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Microbiology and Biotechnology Letters

Research Article(보문)

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Food Microbiology (FM)  |  Food Borne Pathogens and Food Safety

Microbiol. Biotechnol. Lett. 2024; 52(3): 275-287

https://doi.org/10.48022/mbl.2407.07008

Received: July 11, 2024; Revised: August 8, 2024; Accepted: August 12, 2024

Reduction of Insulin Resistance by Momordica Charantia with Lactobacillus Acidophilus CBT-LA1 or Lactiplantibacillus Plantarum CBT-LP3 Improves Hepatosteatosis

Dong-Jin Kim, Ju Sung Lee, Seungwoo Kim, Sang Kyun Park, Yeo-Sang Yoon, Yougku Ryu, and Myung Jun Chung*

R&D Center, Cell Biotech, Co., Ltd., Gimpo 10003, Republic of Korea

Correspondence to :
Myung Jun Chung,          ceo@cellbiotech.com

Abstract

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Insulin resistance is a primary risk factor for developing diabetes. However, diabetes drugs generally focus on regulating and lowering patients’ blood glucose levels. In recent years, diverse materials have been evaluated to improve insulin resistance and hinder the development of diabetes. Momordica charantia extract (MCE) and lactic acid bacteria (LAB) have been considered as potential therapeutic agents against insulin resistance and hyperglycemia. In a streptozotocin-induced type 1 diabetes animal model, treatment with MCE and LAB had no effect on hyperglycemia. To evaluate the effect of MCE and LAB on insulin resistance, we chose a high-fat diet-induced insulin resistance model and co-administered MCE and Lactobacillus Acidophilus CBT-LA1, Lactiplantibacillus plantarum CBT-LP3, or Lacticaseibacillus rhamnosus CBT-LR5. MCE with CBT-LA1 or CBT-LP3 improved insulin resistance and hepatosteatosis. However, the effect of MCE and MCE with CBT-LR5 was weaker than the effect of MCE with CBT-LA1 or CBT-LP3. Momordica charantia induced insulin secretion from RIN-m5F in a dose-dependent manner. Interestingly, CBT-LA1 and CBT-LP3 enhanced the insulin secretion of MCE. These results suggest that the co-administration of MCE and a specific LAB is one approach for overcoming insulin resistance and hyperglycemia.

Keywords: Momordica charantia, lactic acid bacteria, insulin resistance, lipid accumulation

Graphical Abstract


Introduction

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Adopting a Western diet has been linked to an increase in diseases during adulthood, including obesity, hypertension, and diabetes [1]. Excessive intake of energy sources causes insulin resistance, which in turn acts as a risk factor for the development of type 2 diabetes (T2DM) [2]. One of the primary causes of insulin resistance is obesity-induced abnormal lipid accumulation and impaired insulin-mediated signaling pathways [3]. A state of insulin resistance increases the insulin requirement for glucose uptake, and more insulin is produced to regulate blood glucose levels [4]. However, insulin resistance persists for a long time and the balance between insulin and blood glucose levels can be disrupted; that situation can cause blood glucose levels to rise and eventually prompt the development of diabetes [5, 6]. Current blood glucose-lowering drugs can be divided into four types: directly injected insulin or the administration of an insulin substitute, directly or indirectly increasing insulin secretion from the pancreas, inhibiting the breakdown of disaccharides in the intestines to reduce sugar absorption, and preventing the reabsorption of glucose in the kidneys [7, 8]. However, such drugs are targeted at diabetics and are not used by individuals with insulin resistance [7, 8]. Given that improving insulin resistance is one possible approach to reducing the risk of developing diabetes, various studies have identified compounds that reduce insulin resistance [9]. Here, we focus on two of those substances: Momordica charantia (MC) and lactic acid bacteria (LAB).

Momordica charantia is a plant commonly used by indigenous populations in Asia, South America, India, the Caribbean, and East Africa to help regulate blood glucose levels [10]. Lactic acid bacteria have also been suggested to reduce diabetes symptoms in some previous studies [11−13]. The combination of MC and LAB was recently noted to increase anti-diabetic effects; researchers concluded that the efficacy of MC was increased via the fermentation process by LAB [14−16].

In this study, we hypothesized that the co-administration of MC and LAB without fermentation may enhance the effects of MC and LAB on insulin resistance. We accordingly co-administered MC water extract (MCE) and LAB without fermentation to a high-fat diet (HFD)-induced insulin-resistance model and evaluated the synergic effects of these compounds on insulin resistance and lipid metabolism.

Materials and Methods

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Momordica charantia extract and lactic acid bacteria

We purchased MCE from FineFT Co. Ltd., (Republic of Korea). The information of MCE extraction method was offered from FineFT Co. Ltd. Deionized water was mixed equivalent to sixteen times the weight of MC and performed circular extraction at 90℃ for 10 h. The primary extract was filtered by using a 1 um cartridge filter and then concentrated using a continuous decompression concentrator (65 ± 5℃, -700 mmHg, 10.0 Brix%). Afterwards four parts of extract and six parts of malto-dextrin were mixed and sterilized at 80−85℃ for 30 min. To obtain final extract, spray-dry was performed under the following condition (In let: 185 ± 5℃, Out let: 100 ± 5 ℃, nozzle pressure: 70 ± 10 bar). The strains Lactobacillus acidophilus (CBT-LA1), Lactiplantibacillus plantarum (CBT-LP3), and Lacticaseibacillus rhamnosus (CBT-LR5) were from Cell Biotech Co. Ltd., (Republic of Korea); information about these LAB was included in one of our previous studies [17].

Experimental design of streptozotocin-induced Type I diabetes mellitus model

Seven-week-old C57BL6 mice were purchased from Saeron Bio (Republic of Korea) and allowed to adjust to their surroundings for one week. To induce a type 1 diabetes mellitus model, mice were administrated 50 mg/kg of streptozotocin (STZ, Sigma, USA) for five consecutive days. After two weeks of STZ treatment, we checked the animals’ blood glucose levels and HbA1c. The mice were then divided into seven groups: (1) non-diabetic controls (G1, NC, n = 7), (2) diabetic controls (G2, STZ + phosphate buffered saline (PBS), n = 8), (3) diabetic mice with MCE (G3, STZ + MCE, n = 9), (4) diabetic mice with a combination of MCE and CBT-LA1 (G4, STZ + LA1 + MCE, n = 6), (5) diabetic mice with a combination of MCE and CBT-LP3 (G5, STZ + LP3 + MCE, n = 7), (6) diabetic mice with a combination of MCE and CBT-LR5 (G6, STZ + LR5 + MCE, n = 7), and (7) diabetic mice with glibenclamide (Sigma), a known insulin agonist (G7, STZ + Gli, n = 8) [18]. Diabetic mice had blood glucose levels above 250 mg/dl and HbA1c above 6.5%. CBT-LA1, CBT-LP3, or CBT-LR5 was pretreated for 2 weeks to confirm the effect of LAB on glycemic control and body weight and then MCE with or without LAB was treated for 4 weeks. At the end of the treatment cycle, HbA1c was checked.

Experimental design of high fat diet-induced insulin resistance model

Five-week-old C57BL6 mice were purchased from Saeron Bio and allowed to adjust to their surroundings for one week. The animals were pretreated with a 45%HFD (D12451, Research diet Inc., USA) for 8 weeks to induce insulin resistance, and a glucose tolerance test (GTT) was then performed 4 and 8 weeks after pretreatment. Eight weeks after HFD treatment, the mice were divided into seven groups: (1) normal diet-fed controls (G1, ND, n = 10), (2) HFD-fed controls (G2, HFD + PBS, n = 10), (3) HFD-fed mice with MCE (G3, HFD + MCE, n = 10), (4) HFD-fed mice with a combination of MCE and CBT-LA1 (G4, HFD + LA1 + MCE, n = 9), (5) HFD-fed mice with a combination of MCE and CBT-LP3 (G5, HFD + LP3 + MCE, n = 10), (6) HFD-fed mice with a combination of MCE and CBT-LR5 (G6, HFD + LR5 + MCE, n = 10), and (7) HFD-fed mice with glibenclamide (G7, HFD + Gli, n = 10). CBT-LA1, CBT-LP3, or CBT-LR5 was pretreated for 2 weeks and then MCE—with or without LAB—and glibenclamide were orally administrated five times per week. A GTT was performed at 4, 8, and 12 weeks after MCE and LAB treatment. The mice were sacrificed at 12 weeks after the MCE and LAB treatment.

Experimental design for insulin induction by MCE and LAB

To demonstrate the induction of insulin by MCE and MCE plus LAB, it was administrated to eight-week-old C57BL6 mice at one day before and on the day of blood collection. On the day of blood collection, mice were fasted for 6 h and then administrated MCE or MCE plus LAB (n = 6). At 15 min after administration, blood was collected from the inferior vena cava. The levels of serum insulin were measured by ALPCO mouse ultrasensitive insulin ELISA (ALPCO, USA) according to the user manual.

Animal care and handling

The laboratory environment, a specific pathogen-free facility, was maintained at a temperature of 22 ± 2℃with a relative humidity of 40 ± 20% and a light-dark cycle of 12 h (Laboratory Animal Center of Cell Biotech Co., Ltd., Republic of Korea). The animal-use protocol was reviewed and approved by the Institutional Animal Care and Use Committee Board of Cell Biotech (IACUC, Approval No.: CBT-2022-26, CBT-2022-27) based on the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Blood glucose levels

Prior to the GTT, mice were fasted for 16 h. Their blood glucose levels were checked using a G Care Blood Glucose Set Monitoring device (GC Biopharma Corp., Republic of Korea) at 0, 15, 30, 45, 60, 90, and 120 min after the oral administration of 2 g/kg glucose. HbA1c was checked using an A1C EZ 2.0 Test Kit (BioHermes, China).

Histology

Paraffin tissue sections of liver were cut to a thickness of 4 µm. To enable histological assessment of hepatosteatosis (macrovesicular steatosis, microvesicular steatosis, and hypertrophy), the sections were stained with hematoxylin and eosin (H&E). Ten fields were examined in each section at 200x magnification, and a semiquantitative analysis of hepatosteatosis was performed following a grading system for rodent non-alcoholic fatty liver disease [19]. Macrovesicular and microvesicular hepatosteatosis and hypertrophy were graded on a scale of 0−3 based on the percentage of normal hepatocytes and the quantity of hepatocytes contained within a lipid droplet: 0, < 5%; 1, 5−33%; 2, 33−66%; 3, > 66% [19].

Biochemical parameters

Prior to blood collection, the animals were orally administered the test materials and then fasted for 4 h. Plasma was isolated from blood, centrifuged at 2000 ×g for 15 min, and then stored at -70℃ prior to the biochemical analyses. Blood was collected in tubes with anticoagulants (Vacuplast® collection line, Vacuplast GmbH, Austria). Plasma insulin was determined using mouse ultrasensitive insulin ELISA (ALPCO). Plasma lactate dehydrogenase (LDH), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), tri-glycerides (TG), albumin (ALB), and total protein (PRO) were measured using an automatic biochemical analyzer (XL-100TM; Erba Diagnostics Mannheim GmbH, Germany).

In vitro experiments for cell viability by MCE

RIN-m5F was purchased from American type culture collection (ATCC) and cultured in RPMI 1640 medium (ATCC modification, Gibco, USA) with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin. To determine the toxicity of MCE to RIN-m5F, serum-free RPMI 1640 medium containing various concentrations of MCE (i.e., 0, 20, 40, 80, and 160 mg/ml) was treated for 24 h. We then measured cell viability using the cell counting kit-8 (Dojindo, Japan), according to the users’ manual.

In vitro experiments for insulin induction by MCE

To confirm the effect of MCE on stimulating insulin secretion from RIN-m5F, 5 × 105 cells were seeded in a 24-well plate (SPL, Republic of Korea) and maintained for 3 days to stabilize the cells. After stabilization, the media was changed to Krebs-Ringer HEPES buffered solution (Alfa Aesar, USA) with 2.8 mM D-glucose for 1 h, and then Krebs-Ringer HEPES buffered solution with 2.8 mM D-glucose containing various concentrations of MCE (i.e., 0, 2, 4, 8, 16, 20, 40, 80, 120, and 160 mg/ml) was treated for 2 h. Two h after MCE treatment, the upper supernatants were used to measure the concentration of secreted insulin using rat insulin ELISA (Crystal Chem, USA), according to the users’ manual.

In vitro experiments for insulin induction by MCE with LAB

To investigate the effect of combining MCE with CBT-LA1, CBT-LP3 or CBT-LR5 on insulin secretion, RIN-m5F was treated with diverse concentrations of the supernatant of MCE with or without LAB (0, 1.0, 4.5, 9.0, 18.0, 27.0, and 36.0 mg/ml) for 2 h. We then measured the concentration of secreted insulin by rat insulin ELISA according to the users’ manual., LABs were cultured aerobically at 37℃ using Lactobacilli MRS broth (Difco, USA) and harvested 3500 ×g at 4℃ for 10 min. The harvested LABs were washed twice and diluted to 3×104 CFU/ml in sterilized water. 1 × 104 LABs were inoculated in 100 mg/ml MCE solution at 30℃ for 96 h. The cultured mixture was centrifuged at 13,000 ×g at 10℃ for 20 min. We then collected the supernatant, which was filtered through a 0.45-um pore filter (Pall Corp., USA). To calculate the amount of remaining MCE, the supernatant was lyophilized and the final solid product was weighed.

Statistical analysis

The animal study data and in vitro data were statistically analyzed using Prism version 8.0 (GraphPad Software, USA). The results from in vivo experiment are presented as means and standard errors of the mean (mean ± SEM) and the results from in vitro experiment are presents as means and standard deviation (mean ± SD). The data were evaluated using one-way ANOVA followed by Tukey's multiple comparison posttests when significance was observed. A p value less than 0.05 was considered to indicate statistical significance.

Results

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MCE and LAB Do not replace insulin

To demonstrate the effect of MCE and MCE plus CBT-LA1, CBT-LP3, or CBT-LR5 on lowering blood glucose levels by replacing insulin, we made use of an STZ-induced type 1 diabetes model (Fig. 1A). Pre-treatment of LAB had no significantly difference in glycemic control and body weight (Supplementary Fig. S1). The administration of MCE and the co-administration of MCE with CBT-LA1, CBT-LP3, or CBT-LR5 for 4 weeks had no effect on lowering blood glucose levels and HbA1c (Fig. 1B and C). Furthermore, MCE plus CBT-LA1-treated mice exhibited higher HbA1c levels than diabetic mice (Fig. 1C). Furthermore, MCE plus CBT-LA1, CBT-LP3, or CBT-LR5 treatment did not lower blood glucose levels within 2 h of administration (Fig. 1D).

Figure 1.In vivo experiments to investigate insulin replacement by MCE and MCE with LAB. (A) Experimental schedule of an STZinduced type 1 diabetes mellitus model. (B) Changes in blood glucose levels during administration. (C) HbA1c at the beginning of administration and 4 weeks after administration. (D) Changes in blood glucose level ratio at time points ranging from 0-120 min after oral administration of MCE, LAB, and glibenclamide. (E) Changes in blood glucose level ratio at time points ranging from 0- 120 min after intravenous administration of diverse concentration of MCE via the tail vein. Values are means ± SEM. ap < 0.05 versus G1. bp < 0.05 versus 0 week results for each group. cp < 0.05 between two groups.

To determine the potential of MCE for insulin replacement, we injected various concentrations of MCE directly into the animals’ blood streams via the tail vein and checked their blood glucose levels 0, 15, 30, 60, 90, and 120 min after injection. There was no concentration-dependent decrease in blood glucose levels (Fig. 1E). These results reveal that MCE plus LAB cannot replace insulin.

MCE plus LAB improved insulin resistance

To demonstrate the effect of MCE and MCE plus LAB on insulin resistance, we considered an HFD-induced obesity model for inducing insulin resistance: we pre-treated the mice with a 45% HFD for 8 weeks (Fig. 2A). Prior to the pre-treatment of HFD, we performed a GTT to check the induction of insulin resistance. Eight weeks after pre-treatment of HFD, the body weights and area-under-the-curve (AUC) of the GTT were significantly larger than normal diet-fed mice (Supplementary Fig. S2).

Figure 2.In vivo experiments to investigate the effect of MCE plus LAB on insulin resistance. (A) Experimental schedule of a HFDinduced insulin resistance model. (B) The AUC results of glucose tolerance 4 weeks after treatment. (C) The AUC results of a GTT 12 weeks after treatment. (D) Changes in blood glucose levels from a GTT 4 weeks after treatment. (E) Changes in blood glucose levels from a GTT 12 weeks after treatment. Values are means ± SEM. ap < 0.05 versus G1. bp < 0.05 versus G2.

Four weeks after treatment with MCE plus CBT-LP3, the experimental mice exhibited reduced insulin resistance compared with HFD-fed control obese mice (Fig. 2B and D). Twelve weeks after treatment with MCE plus CBT-LA1, the experimental mice exhibited reduced insulin resistance compared with HFD-fed control obese mice (Fig. 2C and E). However, there was no significant difference in insulin resistance between the MCE-treated obese mice and the HFD-fed control obese mice (Fig. 2B and C).

MCE plus LAB improved hepatosteatosis

Hepatosteatosis induced by a HFD is characterized by macrovesicular steatosis, microvesicular steatosis, and hypertrophy (Fig. 3). Treatment with MCE for 12 weeks significantly reduced macrovesicular steatosis but did not significantly change the incidence of microvesicular steatosis or hypertrophy. Additionally treatment with CBT-LA1 or CBT-LP3 did not significantly improve hepatosteatosis (Supplementary Fig. S3). Comparing with HFD-fed control obese mice, treatment with MCE plus CBT-LA1 improved hepatosteatosis including macrovesicular steatosis, microvesicular steatosis and hypertrophy. In particular, the incidences of microvesicular steatosis and hypertrophy in MCE plus CBT-LA1-treated mice were significantly lower than in MCE-treated obese mice. Treatment with MCE plus CBT-LP3 improved macrovesicular steatosis. However, microvesicular steatosis and hypertrophy were not significant differently in treated animals compared with HFD-fed control obese mice. Treatment with MCE plus CBT-LR5 improved microvesicular steatosis and hypertrophy compared with the case of the HFD-fed control obese mice. However, the incidence of macrovesicular steatosis did not significantly differ between the treated and HFD-fed control mice. Microvesicular steatosis was less pronounced in the MCE-treated obese mice and the effects of MCE plus CBT-LR5 on microvesicular steatosis and hypertrophy were weaker than the effects of MCE plus CBT-LA1. Glibenclamide, an insulin-stimulating drug, exhibited similar effects to MCE plus CBT-LA1 on hepatic steatosis (Fig. 3A−D). These results reveal that a synergetic effect of MCE and LAB leads to an improvement in hepatosteatosis; the effect does not derive from LAB alone (Supplementary Fig. S3).

Figure 3.Histological analysis of the livers 12 weeks after treatment. (A) H&E staining for hepatosteatosis. Scoring graphs of (B) macrovesicular steatosis, (C) microvesicular steatosis, and (D) hypertrophy. Values are means ± SEM. ap < 0.05 versus G1. bp < 0.05 versus G2. cp < 0.05 versus G3. dp < 0.05 between two groups.

MCE Plus LAB improved indicators of hepatosteatosis

Given that lipid accumulation in the liver is related to lipid metabolism and damage [20, 21], we performed bio-chemical analyses of blood. We found significantly higher LDH levels in HFD-fed control obese mice and MCE-treated obese mice compared with normal diet-fed mice (Fig. 4A). Furthermore, the levels of LDH were no significant difference between MCE plus CBT-LA1-treated obese mice and MCE-treated obese mice. However, the levels of LDH were lower in MCE plus CBT-LP3 or CBT-LR5-treated obese mice than HFD-fed control obese mice. Although GPT did not differ among the various groups, we observed higher levels of GOT in HFD-fed control obese mice and MCE-treated obese mice compared with normal diet-fed mice (Fig. 4B and C). We also noted lower levels of GOT in mice treated with MCE plus CBT-LA1 or CBT-LR5 compared with HFD-fed control obese mice. In particular, treatment with MCE plus CBT-LP3 resulted in lower GOT levels than those observed in HFD-fed control obese mice. In the case of liver function, we analyzed plasma ALB and PRO (Fig. 4D and E). Both ALB and PRO were significantly higher in HFD-fed control obese mice and MCE-treated obese mice than normal diet-fed mice. Treatment of MCE plus CBT-LA1 or CBT-LR5 significantly reduced ALB and PRO levels. Treatment of MCE plus CBT-LP3 did not affect ALB, but this treatment did reduce PRO levels compared with the levels observed in HFD-fed control obese mice and MCE-treated obese mice. However, treatment of glibenclamide significantly reduced ALB levels. Even so, other indicators of liver damage and function did not differ in those animals compared with HFD-fed control obese mice (Fig. 4A−E).

Figure 4.Blood biochemical parameters of liver function and lipid metabolism 12 weeks after treatment. (A) LDH, (B) GPT, (C) GOT, (D) PRO, (E) ALB, (F) TC, (G) HDL, (H) LDL, and (I) TG. Values are means ± SEM. ap < 0.05 versus G1. bp < 0.05 versus G2. cp < 0.05 versus G3.

Total cholesterol, HDL, and LDL levels in plasma were significantly higher in HFD-fed control obese mice and MCE-treated obese mice compared with normal diet-fed mice (Fig. 4F−H). Triglycerides were higher in HFD-fed control obese mice and MCE-treated obese mice compared with normal diet-fed mice (Fig. 4I). Treatment with MCE plus CBT-LA1 significantly reduced total cholesterol, HDL, and LDL levels (Fig. 4F−H). Treatment with MCE plus CBT-LP3 significantly reduced triglyceride levels and HDL (Fig. 4G). Treatment with MCE plus CBT-LP3 additionally lowered total cholesterol levels and LDL levels in plasma compared with HFD-fed control obese mice (Fig. 4F and H). However, treatment with MCE plus CBT-LR5 did not result in a significant difference in total cholesterol, HDL, or LDL levels compared with HFD-fed control obese mice (Fig. 4F and H). Glibenclamide significantly reduced HDL levels (Fig. 4G), but other indicators of lipid metabolism in glibenclamide-treated obese mice were not significantly different from the levels observed in HFD-fed control obese mice and MCE-treated obese mice (Fig. 4G−I). Although changes were not observed in all of the indicators related to liver and lipid metabolism, some indicators improved with treatment of MCE plus LAB; these results might explain the effect of coadministration of MCE with LAB on improving hepatosteatosis.

MCE induced beta cell insulin secretion

To determine whether MCE was toxic to RIN-m5F, we used a CCK assay to determine cell viability as a function of MCE concentration (Fig. 5A). The viability of RIN-m5F began to decrease at a concentration of 20 mg/ml, and cell viability decreased to less than 20% of its original value at a concentration of 80 mg/ml. To better understand the relationship between MCE and insulin secretion, we confirmed that insulin secretion in RIN-m5F depended on the concentration of MCE (Fig. 5B). At concentrations between 4 and 80 mg/ml, insulin secretion also increased as the concentration of MCE increased (Fig. 5B). However, at concentrations above 80 mg/ml, insulin secretion actually decreased due to toxicity of MCE. Although MCE was toxic to pancreatic beta cells in our study, these results suggest that MCE contains insulin-promoting substances.

Figure 5.The dose-dependent effects of MCE on cell cytotoxicity and insulin secretion. (A) Cell viability of RIN-m5F as a function of MCE concentration. (B) Insulin secretion of RIN-m5F as a function of MCE concentration. Values are means ± SD. ap < 0.05 versus at 0 mg/ml of MCE.

To investigate whether LAB enhanced the insulin-secreting effects of MCE, CBT-LA1, CBT-LP3, and CBT-LR5 were each cultured with MCE; their supernatant was then treated with RIN-m5F. The supernatant of MCE without LAB induced increasing levels of insulin secretion as the concentration of the supernatant increased (Fig. 6). The insulin-stimulating effects of the supernatant of MCE plus CBT-LA1 or CBT-LP3 were significantly higher than those of the supernatant of MCE without LAB (Fig. 6A and B). However, the super-natant of MCE plus CBT-LR5 exhibited no difference in insulin secretion compared with the supernatant of MCE without LAB (Fig. 6C).

Figure 6.Enhancement in insulin secretion in RIN-m5F via MCE with LAB. (A) Comparison of insulin secretion in RIN-m5F with MCE with or without CBT-LA1. (B) Comparison of insulin secretion in RIN-m5F with MCE with or without CBT-LP3. (C) Comparison of insulin secretion in RIN-m5F with MCE with or without CBT-LR5. Values are means ± SD. ap < 0.05 versus MCE.

To confirm insulin secretion by MCE plus LAB, we performed a single administration test in normal mice (Supplementary Fig. S4). A tendency of increasing serum insulin concentration in MCE-treated mice compared with normal mice was shown and it was enhanced by MCE plus CBT-LA1 or CBT-LP3. However, MCE plus CBT-LR5 exhibited no difference in insulin secretion compared with MCE without LAB.

These results revealed that the additional increase in insulin-secretion ability by CBT-LA1 and CBT-LP3 compensated for the weak effect of MCE; this enhancement is one of the possible mechanisms explaining alleviations in insulin resistance and hepatosteatosis. Additionally, lack of increased insulin secretion of CBT-LR5 may derive from its weaker effect on insulin resistance and hepatosteatosis compared with CBT-LA1 and CBT-LP3.

Discussion

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The preferred clinical strategy for treating diabetes is to regulate blood glucose levels and maintain a stable patient condition [22]. Given that almost all anti-diabetic drugs used primarily for diabetic patients have side effects such as inducing hypoglycemia [23], these drugs cannot be prescribed to non-diabetic people with insulin resistance [24]. Therefore, people who are worried about developing diabetes have attempted to inhibit the disease by consuming foods and substances that alleviate insulin resistance, which is one of the causes of diabetes [25].

MC is a plant known to lower blood sugar and alleviate insulin resistance [26]. It contains polypeptide-P, Charantin, a well-known insulin agonist [27]. Previous studies have suggested that MC had a hyperglycemic effect and medical potency [28, 29]. Diverse forms of MC including n-buthanol or methanol extract, dried powder by 60℃ hot air oven, lyophilised aqueous extract, juice, water extract by percolation at room temperature, powder of dried fruit soaked in water, 80% ethanol extract, were used to demonstrate antidiabetic effect and the regulation of lipid metabolism by MC [26, 30, 42−46]. In this study, we used hot water extract of MC from FineFT Co. Ltd., and reconfirmed the insulin secretion effect of MCE; however, we noted that insulin resistance was not significantly different between obese mice and MCE-treated obese mice. Although the accumulation of lipid in the liver was reduced with MCE treatment, factors related to liver injury and lipid metabolism in the blood were not significantly different in MCE-treated obese mice and HFD-fed control obese mice. Interestingly, MCE plus CBT-LA1 or CBT-LP3 enhanced the efficacy of MCE by alleviating insulin resistance and lipid accumulation in the liver, factors responsible for liver injury and lipid metabolism in the blood. Furthermore, the supernatant of MCE plus CBT-LA1 or CBT-LP3 enhanced the secretion of insulin from RIN-m5F compared with the supernatant of MCE without LAB or MCE plus CBT-LR5. These results indicate that an enhancement in MCE efficacy in terms of insulin secretion by CBT-LA1 or CBT-LP3 leads to improved insulin resistance, reduced lipid accumulation in the liver, better prevention of hepatosteatosis, and decreased total cholesterol levels in the blood. On the other hand, there was no enhancement in insulin secretion with MCE plus CBT-LR5, which exhibited a weaker effect than MCE plus CBT-LA1 or CBT-LP3.

Earlier studies have demonstrated the anti-diabetic effects of LAB [31]. L. acidophilus KLDS1.1003 and KLDS1.0901 regulate glucose and lipid metabolism by improving intestinal barrier function and reducing liver and colon inflammation in HFD-fed and STZ-induced diabetic mice [32]. L. acidophilus NCFM preserves insulin sensitivity in humans with impaired glucose tolerance via the injection of lipopolysaccharides [33]. L. plantarum HAC01 ameliorates T2DM in HFD-fed and STZ-induced diabetic mice in association with modulating the gut microbiota and improving hyperglycemia by regulating the gut microbiota-liver axis [34]. L. plantarum Ln4 reduces lipid accumulation in 3T3L1 and decreased body weight, epididymal fat mass, and plasma tri-glycerides in HFD-fed mice by changing the expression of genes involved in the regulation of glucose and lipid metabolism [35]. L. rhamnosus LRa05 exhibited a hypoglycemic effect by regulating the hepatic glucagon signaling pathway and inflammation in HFD-fed and STZ-induced diabetic mice [36]. L. rhamnosus GG induced AMPK activation, GLUT4 expression, adiponectin production, and improved insulin sensitivity in HFD-fed mice [37]. Unfortunately, L. acidophilus CBT-LA1 and L. plantarum CBT-LP3 did not improve insulin resistance and hepatosteatosis; MCE had only a weak effect on glucose metabolism in this study. However, we overcame that limitation by combining MCE and LAB. We suggest that such a route is one possible approach to enhancing efficacy.

Fermentation of MC by the microbiome is one possible way of enhancing the efficacy of MC [38]. Previous studies have suggested that fermentation leads to an increase in MC-secreted anti-hyperglycemic substances [39, 40]. However, we demonstrated that coadminstration of vitamins with CBT-LR5 improved the absorption of water soluble vitamins and enhanced vitamin C-mediated antioxidant response and vitamin B-mediated regulation of lipid metabolism in our previous study [50]. Based on this result, we hypothesized that lactic acid bacteria may increase the absorption of antidiabetic substances in MCE and enhance their anti-diabetic efficacy and finally demonstrated that the co-administration of MC and LAB without fermentation improved insulin resistance and hepatosteatosis by increasing insulin secretion. However, the limitation of this study, 1) we could not find the specific effector molecule from MCE, 2) we could not have the method to measure the effective molecules, 3) we could not demonstrated the elevated levels of insulin in animal model, 4) the different mechanism between CBT-LA1 and CBT-LP3 existed. These limitations will be solved further studies.

Although elevated levels of insulin in the blood were not clearly demonstrated and different mechanisms between CBT-LA1 and CBT-LP3 were expected in this study, a comparative analysis of MCE and MCE plus CBT-LA1, CBT-LP3 or CBT-LR5 in terms of insulin secretion from RIN-m5F revealed a similar pattern of results of insulin resistance and hepatosteatosis in HFD-induced insulin resistance animal models. A relationship between insulin secretagogues and hepatosteatosis is one of possible explanation for our results. Therapy of insulin glargine or insulin secretagogues, including GLP-1 analogues, reduced liver fat fraction and short-term therapy with insulin secretagogues lead to lower 2-h postprandial TG and FFA [46, 47]. Nateglinde, other insulin secretagogues, inhibited postprandial hyperlipidemia [48, 49]. Glycemic control and regulation of lipid metabilsm by insulin secretagogues could be one of possible mechanism to improve insulin resistance [51]. Since previous studies have been already demonstrated that MCE improved insulin sensitivity by stimulating IRS-1 tyrosin phosphorylation, skeletal muscle GLUT4 expression and inhibiting NF-kB and JNK pathways, it could be a strong possible mechanism to explain our results [52−54]. Therefore, it will be continuously studied to identify the different mechaisms between coadminstration MCE with CBT-LA1 and CBT-LP3.

In conclusion, we have demonstrated that the combination of MCE and CBT-LA1 or CBT-LP3 may improve insulin resistance and hepatosteatosis by promoting insulin secretion. However, the substance or mechanism that induced that insulin secretion still remains unclear. Therefore, additional studies are necessary to demonstrate whether the insulin agonist in MCE is increased by LAB or whether LAB are influenced by MCE and secrete substances that function as insulin secretagogues.

Acknowledgments

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This research was supported by Cell Biotech, Co., Ltd. (Republic of Korea).

The information of Momordica charantia extraction was supproted by FineFT Co. Ltd., Republic of Korea).

Author Contributions

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D Kim designed and performed all of the experiments, analyzed the data and wrote manuscript. JS Lee and S Kim prepared samples and performed in vitro experiments. SK Park, Y Yoon and Y Ryu discussed the results and wrote manuscript. MJ Jung devised the project, the main conceptual ideas and proof outline.

Conflict of Interest

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All of Authors were employed by the company Cell Biotech Co., Ltd. This research received no external funding.

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