digestive tract [4], have been investigated for their role in reducing food intake after metabolic bariatric surgery.

Bariatric metabolic surgery influences nutrient digestion and absorption. Undigested (or not well-enough digested) nutrients reach the small intestine by accelerated gastric emptying after the surgery. However, in clinical situations, the administration of alpha-glucosidase inhibitors for carbohydrates or lipase inhibitors for fats has not been shown to suppress appetite or food intake.

Among the nutrients, the digestion of proteins is affected by gastric bypass surgery due to the accelerated gastric transit time and decreased gastric acid production, resulting in undigested (or not well-enough digested) proteins flowing into the small intestine. It is reported that protein-rich diets enhanced satiety and promoted weight loss via an increase in the anorectic hormone PYY [5], and peptone (protein hydrolysate) causes GLP-1 release from primary cultures of the small intestine of mice [6]. However, the direct delivery of undigested proteins into the small intestine without gastric bypass has not been investigated in vivo.

Here, we investigated whether undigested protein has food intake-reducing and antiobesity effects by utilizing “enteric capsules” coated with the functional polymer EUDRAGIT(R), which dissolves only at a certain pH and is often used for delayed release from capsules. This study may shed light on the influx of undigested proteins into the intestine, which is not only a possible mechanism for food intake reduction after bariatric surgery but also a new, useful candidate for weight reduction therapy.

2. Materials and Methods

2.1 Animals

Male C57BL/6J (wild-type) mice were purchased from CLEA Japan. Mice were housed in cages in a room at 22°C with a 12-hour light-dark cycle (lights off from 8:00 a.m. to 8:00 p.m.). This study was approved by the Ethics Review Committee for Animal Experimentation of Osaka University School of Medicine and followed the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health.

2.2 Preparation of protein in enteric capsules

Size M Gelatine capsules for mice were purchased from Torpac, Inc. (USA). Soy and whey protein were purchased from Nichie Inc. (Japan), and pea protein was purchased from JAY&CO Inc. (Japan). For heat-induced aggregation, the proteins were heated at 120°C for 20 minutes in a laboratory autoclave and were dried overnight in a desiccator with silica gel. Approximately 3 milligrams of each protein was packed into the capsule.

For enteric coating, we utilized Eudragit L100-55 (Evonic Industries) according to the manufacturer’s protocol. Briefly, a coating solution (19 g of Eudragit L100-55, 31.6 g of acetone, 47.5 g of isopropanol, and 1.9 g of polyethylene glycol) was prepared at the time of use. A capsule filled with protein was dipped in the coating solution and dried at room temperature. The same procedure was repeated to make a total of two coatings.

2.3 Evaluation of food intake after administration of protein in enteric capsules

For the single-administration experiment, each overnight-fasted mouse was orally administered one capsule at 9:00 a.m. The quantity of food intake was assessed by measuring the weight of the remaining food at 1, 2, 4, and 8 hours after administration. For the multiple-administration experiment, after 4 weeks of high-fat diet (Rodent Diet With 60 kcal% Fat, RESEARCH DIET, #D12492) feeding, each mouse was orally administered one capsule five times a week at 6:00 p.m. The body weight was measured before the administration. The mice were fed a high-fat diet ad libitum. On day 12 (a total of 10 times were administered), the mice were sacrificed. To measure peripheral enteric hormone levels, GLP-1 Active form Assay Kit (IBL, #27784), PYY ELISA Kit (RayBiotech, #EIA-PYY-1), and CCK ELISA Kit (RayBiotech, #EIA-CCK-1) were used according to the manufacturers’ instructions.

2.4 Capsaicin treatment

For the blockade of the vagal afferent nerve, capsaicin treatment was performed as described previously [7]. The composition of the capsaicin solution was 50 mg/mL capsaicin (Sigma-Aldrich, #M2028), 10% ethanol, and 10% Tween-20 in normal saline. Mice were anesthetized by inhalation of isoflurane followed by subcutaneous administration of 25 mg/kg capsaicin. This procedure was conducted for three consecutive days, and the food intake evaluation described above was performed 2 weeks after the capsaicin treatment.

2.5 Liver triglyceride assay

Approximately 100 mg of liver tissue was lysed with cell lysis buffer, and the supernatant was collected after centrifugation. Triglyceride concentrations were measured with a LabAssay^TM Triglyceride kit (Fujifilm Wako, #LABTRIG-M1). Total protein concentrations were measured using a protein assay BCA kit (Nacalai, #06385-00) and were used to adjust the quantity of triglyceride content.

2.6 Statistical analysis

Statistical analyses are performed using JMP Pro 17 (SAS Institute). P values less than 0.05 were considered as statistical significance. The results are indicated as the mean ± SEM unless otherwise specified.

3. Results

3.1 Single administration of protein in the enteric capsule

Fig. 1A, B depicts the protocols for a single administration of the encapsulated protein. An empty capsule was administered to the mice of the control group.

Fig. 1  Soy protein administration by the enteric capsule reduced food intake in mice.

(A) Time course of short-term administration of protein in enteric or non-enteric capsules. After fasting overnight, the wild-type mice were administered capsules. The quantity of food intake was evaluated at 1, 2, 4, and 8 hours after administration.

(B) Differences in protein release sites in capsules without (left) or with (right) enteric coating. The contents in non-coated capsules are released into the stomach (left), whereas the contents in EUDRAGIT-coated capsules (“enteric capsule”) are released in the small intestine (right).

(C, D) Comparison of food intake quantity after administration of non-coated capsules (C) or EUDRAGIT-coated capsules (D) containing nothing (control) or soy protein. All the data points are shown as scatter plots with mean ± standard error. ((C): control = 5, soy = 5. (D): Control = 9, soy = 11. *; p < 0.05, unpaired t-test)

First, we determined the effect of capsule coating with EUDRAGIT L100-55, a pH-dependent eluting polymer (Fig. 1A). The EUDRAGIT coating protects the capsule from being digested in the stomach. As shown in Fig. 1C, the mice administered protein in a non-coated capsule showed a similar or slightly higher quantity of food intake than the control mice administered an empty capsule. On the other hand, the mice administered less than 4 milligrams of soy protein in the EUDRAGIT-coated capsules showed a significant reduction of food intake at 1–2 hours, followed by a trend at 4–8 hours after administration (Fig. 1D).

Next, we examined whether other proteins in enteric capsules had a similar effect on food intake (Fig. 2). We found that not only soy (Fig. 1D) but also pea (Fig. 2A), chicken (Fig. 2B), and whey proteins (Fig. 2C) had similar effects on reducing food intake, although the effects varied depending on the protein.

Fig. 2  Reduction of food intake by the administration of various proteins in enteric capsules in mice (A–C) and the effect of heat aggregation (D) and the vagal nerve pathway (E).

(A–D) Comparison of food intake quantity after the administration of pea (A), chicken (B), whey (C), and heated whey (D) proteins in enteric capsules. The experiment was performed as shown in Fig. 1 (A).

(E) Comparison of food intake quantity after administration of heated whey protein after capsaicin treatment for vagal nerve blockade.

All the data points are shown as scatter plots with mean ± standard error. (A–D) Control = 5, protein = 5. (E) Control = 8, protein = 9. *; p < 0.05, **; p < 0.01, ***; p < 0.001, unpaired t-test

Among the proteins we used, soy and pea proteins were heat-treated because of their allergenicity. As heat treatment of proteins leads to denaturation and aggregation, we also performed heat treatment on whey protein (Fig. 2C, D). Compared with non-heated whey (Fig. 2C), heated whey protein in EUDRAGIT-coated capsules had a stronger and longer (up to 8 hours) reduction effect on food intake in the mice (Fig. 2D).

To investigate the mechanism of food intake reduction observed in this study, we tested the involvement of the capsaicin-sensitive vagal nerve pathway (Fig. 2E) and the enteric hormones GLP-1, PYY and CCK (Supplementary Fig. 1). The food intake reduction in the protein group was not observed after capsaicin treatment (Fig. 2E). The peripheral PYY levels were significantly higher in the protein group than in the control group, although the active concentrations of GLP-1 and CCK were not significantly changed by the single administration of protein in the enteric capsule (Supplementary Fig. 1A–D).

3.2 Multiple administrations of protein in enteric capsules

We next examined the effect of multiple administrations of soy protein in EUDRAGIT-coated (enteric) capsules. After 4 weeks of high-fat diet feeding, the mice were administered soy protein in enteric capsules on days 0, 1, 2, 3, 4, 7, 8, 9, 10, and 11 (Fig. 3A).

Fig. 3  Multiple administrations of soy protein in enteric capsules reduced body weight gain, liver weight, and liver triglyceride levels in mice.

(A) Time course of multiple administrations of the capsules. After being fed a high-fat diet for four weeks, the mice were administered soy protein in enteric capsules a total of 10 times at 6:00 p.m. (indicated by arrowheads) and were sacrificed on day 12.

(B, C) Comparison of (B) body weight (BW) and (C) BW changes from baseline between the control (con) and protein (pro) groups. As one mouse in the “protein” group died on day 6, it was omitted from further analysis.

(D, E) Comparison of the weights of (D) subcutaneous white adipose tissue (subWAT) and (E) epidydimal white adipose tissue (epiWAT) on day 12.

(F, G) Comparison of liver weight and triglyceride (TG) content in the liver on day 12.

All the data points are shown as scatter plots with mean ± standard error. (control = 6, protein = 5–6. *; p < 0.05, **; p < 0.01, ***; p < 0.001, unpaired t test. +: p < 0.05, mixed model ANOVA)

Body weight (Fig. 3B) and body weight changes (Fig. 3C) were significantly lower in the protein capsule group than in the empty capsule group. The weights of subcutaneous and epididymal white adipose tissue did not differ between the two groups (Fig. 3D, E). We found that the liver weight (Fig. 3F) was lower in the protein capsule group, and the quantity of triglyceride content in the liver was lower in the mice administered protein capsules (Fig. 3G).

4. Discussion

We demonstrated that EUDRAGIT-coated (enteric) capsules containing soy protein significantly reduced food intake in mice after overnight fasting, whereas the same amount of protein in non-coated capsules did not alter food intake (Fig. 1C, D). A similar food intake reduction by enteric encapsulation was observed for other proteins, such as peas, chicken, and whey (Fig. 2A–C). While whey protein showed smaller effects than the other proteins, the heating dramatically increased its efficacy at reducing food intake (Fig. 2D), suggesting that protein aggregation by heating or relatively high average molecular weights (soy (Fig. 1D) and pea (Fig. 2A)) might reduce food intake. Moreover, capsaicin pretreatment of the mice abolished the food intake reduction caused by soy protein in enteric capsules, suggesting the involvement of vagus nerve-mediated afferent inputs (Fig. 2E). Finally, two consecutive weeks of daily administration of soy protein in enteric capsules significantly reduced body weight gain and liver triglyceride accumulation under high-fat diet conditions (Fig. 3).

In this study, we found that protein that was not digested in the stomach reduced food intake in mice and that heat treatment of the protein enhanced the reduction in food intake. Heat treatment causes proteins to form high-molecular-weight aggregates that are resistant to digestive enzyme degradation [8]. Moreover, aggregated proteins have been reported to bind bile acids [9], which causes their pH to deviate slightly from the optimal pH values for digestive enzymes, which will decrease the activity of digestive enzymes and will alter the overall digestive state of proteins. Although the impacts on digestive enzymes and pH in the intestine were not fully determined in this study, the soy, pea, chicken, or heat-treated whey in the enteric capsules are likely to reach the distal intestine as peptides or larger polypeptides and contribute to reducing food intake.

High-protein meals have been shown to increase satiety and decrease food intake [10], improving both weight loss and weight loss maintenance [11, 12]. However, 65% of the total calories of the “high-protein diet” in their study were derived from protein, which is difficult to achieve in our daily diet, and these studies did not focus on the digestive status of nutrients in post-bariatric surgery patients or the quantity of protein administered to the subjects. In addition, the influx of the undigested protein into the intestine will occur in patients with chronic pancreatitis, celiac disease, hypochlorhydria (low stomach acid), and so on. These patients suffer from several symptoms, such as stomach pain, diarrhea, and heartburn, all of which impair their quality of life. In our study, the quantity of protein used in the single administration was less than 4 milligrams, which would have little effect on total calorie intake and would not induce severe adverse effects on the digestive tract, at least in mice.

Furthermore, our results suggested that the single administration of protein in enteric capsules may not only affect enteric hormones (especially PYY) but also capsaicin-sensitive vagal afferent nerve input to reduce food intake. On the other hand, it is reported that there are differences in the microbiome composition of obese, overweight, and lean subjects [13, 14], and that undigested nutrients affect the intestinal microbiota [15]. It has also been reported that rats consuming diets with resistant proteins presented increased cecal butyrate levels and decreased succinate levels, indicating alterations in the gut microbiota [16]. The microbiome may have been altered in the protein group and be one of the mechanisms responsible for the weight loss observed with the multiple administrations of protein in enteric capsules in this study.

Metabolic/bariatric surgery has been conducted worldwide; however, the medical indication for metabolic/bariatric surgery is strictly controlled by current guidelines worldwide [17], including in Japan [18]. Patients with eating disorders experience less postoperative weight loss and greater psychosocial distress after surgery [19]. We reported that the “emotional eating behavior” score (e.g., “Do you have the desire to eat when you are irritated?”) gradually returned to the preoperative level and was associated with poor weight loss [20]. Thus, patients with severe complications or mental health problems with eating disorders may be considered to have a high risk of surgery. Although GLP-1 analogs or GIP/GLP-1 agonists have been used for the treatment of morbid obesity in recent years, the costs of both surgery and analogs/agonists are too high to treat all patients with obesity worldwide. The materials that we used in this study were safe and cost-effective. The proteins in enteric capsules might provide a cost-effective means for larger populations.

There are several limitations in this study. First, compared with humans, mice have longer digestive tracts and increased food transit times. Additionally, mice and humans exhibit differences in feeding frequency (mice eat essentially throughout the day, whereas humans typically eat three times a day). These factors should be considered when assessing clinical applicability. How undigested proteins in the digestive tract are sensed and the molecular mechanism underlying food intake reduction need to be investigated further.

In conclusion, administration of protein in enteric capsules reduced food intake and inhibited high-fat diet-induced weight gain in mice (Graphical Abstract). Heat-induced aggregation of the protein and the capsaicin-sensitive vagal afferent nerve might play a role in this effect.

Graphical Abstract 

Data Availability

All the raw datasets required to reanalyze are available from the lead contact upon request.

CRediT Authorship Contribution Statement

KK: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing-original draft; SF: Funding acquisition, Methodology, Project administration, Writing-original draft, Writing-review & editing; SK: Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Writing-original draft, Writing-review & editing; HNa: Funding acquisition, Writing-review & editing; YF: Funding acquisition, Writing-review & editing; HNi: Funding acquisition, Writing-review & editing; IS: Funding acquisition, Writing-review & editing.

Acknowledgments

We thank all members of the Third Laboratory (Adiposcience Laboratory), Department of Metabolic Medicine, Osaka University, for helpful discussion of the project.

Disclosure

1. Funding

This work was supported by Grants-in-Aid for Scientific Research [JP22K08669 to SK, JP23K08006 to HNi], Grants-in-Aid for Early Career Scientists [JP21K16353 to YF, JP21K16340 to SF, JP23K19589 to HNa, and JP24K19305 to HNa], MSD Life Science Foundation, Public Interest Incorporated Foundation [to SF], Japan Diabetes Society Junior Scientist Development Grant supported by Novo Nordisk Pharma Ltd. [to SF], Lotte Research Promotion Grant, [to HNa], Joint Research Grants with Kowa Pharmaceutical [to IS], and the Uehara Memorial Foundation [to IS]. The funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.

2. Declaration of competing interest

The authors declare no relationships or activities to disclose that might bias this work.

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