The Metabolite β-Hydroxybutyrate of Lactobacillus Plantarum YZX21 Improves Type 2 Diabetes By Promoting Intestinal Secretion of GLP-1

doi:10.21203/rs.3.rs-850464/v1

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The Metabolite β-Hydroxybutyrate of Lactobacillus Plantarum YZX21 Improves Type 2 Diabetes By Promoting Intestinal Secretion of GLP-1

https://doi.org/10.21203/rs.3.rs-850464/v1

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Background: Probiotics and their metabolites regulate type 2 diabetes mellitus (T2DM) by promoting GLP-1 secretion, but the mechanism has not yet been fully clarified.

Results: In order to reveal the effect of Lactobacillus plantarum（L. plantarum）YZX21 on the regulation of T2DM, type 2 diabetic C57BL/6 mice induced by a high-fat diet and streptozotocin (STZ) were divided into different groups and were daily treated with L. plantarum YZX21 for 8 weeks. We identified that L.plantarum YZX21 reduced blood glucose, insulin levels and HOME-IR. Histopathology was found that L.plantarum YZX21 restored the mouse islet cells morphology and increased insulin secretion. The Elisa and immumohistochemical staining revealed concentration of glucagon-like peptide-1 (GLP-1) was increased in the mice colon. UPLC-MS/MS based widely-targeted metabolomics analysis was used to identify the differential intestinal metabolites in the mice colon. It was found that the metabolite β-Hydroxybutyrate(BHB) of L.plantarum YZX21 had a negative associated with T2DM. Subsequent, type 2 diabetic C57BL/6 mice was established and used to verify hpyerglycemic effest by daily treated with BHB for 8 weeks. It was found that BHB improved pathoglycemia, insulin resistance and increased the intestinal GLP-1 levels, especially reduced free fatty acid (FFA) levels. The expression of receptors G protein-coupled receptor (GPR) was verified by RT-PCR. The GPR109a receptor was significantly stimulated which was up-regulated the expression of GLP-1, but not GPR41 and GPR43 in the mice colon.

Conclusions: We found that the hypoglycemic effect of L.plantarum YZX21 was demonstrated by increasing the intestinal GLP-1 levels in the diabetic mice. Through the widely-targeted metabolomics, we identify the close correlation between serum concentrations of BHB and T2DM. In T2DM mice model, BHB reduced the FFA levels and increased the intestinal GLP-1 levels, which is associated with the downregulated expression of GPR109a. These results demonstrated that L.plantarum YZX21 alleviated T2DM by upregulating BHB/GPR109a/GLP-1 associated pathway.

General Microbiology

Probiotics

Glucose Homeostasis

Metabolomics

GLP-1 Secretion

Insulin Resistance

Type 2 diabetes mellitus (T2DM) was the third independent risk factor for premature mortality and disability in individuals [1]. Previous studies showed that hormone glucagon like peptide-1 (GLP-1) secreted by intestinal L cells stimulated insulin secretion and controlled blood glucose stability [2]. Therefore, increasing the production and secretion of endogenous GLP-1 had been widely concerned [3].

It is reported that probiotics can colonize in the intestine to regulate blood glucose by increasing the secretion of GLP-1 [4, 5]. The probiotics regulated the expression of GLP-1 through metabolites such as bile acids, short chain fatty acids and tryptophan derivatives [6]. However, there was no direct correlation between these metabolites and FFA concentrations. It was report that prolonged FFA stimulation or long-term high-fat diet leaded to impaired secretion of GLP-1 [7]. Since more than half of insulin secretion after eating is induced by GLP-1, impaired GLP-1 secretion may be an important mechanism of lipotoxicity causing blood glucose disorders [8, 9]. However, the mechanism which probiotics regulated FFA to increasing secretion of GLP-1 was not fully understood.

Metabolomics has been widely used in research related to disease diagnosis, as well as the exploration of drug action mechanisms and new biomarkers [10]. Widely-targeted metabolomics can reflect the metabolic pathways and be commonly used in research on the response mechanisms of different diseases and microorganisms [11]. With the help of Metabolomics, it was expected to reveal the mechanism of probiotics regulating GLP-1.

This paper used T2DM mice model to study the effects of Lactobacillus plantarum (L.plantarum)YZX21 on the glucose homeostasis as well as the secretion of intestinal GLP-1. And through the widely-targeted metabolomic analyses was to identify the target metabolites that stimulate GLP-1 secretion and reveal the regulatory mechanism of probiotics.

The effect of L.plantarum YZX21 on body weight, food intake and blood glucose of mice

From the fifth week, diabetic mice showed typical symptoms of diabetes. The body weight and food intake of mice results are shown in Fig. 1A and 1B. Compared with control group, all diabetic mice showed significant weight loss from fifth week to twelfth week (p < 0.05). After supplementing with L.plantarum YZX21, the weight of diabetic mice recovered significantly from 20.34g to 24.24g, which was not significantly different from the M group. The food intake of diabetic mice increased significantly from fifth week to twelfth week. After the supplement of L.plantarum YZX21, the diet of mice was relieved from 13.28g to 7.03g (p < 0.05). The FBG and PBG levels are shown in Fig. 1C and 1D. Compared with control group, the FBG levels of diabetic mice were higher than 7.0 mmol/L and the PBG levels were higher than 11.1mmol/L. The FBG of mice in the L.plantarum YZX21 group decreased from 12.26 mmol/L to 7.13 mmol/L (p < 0.05). The PBG levels was significantly reduced, from 24.68 mmol/L to 12.95 mmol/L (p < 0.05). The OGTT results are shown in Fig. 1E. Compared with control group, the curve area was almost the 3 times larger than the control group. In the L.plantarum YZX21 supplement group, The area under the blood OGTT curve was significantly reduced, which was no different from the M group (Fig. 1F).

The effects of L.plantarum YZX21 on insulin secretion and pancreatic islets

The results of insulin intraperitoneal injection (ITT) are shown in Fig. 2A. Compared with control group, the area of AUCglucose was 3 times than the control group. The insulin sensitivity of mice in the L.plantarum YZX21 group was significantly improved, and the area of AUCglucose decreased significantly (Fig. 2B), down 2.4 times (p < 0.05). Figure 2C and D show the fasting insulin levels and HOMA-IR. Compared with the control group, the fasting insulin levels of diabetic mice were significantly higher, and the HOMA-IR were also at high levels. After L.plantarum YZX21 intervention, serum insulin levels were significantly reduced from 38.42 mmol/L to 24.14 mmol/L and the HOMA-IR was decreased from 17.60 to 7.65, which was no different from the M group. The effects of L.plantarum YZX21 on islet cells was further verified by histopathology. The histopathological staining results of the pancreas are shown in Fig. 2E. The pancreatic islet cells were round or oval, with clear cells, rich and uniform cytoplasm in control group. In diabetic mice, the area ratio of islets to pancreas decreased significantly, the cells showed irregular structures, and the volume of islet cells decreased. In the L.plantarum YZX21 group, some pancreatic islet cells shrank and stained very lightly, but the cell morphology was complete, and the morphology was close to normal pancreas. The results of immunofluorescence staining showed (Fig. 2F) that the insulin of the control group occupied most of the pancreatic islet area, and insulin secretion was normal. While the insulin signal of diabetic mice was greatly reduced and the glucagon signal was significantly increased. In the L.plantarum YZX21 group, insulin secretion was significantly increased, glucagon secretion was decreased, and the release pressure of islet hormone was significantly relieved.

The colonize site of L.plantarum YZX21 and its effects on the intestinal GLP-1 secretion

Through the fluorescence staining experiment (Fig. 3A), it was found that L.plantarum YZX21 gavaged for 6 h showed high fluorescence intensity in the colon, and at 12 h, the colon still maintained high fluorescence intensity. This suggested that L.plantarum YZX21 was likely to mainly colonize the colon. As shown in Fig. 3B, the number of GLP-1 positive cells in the colonic mucosa of diabetic mice decreased. While after supplementation with L.plantarum YZX21, the colonic GLP-1 positive cells increased significantly from 17 to 31 (Fig. 3C). Consistent with this finding, in diabetic mice, the content of GLP-1 in the colon was significantly reduced (Fig. 3D). After supplementing with L.plantarum YZX21, the content of GLP-1 was significantly increased from 4.23 pmol/L to 11.07 pmol/L. Further the GLP-1 regulator gene GCG and PC3 mRNA levels were revealed in Fig. 3E and F. It was found that the expressions of GCG and PC3 were significantly increased in the colon after supplement of L.plantarum YZX21, where the expression was increased both about 3 times.

Metabonomics analysis of intestinal metabolites of L.plantarum YZX21

A significant difference between the metabolites of the diabetes group and the L.plantarum YZX21 group was observed in the OPLS-DA score chart (Fig. 4A), which suggested that there was a significant difference between the intestinal metabolism profiles of the two groups. Based on the variable importance in the projection value (VIP) greater than 1, the fold change value than 2 and p-value less than 0.05, it revealed that there were 87 metabolites that are significantly different between the diabetes group and the L.plantarum YZX21 intervention group(Fig. 4B). Based on the VIP score and the fold change value, we listed the top 10 metabolites in Table S3. FFA was the most changed metabolite, which was significantly reduced in YZX21 group. Through human metabolomic database (HMDB) and association analysis of metabolites with type 2 diabetes (Fig. 4C), it is found that BHB was closely related to FFA metabolic pathway and T2DM. It was found that free fatty acids (FFA) were highly increased in D group, which was nearly 4 times that of the YZX21 group (Fig. 5D). And BHB of L.plantum YZX21 group increased significantly, which was more than 3.5 times higher than that of D group (Fig. 5E).

Effects of BHB supplement on blood glucose and pancreatic islets in type 2 diabetic mice

To further characterize the effects of BHB on T2DM and FFA, we measured effects of BHB supplement on diabetes-related indicators through the diabetic mice. The FBG levels of mice are shown in Fig. 5A. Compared with control group, the FBG levels of diabetic mice were higher than 7.0 mmol/L. The FBG of mice in the BHB group decreased from 9.72mmol/L to 7.52mmol/L (p < 0.05). The PBG levels were shown in Fig. 5B. The PBG levels of diabetic mice were higher than 11.1mmol/L. After supplementing BHB, the PBG decreased significantly, from 21.15mmol/L to 11.88mmol/L (p < 0.05). Fasting insulin levels and HOMA-IR are shown in Fig. 5C and D. Compared with the control group, the fasting insulin levels of diabetic mice were significantly higher, and the HOMA-IR levels were also at high levels. However BHB intervention significantly reduced the serum insulin levels from 37.67 mIU/L to 21.19 mIU/L and HOMA-IR from 16.57 to 7.32, which were no difference from the M group. The histopathology was shown in Fig. 5E. The islet cells in the control group were rich and uniform. In diabetic mice, the cells presented irregular cell structure. In the BHB group, except for occasional lymphocytes and hemorrhage, the morphology and structure of the islet cells were similar to normal pancreas. Immunofluorescence staining results (Fig. 5F) the insulin signal of diabetic mice was significantly decreased and glucagon signal was significantly increased. In the BHB treatment group, insulin secretion was significantly increased, glucagon secretion was decreased.

Effects of BHB on intestinal GLP-1 hormone

The number of GLP-1 positive cells in the colonic mucosa of diabetic mice was reduced. While the supplementation of BHB, the positive cells was restored to the level in control group (Fig. 6A and B). Intestinal GLP-1 levels were significantly reduced in diabetic mice (Fig. 6C) and increased significantly after BHB supplementation, from 3.98 to 7.61 pmol/L.

Effect of BHB on FFA and TG levels

The experimental results showed that the diabetic mice showed significant FFA metabolic disorder, and the serum FFA level was significantly increased, which was 2.73-fold that of the control group (Fig. 7A). Serum TG level was also significantly increased, 2.41 times that of control mice (Fig. 7B). After BHB intervention, FFA disorder of diabetic mice were significantly improved, FFA level decreased to 187.56 µmol/L, significantly lower than the diabetic group. And TG level decreased to 1.56 mmol/L, also lower than the diabetic group.

Effect of BHB on GPR109a, GPR41 and GPR43 in colon

In order to further explain the way BHB regulates FFA, fluorescence immunoassay was used to detect the expression of GPR109a in colon. The results in Fig. 8A showed that the expression of GPR109a in the colon of the diabetic mice were significantly reduced. After BHB intervention, its levels were significantly restored, and the fluorescence intensity was increased, and basically returned to a high level. GPR41 and GPR43 which highly expressed in colon tissue were also possibly receptors of BHB. Further testing the expression of other GPRs in the colon (Fig. 8B), it found that the diabetic group has a significant inhibiting effect on GPR41, GPR43 and GPR109a. After supplementation with BHB, it had a significant up-regulation effect on the expression of GPR109a, while the influence of GPR41 and GPR43 was not obvious.

Increased appetite and weight loss were the characteristics of T2DM [12]. After supplementation with L.plantarum YZX21, the weight of diabetic mice was restored, while food intake decreased. It was suggested that the glucose utilization was increased and glycogen and fat conversion was reduced. FBG and PBG were the criteria for diagnosing diabetes [13]. After supplementing L.plantarum YZX21, FBG and PBG were all relieved, and basically returned to normal levels. OGTT and its AUCglucose were the indicators of ability of pancreatic β-cells to regulate blood glucose [14]. The blood glucose of diabetic mice climbed rapidly and decreased slowly. The area of AUCglucose of diabetes was significantly higher. However supplementing L.plantarum YZX21, the peak blood glucose level decreased, and the blood glucose level dropped smoothly. This suggested that after supplementing with L.plantarum YZX21, the ability of pancreatic β-cells to regulate blood glucose was significantly enhanced.

Insulin is the only hypoglycemic hormone secreted by β cells. The main reason for the increase blood glucose in T2DM is insulin parasecretion [15]. The ITT experiment reflected the sensitivity of insulin in body [16]. After supplementing L.plantarum YZX21, the insulin sensitivity of the mice was obviously restored, and the area of AUCglucose was reduced. Fasting insulin level and HOME-IR are other key indicators of the body’s sensitivity to insulin [17]. After supplementing L.plantarum YZX21, fasting insulin levels and HOME-IR were adjusted. These results suggested that L.plantarum YZX21 improved insulin sensitivity in T2DM mice. Histopathology also found the L.plantarum YZX21 supplement group reduced insulin resistance, protected the morphology of islet cells, restored insulin secretion, and inhibited insulin secretion. These results suggested that L.plantarum YZX21 reduced blood glucose levels in mice by improving insulin secretion. Improving insulin secretion and insulin sensitivity were the key of probiotics to play the role of hypoglycemia.

Through the fluorescence colonization experiment, we found that L.plantarum YZX21 reached the colon and maintained with a high intensity. This suggested that L.plantarum YZX21 was likely to colonize in colon, which were a large number of L cells. GLP-1 is the intestinal hormone that protects the morphology of pancreatic islets, increase insulin secretion, and reduce foreign insulin resistance [18]. We also found that the number of GLP-1 positive cells and the GLP-1 content increased significantly in the colon. This suggested that L.plantarum YZX21 was likely to increase and regulate the content of GLP-1 by colonizing the colon.

Intestinal metabolites are the key to the connection between strains and intestinal cells [19]. The increase or decrease of specific metabolites will affect the metabolism of the host [20, 21]. Specific metabolites are likely to be key components of hypoglycemic function. Through metabolomics techniques, we found that the FFA content in the intestine was significantly reduced in the L.plantarum YZX21 group. FFA has lipotoxic effects and is an important risk factor for diabetes[22]. As a kind of ketone body metabolite, BHB was closely related to metabolic diseases. In the L.plantarum YZX21 group, the BHB level increased significantly. The HMDB and correlation analysis suggested a close relationship between BHB and diabetes. However, the relationship between BHB, FFA and GLP-1 was not fully understood.

BHB was a common metabolite and the most important member of the ketone body family [23]. BHB was closely related to major diseases such as energy metabolism disorders in the organism and diabetes [24]. The relationship between BHB and GLP-1 had not been studied. In particular, whether probiotics could improve GLP-1 through BHB was still unclear. We constructed T2DM mice and gavage BHB to detect the mechanism and pathway of blood glucose regulation. After intragastric administration of BHB, the FBG and PBG of diabetic mice was decreased significantly and OGTT was improved. In the same way, insulin levels and HOME-IR in diabetic mice was also significantly improved. These results indicated that BHB intervention in type 2 diabetic mice reduced blood glucose by improving insulin resistance, which undoubtedly affects the process of T2DM. In addition, the results from pancreas histopathology and immunofluorescence analysis suggested that BHB contributes to increasing the insulin secretion, protecting islet cell morphology and inhibiting apoptosis. Hypoglycemic effects of BHB came down to increase the intestinal GLP-1 secretion. It was worth noting that BHB reduced TG and FFA levels, respectively. Metabolomics prompted that FFA was the greatest decrease in intestinal contents in diabetic mice. These results again suggested that BHB was likely to change intestinal GLP-1 content by influencing FFA content.

Circulating FFA is believed to play a causal role in the pathogenesis of liver and peripheral insulin resistance [25, 26] and is also a hallmark of T2DM [27]. Too much FFA aggravates insulin resistance, and is called lipotoxicity [28, 29]. Studies found that high levels of FFA inhibit GLP-1[30]. Inhibiting FFA levels can increase GLP-1 secretion, thereby improving T2DM [7, 31]. Studies have shown that BHB significantly improved tissue damage, metabolic disorders and protein metabolism [32]. As an endogenous signaling molecule, BHB binds to an activate the intestinal hydroxyl carboxylic acid receptor (HCA2) [33, 34], also known as GPR109a. BHB reduces the levels of FFAs and triglycerides in the circulation [35]. Our experiments found that after BHB intervention, serum FFA and TG levels were significantly reduced, while GLP-1 levels were significantly increased. The expression levels of its possible receptors GPR109a, GPR41 and GPR43 showed that GPR109a was mainly activated, but the effect on GPR41 and GPR43 was not obvious. Fluorescence histochemical experiments also found that BHB significantly increased the expression of GPR109a in the intestine. GPR109a in the intestine could inhibit the hydrolysis of TG into FFA and reduce its inhibitory effect on GLP-1 [36, 37]. Thereby BHB restored the secretion of GLP-1 by reducing the inhibition of FFA through activating GPR109a receptor.

In summary, through the widely-targeted metabolomic analyses of intestinal contents, we found that L.plantarum YZX21 increased the content of BHB in the intestine. As the endogenous ligand of GPR109a, BHB inhibited fat degradation and reduce FFA levels, thereby increasing GLP-1 secretion and regulating diabetes. These results provide a new theoretical basis for the use of L.plantarum YZX21 to intervene in T2DM.

Experimental strains

The L.plantarum YZX21 was stored in the Functional Dairy and Probiotic Engineering Laboratory of Ocean University of China (preservation number CCTCC M 2020829). The strain was activated in de Man, Rogosa and Sharpe (MRS) broth at 37 ℃ for 48 h for twice, then inoculated in MRS broth at an inoculum size of 2% (v/v) and incubated at 37℃ for 24 h. Following that, the bacteria were centrifuged at 4 ℃ and 5000 ×g for 5 min. Sediments washed twice with sterile PBS buffer and resuspended in PBS to a final concentration of 1×10⁸ CFU/mL, stored at 4 ℃ before use.

Animal experiment

Experimental animals and model construction

6-week-old SPF C57BL/6J male mice (weighing 18-20g, n=80) were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. The mice were fed in a single cage with normal or high-fat feed. The specific ingredients are shown in Table S1. The animal room had constant temperature and humidity (temperature 21-25°C, humidity 40%-60%, light from 8:00-20:00 for 12 h).

Before the experiment, the mice were adaptively fed in the animal room for 1 week. The control mice (n=8) was fed with normal diet and diabetic mice (n=24) was fed with high-fat diet for 4 weeks. After 4 weeks of dietary manipulation, the control mice were treated with 50 mmol/L citrate buffer (pH 4.5) by intraperitoneal injection. And diabetic mice were given intraperitoneal injection of Streptozotocin（STZ）at a dose of 50 mg/ (kg bw) [38]. After 7 days of STZ injection, the blood glucose of the mice was measured. The fasting blood glucose of the mice ≥7.0 mmol/L or the PBG level ≥11.1 mmol/L was considered as a successful model of diabetes[13].

Animal grouping and handling

L.plantarum YZX21 supplementing experiment of mice by gavage

Control group (C group): normal mice, gavage 0.25 ml 3% PBS solution; diabetic group (D group): diabetic mice, gavage 0.25 ml 3% PBS solution; Metformin group (M group): diabetic mice, gavage 0.25 ml of 10 mg/kg.bw metformin; L.plantarum YZX21 group (YZX21 group): diabetic mice, gavage 1×10⁸ CFU/mL.bw. The mice were given intragastric administration once daily from weeks 1 to 12.

β-Hydroxybutyrate supplementing experiment of mice by gavage

Control group (C group): normal mice, gavage 0.25 ml 3% PBS solution; diabetic group (D group): diabetic mice, gavage 0.25 ml 3% PBS solution; Metformin group (M group): diabetic mice, gavage 0.25 ml of 10 mg/kg.bw metformin; β-Hydroxybutyrate group (BHB group): diabetic mice, gavage 40mg/kg.bw [39]. The mice were given intragastric administration once daily from weeks 1 to 12.

Blood glucose testing

The blood was collected from the tail using a rapid blood glucose meter (Sannuo, China). Fasting blood glucose (FBG) was measured in mice overnight fasting for 12 h. Postprandial 2 h blood glucose (PBG) was measured in mice after eating for 2 h.

Oral glucose tolerance test (OGTT)

The mice were fasted overnight for 12 h. The initial blood glucose was measured (0 min), and then a 2 g/kg glucose solution was given to the stomach. The blood glucose values were measured at 30, 60, 90 and 120 min.

Samples collection

At the end of the experiment, the mice were fasted for 12 h and injected 100 mg/kg bw of ketamine into the intraperitoneal cavity after anesthesia. Blood was taken from the eyeballs, and then dislocated and sacrificed. The blood was centrifuged for 10 min at 3000×g at 4°C, and then the supernatant liquid was drawn and frozen at -80°C. The collected pancreatic tissues were fixed in 4% formaldehyde solution. The liver, intestinal tissue and intestinal contents were collect and freeze it at -80°C.

GLP-1 content detection

The colon contents were added the sample diluent and centrifuged for 10 min at 3000×g at 4°C. The supernatant liquid was drawn and the GLP-1 level was detected by ELISA kit (Nanjing Jiancheng, China), according to the instructions.

Determination of colonization of probiotic agents in intestinal tract of mice

L.plantarum YZX21 was incubated with 1 mL FITC fluorescent dye with a concentration of 1 mg/mL for 30 min at 37°C, washed with PBS until the liquid had no color, and diluted to 1×10⁸ CFU/mL. The YZX21 group was gavaged the 0.5 mL of bacteria solution treated with dye. The mice were sacrificed at 1 h, 3 h, 6 h, and 12 h, and the entire digestive tract of the mice was taken and stored in liquid nitrogen. Intestinal tissue was photographed by IVIS Lumina XRMS Series III (PerkinElmer, Waltham, MA).

Widely-targeted metabolomics metabolic testing

The colon contents was thawed on ice. 50 mg of sample was taken and homogenized it with 500 ul of ice-cold methanol/water (70%, v/v). The sample was vortexed for 3 min, sonicated for 10 min in an ice water bath, and vortexed for 1 min. Then centrifuge it with 12,000×g at 4°C for 10 min.

The colon contents of the mice were analyzed using an LC-ESI-MS/MS system. The analytical conditions were as follows, UPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm*100 mm); column temperature, 40°C; flow rate, 0.4 mL/min; injection volume, 5μL [40]. LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). Metabolome measurements were carried out through a facility service at Metware Biotechnology Co., Ltd.

Determination of FFA in the colon

The contents of the mouse colon were taken, and a commercial kit (Solarbio, China) was used to detect the levels of FFA according to the instructions.

Histological analysis

HE staining: The mouse pancreas was fixed in tissue fixative for 24 h, and then stored in 70% ethanol solution. Part of the tissue was embedded in paraffin, and then stained with hematoxylin and eosin for histopathological analysis. The morphology was observed through a microscope (Olympus, Japan).

Immunofluorescence double labeling: The pancreas tissue was added insulin and glucagon primary antibody and incubated overnight at 4°C. DAPI was added to counter-stain the nucleus. The slides were mounted and sliced under a fluorescent microscope (NIKON ECLIPSE TI-SR, Japan) to observe and collect images.

Immunohistochemistry: The primary antibody GLP-1 was added to incubate overnight, and the secondary antibody was added to incubate at room temperature. The hematoxylin stained nuclei were thick, dehydrated, transparent, and mounted. The sections were observed under an upright microscope (NIKON ECLIPSE TI-SR, Japan) and the number of positive cells was recorded.

Detection of related genes in diabetic mice

Take the colon tissue of mice added Trizol (Invertrogen, USA) lysate to extract total RNA from the tissues. CDNA was synthesized using ReverTra Ace®qPCR RT Master Mix Reverse Transcription Kit (Invertrogen, USA). The primer forward and reverse sequences are shown in Table S2[41]. After adding SYBR Green (Invertrogen, USA), perform the reaction in a real-time PCR (Bio-Rad, USA). The relative levels of gene expression was detected by using the 2^-ΔΔCt method.

Statistical analysis

Data were presented as mean ± standard deviation. SPSS 21.0 and Prism 7.0 (GraphPad Software) were used for statistical analysis and figure drawing. One-way analysis of variance and Turkey’s multiple comparison test were used to analyze the differences among the groups. p < 0.05 was considered statistically significant.

Type 2 diabetes mellitus (T2DM), Lactobacillus plantarum（L. plantarum）, streptozotocin (STZ), Glucagon-like peptide-1 (GLP-1), β-Hydroxybutyrate(BHB), Free fatty acid (FFA), G protein-coupled receptor (GPR), Insulin intraperitoneal injection (ITT), Hydroxyl carboxylic acid receptor (HCA2), De Man, Rogosa and Sharpe (MRS), Fasting blood glucose (FBG), Postprandial 2 h blood glucose (PBG), Oral glucose tolerance test (OGTT)

Ethics approval and consent to participate

The experiment was carried out in strict accordance with the “British Animal (Scientific Procedure) Act of 1986” (PPL 70/7652) and was approved by the Ethics Committee of Ocean University of China (SPXY2020060503).

Consent for publication

Not applicable.

Availability of data and materials

The data reported in this paper have been deposited in Mendeley Data: DOI: 10.17632/3hj8cbj32y.1

Competing interests

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.

Funding

This work was supported by Shandong Taishan industry leading talent project（tscy2020xxxx）and the National Key R & D of China (2018YFC0311201).

Authors' contributions

Lanwei Zhang designed and guided the experiments; Zhe Zhang wrote the original draft; Zhe Zhang, Xi Liang and Jingjun Tong performed the experiments; Youyou Lv, Haiyan Lu, and Maozhen Zhao analyzed the data; Pimin Gong, Tongjie Liu and Huaxi Yi revised the manuscript.

Acknowledgements

We acknowledge the laboratory of functional dairy and probiotics engineering and marine bioactive substances that have contributed to the research results reported in this work.

Authors' information

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

Cho NH, Shaw JE, Karuranga S, Huang Y, Fernandes JDDR, Ohlrogge AW, Malanda B. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res Clin Pr. 2018;138:271–81.
Meloni AR, DeYoung MB, Lowe C, Parkes DG. GLP-1 receptor activated insulin secretion from pancreatic ss-cells: mechanism and glucose dependence. Diabetes Obes Metab. 2013;15(1):15–27.
Gribble FM, Reimann F. Enteroendocrine cells: chemosensors in the intestinal epithelium. Annu Rev Physiol. 2016;78:277–99.
Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature. 2006;444(7121):840–6.
Boden G. Effects of free fatty acids (FFA) on glucose metabolism: significance for insulin resistance and type 2 diabetes. Experimental clinical endocrinology diabetes. 2003;111(03):121–4.
Visconti A, Le Roy CI, Rosa F, Rossi N, Martin TC, Mohney RP, Li W, de Rinaldis E, Bell JT, Venter JC. Interplay between the human gut microbiome and host metabolism. Nature communications. 2019;10(1):1–10.
Bjørnshave A, Holst JJ, Hermansen K. A pre-meal of whey proteins induces differential effects on glucose and lipid metabolism in subjects with the metabolic syndrome: a randomised cross-over trial. Eur J Nutr. 2019;58(2):755–64.
Kasińska MA, Drzewoski J: Effectiveness of probiotics in type 2 diabetes: a meta-analysis. Polish Archives of Internal Medicine 2015, 125(11):803–813.
Lee E, Jung S-R, Lee S-Y, Lee N-K, Paik H-D, Lim S-I. Lactobacillus plantarum strain Ln4 attenuates diet-induced obesity, insulin resistance, and changes in hepatic mRNA levels associated with glucose and lipid metabolism. Nutrients. 2018;10(5):643.
Yang M, Lao L. Emerging applications of metabolomics in traditional chinese medicine treating hypertension: Biomarkers, pathways and more. Front Pharmacol. 2019;10:158.
Wu D, Shu T, Yang X, Song J-X, Zhang M, Yao C, Wen L, Huang M, Yu Y, Yang Q. Plasma Metabolomic and Lipidomic Alterations Associated with COVID-19. National Science Review. 2020;7(7):1157–68.
Nie Q, Hu J, Gao H, Fan L, Chen H, Nie S. Polysaccharide from Plantago asiatica L. attenuates hyperglycemia, hyperlipidemia and affects colon microbiota in type 2 diabetic rats. Food Hydrocolloids. 2017;86:34–42.
Qu L, Ren J, Huang L, Pang B, Liu X, Liu X, Li B, Shan Y. Antidiabetic Effects of Lactobacillus casei Fermented Yogurt through Reshaping Gut Microbiota Structure in Type 2 Diabetic Rats. J Agr Food Chem. 2018;66(48):12696–705.
Xu J, Liu T, Li Y, Yuan C, Ma H, Seeram NP, Liu F, Mu Y, Huang X, Li L: Hypoglycemic and hypolipidemic effects of triterpenoid-enriched Jamun (Eugenia jambolana Lam.) fruit extract in streptozotocin-induced type 1 diabetic mice. Food & Function 2018, 9(6):3330–3337.
Tahrani AA. Novel therapies in type 2 diabetes: insulin resistance. Practical Diabetes. 2017;34(5):161–6a.
Pei C, Zhang Q, Hui D, Liu X, Tian F, Zhao J, Chen Y, Hao Z, Wei C. Oral administration of Lactobacillus rhamnosus CCFM0528 improves glucose tolerance and cytokine secretion in high-fat-fed, streptozotocin-induced type 2 diabetic mice. J Funct Foods. 2014;10:318–26.
Dang F, Jiang Y, Pan R, Zhou Y, Wu S, Wang R, Zhuang K, Zhang W, Li T, Man C: Administration of Lactobacillus paracasei ameliorates type 2 diabetes in mice. Food & Function 2018, 9(7):3630–3639.
Liang YL, Khoshouei M, Glukhova A, Furness SGB, Wootten D. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor–Gs complex. Nature. 2018;555(7694):121–5.
Miani M, Le Naour J, Waeckel-Enée E, chand Verma S, Straube M, Emond P, Ryffel B, Van Endert P, Sokol H, Diana J. Gut microbiota-stimulated innate lymphoid cells support β-defensin 14 expression in pancreatic endocrine cells, preventing autoimmune diabetes. Cell Metab. 2018;28(4):557–72.
Wollam J, Riopel M, Xu Y-J, Johnson AM, Ofrecio JM, Ying W, El Ouarrat D, Chan LS, Han AW, Mahmood NA. Microbiota-produced N-Formyl Peptide fMLF promotes obesity-induced glucose intolerance. Diabetes. 2019;68(7):1415–26.
Quan L-H, Zhang C, Dong M, Jiang J, Xu H, Yan C, Liu X, Zhou H, Zhang H, Chen L. Myristoleic acid produced by enterococci reduces obesity through brown adipose tissue activation. Gut. 2020;69(7):1239–47.
Belfort R, Mandarino L, Kashyap S, Wirfel K, Pratipanawatr T, Berria R, DeFronzo RA, Cusi K. Dose-response effect of elevated plasma free fatty acid on insulin signaling. Diabetes. 2005;54(6):1640–8.
Miyamoto J, Ohue-Kitano R, Mukouyama H, Nishida A, Watanabe K, Igarashi M, Irie J, Tsujimoto G, Satoh-Asahara N, Itoh H: Ketone body receptor GPR43 regulates lipid metabolism under ketogenic conditions. Proceedings of the National Academy of Sciences 2019, 116(47):23813–23821.
Puchalska P, Crawford PA. Multi-dimensional roles of ketone bodies in fuel metabolism, signaling, and therapeutics. Cell Metabol. 2017;25(2):262–84.
Gormsen LC, Jessen N, Gjedsted J, Gjedde S, Nørrelund H, Lund S, Christiansen JS, Nielsen S, Schmitz O, Møller N. Dose-response effects of free fatty acids on glucose and lipid metabolism during somatostatin blockade of growth hormone and insulin in humans. The Journal of Clinical Endocrinology Metabolism. 2007;92(5):1834–42.
Randle P. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet. 1963;1:785–9.
Tolhurst G, Heffron H, Lam YS, Parker HE, Habib AM, Diakogiannaki E, Cameron J, Grosse J, Reimann F, Gribble FM. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein–coupled receptor FFAR2. Diabetes. 2012;61(2):364–71.
Zhang D-M, Jiao R-Q, Kong L-D. High dietary fructose: direct or indirect dangerous factors disturbing tissue and organ functions. Nutrients. 2017;9(4):335.
Rada P, González-Rodríguez Á, García-Monzón C, Valverde ÁM. Understanding lipotoxicity in NAFLD pathogenesis: is CD36 a key driver? Cell Death Dis. 2020;11(9):1–15.
Vestergaard ET, Hjelholt AJ, Kuhre RE, Møller N, Larraufie P, Gribble FM, Reimann F, Jessen N, Holst JJ, Jørgensen JOL. Acipimox acutely increases GLP-1 concentrations in overweight subjects and hypopituitary patients. The Journal of Clinical Endocrinology Metabolism. 2019;104(7):2581–92.
Ma N, Ma R, Tang K, Li X, He B: Roux-en-Y Gastric Bypass in Obese Diabetic Rats Promotes Autophagy to Improve Lipid Metabolism through mTOR/p70S6K Signaling Pathway. Journal of Diabetes Research 2020, 2020.
Rahman M, Muhammad S, Khan MA, Chen H, Ridder DA, Müller-Fielitz H, Pokorná B, Vollbrandt T, Stölting I, Nadrowitz R. The β-hydroxybutyrate receptor HCA 2 activates a neuroprotective subset of macrophages. Nature communications. 2014;5(1):1–11.
Tunaru S, Kero J, Schaub A, Wufka C, Blaukat A, Pfeffer K, Offermanns S. PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect. Nat Med. 2003;9(3):352–5.
Offermanns S. Hydroxy-carboxylic acid receptor actions in metabolism. Trends in Endocrinology Metabolism. 2017;28(3):227–36.
Haemmerle G, Zimmermann R, Hayn M, Theussl C, Waeg G, Wagner E, Sattler W, Magin TM, Wagner EF, Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis. J Biol Chem. 2002;277(7):4806–15.
Lee AK, Kim DH, Bang E, Choi YJ, Chung HY. beta-Hydroxybutyrate Suppresses Lipid Accumulation in Aged Liver through GPR109A-mediated Signaling. Aging Dis. 2020;11(4):777–90.
Koh A, De Vadder F, Kovatcheva-Datchary P, Bäckhed F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell. 2016;165(6):1332–45.
Li X, Wang E, Yin B, Fang D, Chen P, Wang G, Zhao J, Zhang H, Chen W. Effects of Lactobacillus casei CCFM419 on insulin resistance and gut microbiota in type 2 diabetic mice. Beneficial Microbes. 2017;8(3):421–32.
Nishitani S, Fukuhara A, Shin J, Okuno Y, Otsuki M, Shimomura I. Metabolomic and microarray analyses of adipose tissue of dapagliflozin-treated mice, and effects of 3-hydroxybutyrate on induction of adiponectin in adipocytes. Scientific reports. 2018;8(1):1–11.
Xiao Y, Li X, Zeng X, Wang H, Mai Q, Cheng Y, Li J, Tang L, Ding H. A low ω-6/ω-3 ratio high-fat diet improves rat metabolism via purine and tryptophan metabolism in the intestinal tract, while reversed by inulin. J Agr Food Chem. 2019;67(26):7315–24.
Hu E, Du H, Zhu X, Wang L, Shang S, Wu X, Lu H, Lu X. Beta-hydroxybutyrate promotes the expression of BDNF in hippocampal neurons under adequate glucose supply. Neuroscience. 2018;386:315–25.

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The Metabolite β-Hydroxybutyrate of Lactobacillus Plantarum YZX21 Improves Type 2 Diabetes By Promoting Intestinal Secretion of GLP-1

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