Chungkookjang with High Contents of Poly-γ-Glutamic Acid Improves Insulin Sensitizing Activity in Adipocytes and Neuronal Cells

Abstract

We hypothesized that soybeans fermented with Bacillus spp. for 48 h (chungkookjang) would be rich in poly-γ-glutamate (γ-PGA) and would have greater efficacy for improving insulin sensitivity and insulin secretion in 3T3-L1 adipocytes, min6 cells, and PC12 neuronal cells. We screened 20 different strains of B. subtillus and B. amyloliquefaciens spp. for γ-polyglutamate (PGA) production and their anti-diabetic and anti-dementia activities in cell-based studies. Chungkookjang made with two B. amyloliquefaciens spp. (BA730 and BA731) were selected to increase the isoflavonoid and γ-PGA. Insulin-stimulated glucose uptake was higher in 3T3-L1 adipocytes given both chungkookjang extracts than in the cells given vehicle (control). The ethanol extract of BA731 (BA731-E) increased the uptake the most. Triglyceride accumulation decreased in BA731-E and BA731-W and the accumulation increased in BA730-W and BA730-E. The mRNA expression of fatty acid synthetase and acetyl CoA carboxylase was much lower in BA731-E and BA731-W and it was higher in BA730-W than the control. BA730-E and BA730-W also increased peroxisome proliferator-activated receptor (PPAR)-γ activity. Glucose-stimulated insulin secretion increased with the high dosage of BA730-W and BA730-E in insulinoma cells, compared to the control. Insulin contents and cell survival in high glucose media were higher in cells with both BA731-E and BA730-E. Triglyceride deposition and TNF-α mRNA expression were lower in BA731 than the control. The high-dosage treatment of BA730-E and BA731-E increased differentiated neuronal cell survival after treating amyloid-β(25-35) compared to the control. Brain-derived neurotrophic factor and ciliary neurotrophic factor, indices of neuronal cell proliferation, were higher in BA730 and BA731 than in the control. Tau expression was also reduced in BA731 more than the control and it was a similar level of the normal-control. In conclusion, BA730 increased PPAR-γ activity and BA731 enhanced insulin sensitivity in the brain and periphery. BA730 and BA731 prevented and alleviated the symptoms of type 2 diabetes and Alzheimer's disease with different pathways.

Keywords: Chungkookjang; glucose uptake; insulin resistance; insulin secretion; neuronal cell survival.

Figure 1

Photo of cooked soybeans (CSB), chungkookjang made with B. amyloliquefaciens SRCM100730 (BA730), chungkookjang made with B. amyloliquefaciens SRCM100731 (BA731).

Figure 2

Insulin-stimulated glucose uptake, triglyceride deposition and expression of genes related to fatty acid synthesis in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pre-treated with the 48 h-treatment of the ethanol (-E) and water (-W) extracts (1 and 5 μg/mL) of chungkookjang fermented with B. amyloliquefaciens SRCM100730 (BA730) and SRCM10073 (BA731). (A) Insulin-stimulated glucose uptake was measured with low dosage (1 ng/mL) of insulin. Insulin treatment of 50 ng/mL was considered as normal-control. (B) Triacylglycerol contents were determined in 3T3-L1 adipocytes after 9–10 days of differentiating from 3T3-L1 fibroblast with differentiation inducers and vehicle, 1 or 5 μg∙mL⁻¹ of ethanol (-E) or water (-W) extracts of BA730 and BA731. (C,D) The fold changes of mRNA levels of genes associated with fatty acid metabolism (ACC1 and FAS) from the control were calculated by the 2^−ΔΔCT method after conducting real-time PCR. The control and positive control were used as ethanol extracts of cooked soybeans and rosiglitazone (0.5 or 2 μM), respectively. Values are means ± SD (n = 7). ^a,b,c,d,e Different letters above the bars indicate significant differences among the groups by Tukey’s test at p < 0.05.

Figure 3

Peroxisome proliferator-activated receptor (PPAR)-γ activation in human embryonic kidney 293 cells. 3T3-L1 adipocytes were pre-treated with the 48 h-treatment of the ethanol (-E) and water (-W) extracts (1 and 5 μg/mL) of cooked soybeans (CSB), chungkookjang fermented with B. amyloliquefaciens SRCM100730 (BA730) and SRCM100731 (BA731) in human embryonic kidney 293 cells. PPAR-γ activity was measured by luciferase activity after transfection of PPAR response element (PPRE)-luciferase construct, pSV-SPORT-PPAR-γ expression vector, pSV-SPORT-retinoid X receptor (RXR)-α vector, and renilla phRL-TK vector. The control and positive control used was the ethanol extracts of CSB and rosiglitazone (0.5 or 2 μM), respectively. Values are means ± SD (n = 7). ^a,b,c Different letters above the bars indicate significant differences among the groups by Tukey’s test at p < 0.05.

Figure 4

Glucose-stimulated insulin secretion, cell survival, insulin and triglyceride contents and TNF-α expression in insulinoma Min6 cells. Cell survival (A) was measured after 24 h treatment of vehicle or 5 μg·mL⁻¹ of 70% ethanol (-E) or water extracts (-W) of chungkookjang fermented with B. amyloliquefaciens SRCM100730 (BA730) and SRCM100731 (BA731). Glucose-stimulated insulin secretion (B) measured in high glucose (20 mM) Krebs–Ringer–Hepes buffer for 30 min after treatment with vehicle or 5 μg·mL⁻¹ of BA730-E, BA730-W, BA731-E and BA731-W. The contents of insulin (C) and triglyceride (D) in the Min6 insulinoma cells measured in high glucose (20 mM) DMEM media for 48 h with CSB-E, ethanol or water extracts of different chungkookjangs. TNF-α mRNA expression (E) in the Min6 cells treated with the chungkookjang was determined by real-time PCR. The positive control was exendin-4 (Ex-4). Values are means ± SD (n = 7). ^a,b,c,d,e Different letters above the bars indicate significant differences among the groups by Tukey’s test at p < 0.05.

Figure 5

Cell survival and mRNA expression of genes associated with neurogenesis in differentiated PC12 cells. PC12 cells were differentiated with nerve growth factor (NGF) for 2 days and differentiated PC12 cells were administered with amyloid-β (25–35) for additional 2 days. The PC12 cells had treatments with a vehicle or 5 μg∙mL⁻¹ of 70% ethanol (-E) or water extracts (-W) of chungkookjang fermented with B. amyloliquefaciens SRCM100730 (BA730) and SRCM100731 (BA731). Cell survival (A) was measured after 24 h treatment. The cDNA was generated from the cells and the fold changes of mRNA levels of BDNF (B), CNTF (C), and tau (D) from the control were calculated by the 2^−ΔΔCT method after conducting real-time PCR. The phosphorylation of Akt and GSK-3β was conducted by immunoblooting and intensity of the blots was measured (E). Immunoblot assay was performed with high dosage of assigned chungkookjang extracts. The relative intensity of phosphorylation of Akt and GSK-3β was given (F). Values are means ± SD (n = 7). ^a,b,c,d Different letters above the bars indicate significant differences among the groups by Tukey’s test at p < 0.05.