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Previous studies have shown that RBE contained 13.94% ± 1.05% crude protein, 56.61% ± 2.34% total sugar, 8.96 ± 0.21 mg/g total phenol (expressed as gallic acid equivalent, mg gallic acid/g dried extract), and 4.83% ± 0.43% β-glucan. Compared to unfermented barley, after 24 h of incubation with Lactobacillus plantarum dy-1 (initial cell density 4 × 108 cfu/g) at 30 °C, the protein content in LFBE showed a significant increase to 34.94% ± 1.51%, and the sugar content markedly decreased to 34.35% ± 2.59%. The total phenol content of LFBE increased to 13.61 ± 0.15 mg/g. In addition, the concentration of β-glucan significantly increased to 13.44% ± 0.63%[16].
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The main compositions of LFBE and RBE are included in Supplementary Table S1 (available in www.besjournal.com). Fermentation changes the components of the aqueous extracts of barley. To study the therapeutic effects of LFBE in obese rats, a diet-induced obesity model was first established. Seven-week-old male SD rats were maintained on HFD for 8 weeks to induce obesity. At the end of the treatment period, the HFD group had gained about 30% more body weight than the NC group (554.01 ± 17.19 g vs. 460.13 ± 15.90 g), indicating that the obesity model has been successfully established. The rats in the diet-induced obesity model were then divided into two groups (HFD group and LFBE group). Combined with our previous study, in which the treatment of 3T3-L1 and HepG2 cells with RBE had no effect on inhibition of adipogenesis (data not shown), and to focus solely on exploring the anti-obesity action and lipid metabolism effects of LFBE in HFD-induced obesity rats, an RBE group was not created. In addition, it was also clearly shown that L. plantarum dy-1 fermentation significantly improves the anti-obesity properties of barley[9]. At the end of the 8-week intervention period, the weight of the NC group was 546.83 ± 15.90 g and that of the rats in the HFD group was 710.50 ± 47.19 g (P < 0.05, as shown in Figure 1). Generally, high-fat diets significantly increase body weight and liver weight, which leads to obesity, hyperlipidemia, and fatty liver. It is worth noting that the group treated with LFBE had significantly lower body weights (641.50 ± 30.49 g) than the HFD group (P < 0.05), as can also be seen from the epididymal fat mass and abdominal fat mass. However, the food intake in all groups did not differ significantly during the experimental period (Table 1). As Table 1 also shows, the liver weight in the rats from the HFD group was statistically (P < 0.05) higher than that in the NC group. However, at the end of the study, the liver weight of the rats treated with LFBE was significantly (P < 0.05) lower than that of the HFD group. As for the kidneys, spleen, and pancreas, no significant differences were seen between the experimental groups. Moreover, body fat was higher in the HFD group (8.52 ± 1.44 g/100 g) than in the NC group (2.83 ± 0.48 g/100 g) but, interestingly, was lower in the LFBE group (6.87 ± 1.05 g/100 g) than in the HFD group (Table 1).
Table S1. Macronutrient and selected micronutrient content of RBE and LFBE (dry-weight)
Component RBEa LFBEa Extraction ratio (g/100 g) 8.86 ± 0.48 14.51 ± 0.28 Proteinb (g/100 g) 13.94 ± 1.05 34.94 ± 1.51 Total sugar (g/100 g) 56.61 ± 2.34 34.35 ± 2.59 Total phenols (mg/g) 8.96 ± 0. 21 13.61 ± 0.15 β-glucan (g/100 g) 4.83 ± 0.43 13.44 ± 0.63 Gallic acid (mg/kg) 1.98 ± 0.08 8.30 ± 0.08 Coumaric acid (mg/kg) 5.39 ± 0.05 49.99 ± 0.51 Vanillic acid (mg/kg) 22.35 ± 0.21 117.99 ± 1.15 Caffeic acid (mg/kg) 2.27 ± 0.07 1.31 ± 0.02 Ferulic acid (mg/kg) 23.52 ± 0.07 112.79 ± 1.21 Note. aRBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; all samples were freeze-dried into a powder. bConversion factor used to calculate protein: N = 5.83. The results are expressed as the mean (± SD), n = 3. Figure 1. Effect of LFBE on preventing increase in body weight in rats with HFD-induced obesity. Mean weekly body weight in obese rats from different groups. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obesity rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. During the 8-week treatment, body weight was measured every 2 weeks. Values are expressed as means ± SEM, n = 8 per group. *Significant difference between LFBE group and HFD group (P < 0.05).
Table 1. Serum, liver lipid contents, and relative size of viscera and total fat of three groups of rats
Variables NC group HFD group LFBE Food intake (g/d) 27.66 ± 1.83a 28.84 ± 1.98a 28.12 ± 1.48a Fat mass (g) 15.51 ± 1.75c 59.10 ± 6.78a 29.86 ± 3.56b Epididymal fat mass (g) 6.05 ± 0.79c 18.52 ± 2.15a 12.48 ± 1.44b Abdominal fat mass (g) 8.70 ± 0.92c 39.61 ± 4.46a 26.82 ± 2.06b Serum TG (mmol/L) 1.06 ± 0.32c 3.31 ± 0.89a 1.96 ± 0.25b TC (mmol/L) 1.30 ± 0.11b 1.68 ± 0.14a 1.29 ± 0.20b HDL-C (mmol/L) 1.12 ± 0.15a 0.87 ± 0.14b 1.02 ± 0.14ab LDL-C (mmol/L) 0.27 ± 0.05a 0.14 ± 0.06b 0.19 ± 0.06ab Insulin (mIU/L) 17.66 ± 0.82b 21.17 ± 0.89a 18.30 ± 0.67b Liver TG (mmol/gprot) 0.90 ± 0.08c 2.26 ± 0.32a 1.49 ± 0.14b TC (mmol/gprot) 0.42 ± 0.09b 0.67 ± 0.02a 0.42 ± 0.07b HDL-C (mmol/gprot) 1.65 ± 0.06a 0.85 ± 0.04b 1.52 ± 0.07a LDL-C (mmol/gprot) 0.44 ± 0.09c 0.92 ± 0.03a 0.65 ± 0.05b Relative size (g/100 g body weight) Liver 2.49 ± 0.09c 3.02 ± 0.22a 2.68 ± 0.07b Spleen 0.16 ± 0.01a 0.14 ± 0.01a 0.15 ± 0.02a Kidney 0.50 ± 0.03a 0.45 ± 0.02a 0.47 ± 0.04a Pancreas 0.10 ± 0.02a 0.08 ± 0.01a 0.09 ± 0.02a Body fat 2.83 ± 0.48c 8.52 ± 1.44a 6.87 ± 1.05b Epididymal fat 1.12 ± 0.35c 2.60 ± 0.41a 2.10 ± 0.32b Abdominal fat 1.61 ± 0.28c 5.57 ± 0.87a 4.22 ± 0.65b Note. NC: group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet; HFD: group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD; LFBE: group consisted of diet-induced obesity rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. Values are means ± SEM. n = 8. Different superscript letters indicate a significant difference (P < 0.05). The adipocytes in the LFBE-treated group were also smaller than those in the HFD group (Figure 2A). Furthermore, hepatic steatosis induced by obesity was greatly alleviated in the LFBE-treated group, as shown by H&E staining (Figure 2B). Therefore, we speculate that in addition to its inhibition of diet-induced weight gain, LFBE may reduce the increase in liver size and fat mass by modulation of lipid homeostasis.
Figure 2. LFBE prevents obesity in diet-induced obese SD rats. (A) Representative H&E sections of epididymal fat tissues and (B) livers from HFD-fed rats. Fat cells from epididymal fat tissues in LFBE were smaller in size than those of control HFD-fed rats as shown by H&E staining (scale bar = 100 μm). Accumulation of lipid in the liver reduced following LFBE treatment (scale bar = 100 μm). NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD.
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Glucose content is an important indicator of tissue and cells in obesity models. With the aim to assess the effect of oral administration of LFBE on systemic glucose homeostasis, we performed an oral glucose tolerance test (OGTT) in conscious fasted rats. Figure 3 presents the effect of LFBE on OGTT levels. At the beginning of the study, the rats in the HFD and LFBE groups started with similar oral glucose tolerance. The area under the curve for glucose during the OGTT was 710 ± 29 mmol·min/mL in the NC group, 888 ± 29 mmol·min/mL in the HFD group, and 895 ± 31 mmol·min/mL in the LFBE group (Figure 3).
Figure 3. (A) Results of OGTT before supplementation with LFBE. No significant differences in the area under the curve (AUC) of OGTT was observed in the HFD and LFBE treated groups. (B) Results of OGTT supplementation with LFBE for 8 weeks. Significant differences in the area under the curve (AUC) of oral glucose tolerance was seen in the HFD and LFBE treated groups. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. Data are means ± SEM, n = 8 per group. Data were analyzed by analysis of variance (ANOVA) with different letters (a, b, c) indicating a significant difference (P < 0.05).
After treatment with LFBE for 8 weeks, the serum glucose levels at 0, 30, 60, and 120 min in the HFD group were higher than those in NC group (P < 0.05). The serum glucose levels at 0, 30, 60, and 120 min in the LFBE group were lower than those in the HFD group (Figure 3). The area under the curve for glucose during the OGTT was 701 ± 34 mmol·min/mL in NC group, 878 ± 17 mmol·min/mL in the HFD group, and 781 ± 17 mmol·min/mL in the LFBE group. From these results, we can conclude that oral glucose tolerance of the LFBE rats showed improvement.
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Because obesity has a positive association with higher serum glucose levels and increased lipid contents, we measured levels of TC, TG, HDL-C, and LDL-C in serum and liver. The serum lipid profile is an important metabolic variable altered in obesity.
The mean changes in TG, TC, HDL-C, LDL-C, and insulin in the different treatment groups are shown in Table 1. There was a tendency toward higher serum and liver TG levels in the HFD group than in the NC group, whereas gavage of LFBE could reduce them. Significantly increased (P < 0.05) levels of serum and liver TC were observed in the HFD-fed rats, but gavage of LFBE significantly decreased (P < 0.05) these levels to values similar to those in the NC group. The HFD group showed a significant reduction in serum HDL-C (P < 0.05) compared with the NC group, and although the difference in serum HDL-C levels between the LFBE and NC groups was not significant (P > 0.05), there was a tendency toward a higher serum HDL-C level in the LFBE group. The HFD group showed a significant increase in serum LDL-C (P < 0.05) compared with the NC group, and although there were no significant differences (P > 0.05) in serum LDL-C levels between the LFBE and control groups, there was a tendency toward a lower serum LDL-C level in the LFBE group. A significantly reduced (P < 0.05) level of liver HDL-C was observed in the HFD group, but gavage with LFBE significantly increased (P < 0.05) this level to values similar to that in the NC group. A significantly increased (P < 0.05) level of liver LDL-C was observed in the HFD group, but gavage with LFBE significantly decreased (P < 0.05) this level similar to that in the NC group. At week 8, the serum levels of insulin were significantly greater in the HFD group (21.17 ± 0.89 mIU/L) than in the NC group (17.66 ± 0.82 mIU/L). After being treated with LFBE for 8 weeks, the LFBE group (18.30 ± 0.67 mIU/L) showed a significant reduction in serum insulin compared with the HFD group.
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To identify miRNAs associated with the anti-obesity action of LFBE, miRNAs with at least a 1.5-fold change in expression were selected. Subsequent to data processing and analysis, specific miRNAs were identified to be differentially expressed. Ten miRNAs (miR-23a-5p, miR-466c-3p, miR-345-3p, miR-421-3p, miR-212-3p, miR-3572, miR-195-3p, miR-32-3p, miR-130b-5p, and miR-485-3p) were upregulated and five miRNAs (miR-340-5p, miR-542-3p, miR-3561-3p, miR-34a-5p, and miR-3571) were downregulated in the LFBE group compared with the HFD group (Figure 4A). In addition, seven miRNAs (miR-92a-2-5p, miR-292-3p, miR-382-3p, miR-155-3p, miR-122-5p, miR-3571, and miR-34a-5p) were increased and 11 miRNAs (miR-351-5p, miR-210-3p, miR-299b-5p, miR-224-3p, miR-219a-5p, miR-24-2-5p, miR-32-5p, miR-96-5p, miR-33-3p, miR-338-3p, and miR-143-3p) were decreased in the HFD group compared with the NC group (Figure 4B).
Figure 4. Hierarchical cluster analysis of miRNAs in livers of (A) LFBE rats and (B) NC rats vs. HFD rats (n = 8). Red indicates high relative expression and green indicates low relative expression. miRNAs with expression fold change > 1.5 and with FDR < 0.05 were considered statistically significant. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD.
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To confirm the trends observed in microarray assays, six miRNAs (miR-3571, miR-34a-5p, miR-421-3p, miR-130b-5p, miR-195-3p, and miR-212-3p) from the LFBE group were randomly selected for further validation using RT-qPCR, as compared to the HFD group. Three of the six selected miRNAs (miR-421-3p, miR-130b-5p, and miR-195-3p) were increased in the LFBE group compared with the HFD group (Figure 5A). Three of the six selected miRNAs (miR-3571, miR-212-3p, and miR-34a-5p) were significantly downregulated in the LFBE group compared with the HFD group (Figure 5B) and in the NC group compared with the HFD group (P < 0.05). The results were generally consistent with the microarray data (Figure 5C, D). Interestingly, miR-212-3p was downregulated in both the LFBE group and NC group compared with the HFD group (Figure 5A). However, this trend was opposite to that observed in the microarray data.
Figure 5. Validation of microarray data by real-time qRT-PCR. (A, B): Real-time qRT-PCR analysis of differentially expressed miRNAs in liver tissue. (C, D): Expression fold changes in the validated miRNAs by SAM algorithm in microarray analysis from NC and LFBE rats compared with HFD rats. Triplicate assays were carried out for each RNA sample, and the relative amount of each miRNA was normalized to U6 miRNA. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. *Significant difference between NC group and HFD group (P < 0.05), #Significant difference between LFBE group and HFD group (P < 0.05)
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To further verify the results from the animal experiments, HepG2 cells were selected for in vitro assays for checking the glucose consumption and TG content. As can be seen from Table 2, LFBE treatment showed significant enhancement of glucose consumption in a dose-dependent manner in HepG2 cells. In addition, treatment of cells with LFBE (50 to 100 μg/mL) and RBE (50 to 200 μg/mL) did not result in toxicity except at high concentrations of LFBE (200 μg/mL), which inhibited HepG2 cell growth (data not shown). However, RBE hardly increased the glucose consumption at any of the tested concentrations. As can be seen from Table 3, miR-212 inhibitor and miR-34a inhibitor showed significant enhancement of glucose consumption in HepG2 cells.
Table 2. Effect of LFBE and RBE on glucose consumption and relative TG contents after 24 h treatment of HepG2 cells
Concentration (μg/mL) Glucose consumption (mmol/L) TG contents (%) Control 2.64 ± 0.24** 100.00 ± 1.41** Model 1.26 ± 0.25 135.97 ± 3.25 LFBE 200 2.02 ± 0.81** 104.05 ± 2.15** 100 2.51 ± 0.36** 106.21 ± 2.31** 50 2.18 ± 0.17** 112.63 ± 3.57** RBE 200 1.42 ± 0.26 129.45 ± 4.29 100 1.34 ± 0.22 130.58 ± 3.25 50 1.23 ± 0.13 132.85 ± 2.43 Note. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, compared to model. RBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; Control: normal cells without PA treatment; Model: HepG2 cells treated with PA. Table 3. Effect of miR-212 and miR-34a on glucose consumption and relative TG contents after 24 h treatment of HepG2 cells
Concentration (nmol/L) Glucose consumption (mmol/L) TG contents (%) Control 3.05 ± 0.22** 100.00 ± 1.41** Model 1.68 ± 0.15 135.97 ± 3.25 miR-212 mimic 1.71 ± 0.23 147.89 ± 4.14 miR-212 inhibitor 2.95 ± 0.14** 107.14 ± 2.88** miR-34a mimic 1.52 ± 0.13 153.45 ± 4.89 miR-34a inhibitor 2.88 ± 0.24** 102.85 ± 1.73** Note. Data are presented as mean ± SD (n = 3). **P < 0.01, compared to model. Control: normal cells without PA treatment; Model: HepG2 cells treated with PA. Dietary fat and free fatty acids are known to enhance hepatic glucose production and the accumulation of TG within lipid droplets in hepatocytes. LFBE treatment in concentrations of 50 to 100 μg/mL significantly reduced the accumulation of TG in PA-induced HepG2 cells (Table 2). However, RBE hardly reduced the TG contents at any of the tested concentrations. miR-212 inhibitor and miR-34a inhibitor significantly reduced the accumulation of TG in PA-induced HepG2 cells (Table 3).
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In probing the mechanism by which LFBE improves high levels of glucose and TG in PA-induced HepG2 cells, we found that the upregulation of miR-212 and miR-34a was diminished by LFBE in HepG2 cells (Figure 6).
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Because the number of experimentally validated miRNA targets is very limited, we predicted their potential target genes to explore the functions of the 15 differentially expressed miRNAs among LFBE and HFD groups. Three computer-aided algorithms, including TargetScan, miRanda, and miRBase, were used. The biology of the predicted target genes was further explored in the context of their molecular functions and involvement in known regulatory pathways. The predicated miRNA targets were categorized into several biological functions, and GABAergic synapse were shown to be the most enriched ones (Figure 7A, B).
Figure 7. GO and KEGG pathway analysis of the deregulated miRNAs. Enrichment score is equal to log10 (P-value). The higher the enrichment score, the more specific is the corresponding function. (A) significant GOs and (B) significant signaling pathways. KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene ontology. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD.
doi: 10.3967/bes2019.095
Anti-obesity Action of Fermented Barley Extracts with Lactobacillus plantarum dy-1 and Associated MicroRNA Expression in High-fat Diet-induced Obese Rats
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Abstract:
Objective To further explore associated effects of Lactobacillus plantarum dy-1 (LFBE) on obesity and lipid metabolism at the gene expression level, the expression of microRNAs (miRNAs) was investigated in the liver of high-fat diet (HFD) induced obese rats. Methods Three groups of animal models were established. Changes in miRNA expression in the liver of each group were analyzed by microarray and RT-qPCR, complemented by bioinformatics. Palmitate-induced hepatocellular carcinoma (HepG2) cells were used as a model to validate the test. Results LFBE treatment groups and HFD groups were observed to be distinctly different with respect to rates of increase in body weight and body fat percentage and triglyceride (TG) and total cholesterol (TC) levels in serum and liver. In addition, the LFBE group showed upregulation of ten miRNAs and downregulation of five miRNAs in the liver. Downregulation of miR-34a and miR-212 was observed in the livers of the LFBE group. Gene ontology and kyoto encyelopedia of geues and genomes (KEGG) pathway analysis showed that possible target genes of the deregulated miRNAs were significantly enriched in the adrenergic and HIF-1 signaling pathways. Conclusion These results demonstrate that LFBE might regulate the expression of miRNAs in order to inhibit obesity and fatty liver. -
Key words:
- Fermented barley extract /
- Lactobacillus plantarum dy-1 /
- Anti-obesity /
- Lipid metabolism /
- miRNAs /
- Microarray
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Figure 1. Effect of LFBE on preventing increase in body weight in rats with HFD-induced obesity. Mean weekly body weight in obese rats from different groups. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obesity rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. During the 8-week treatment, body weight was measured every 2 weeks. Values are expressed as means ± SEM, n = 8 per group. *Significant difference between LFBE group and HFD group (P < 0.05).
Figure 2. LFBE prevents obesity in diet-induced obese SD rats. (A) Representative H&E sections of epididymal fat tissues and (B) livers from HFD-fed rats. Fat cells from epididymal fat tissues in LFBE were smaller in size than those of control HFD-fed rats as shown by H&E staining (scale bar = 100 μm). Accumulation of lipid in the liver reduced following LFBE treatment (scale bar = 100 μm). NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD.
Figure 3. (A) Results of OGTT before supplementation with LFBE. No significant differences in the area under the curve (AUC) of OGTT was observed in the HFD and LFBE treated groups. (B) Results of OGTT supplementation with LFBE for 8 weeks. Significant differences in the area under the curve (AUC) of oral glucose tolerance was seen in the HFD and LFBE treated groups. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. Data are means ± SEM, n = 8 per group. Data were analyzed by analysis of variance (ANOVA) with different letters (a, b, c) indicating a significant difference (P < 0.05).
Figure 4. Hierarchical cluster analysis of miRNAs in livers of (A) LFBE rats and (B) NC rats vs. HFD rats (n = 8). Red indicates high relative expression and green indicates low relative expression. miRNAs with expression fold change > 1.5 and with FDR < 0.05 were considered statistically significant. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD.
Figure 5. Validation of microarray data by real-time qRT-PCR. (A, B): Real-time qRT-PCR analysis of differentially expressed miRNAs in liver tissue. (C, D): Expression fold changes in the validated miRNAs by SAM algorithm in microarray analysis from NC and LFBE rats compared with HFD rats. Triplicate assays were carried out for each RNA sample, and the relative amount of each miRNA was normalized to U6 miRNA. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. *Significant difference between NC group and HFD group (P < 0.05), #Significant difference between LFBE group and HFD group (P < 0.05)
Figure 7. GO and KEGG pathway analysis of the deregulated miRNAs. Enrichment score is equal to log10 (P-value). The higher the enrichment score, the more specific is the corresponding function. (A) significant GOs and (B) significant signaling pathways. KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene ontology. NC group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet. HFD group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD. LFBE group consisted of diet-induced obese rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD.
S1. Macronutrient and selected micronutrient content of RBE and LFBE (dry-weight)
Component RBEa LFBEa Extraction ratio (g/100 g) 8.86 ± 0.48 14.51 ± 0.28 Proteinb (g/100 g) 13.94 ± 1.05 34.94 ± 1.51 Total sugar (g/100 g) 56.61 ± 2.34 34.35 ± 2.59 Total phenols (mg/g) 8.96 ± 0. 21 13.61 ± 0.15 β-glucan (g/100 g) 4.83 ± 0.43 13.44 ± 0.63 Gallic acid (mg/kg) 1.98 ± 0.08 8.30 ± 0.08 Coumaric acid (mg/kg) 5.39 ± 0.05 49.99 ± 0.51 Vanillic acid (mg/kg) 22.35 ± 0.21 117.99 ± 1.15 Caffeic acid (mg/kg) 2.27 ± 0.07 1.31 ± 0.02 Ferulic acid (mg/kg) 23.52 ± 0.07 112.79 ± 1.21 Note. aRBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; all samples were freeze-dried into a powder. bConversion factor used to calculate protein: N = 5.83. The results are expressed as the mean (± SD), n = 3. Table 1. Serum, liver lipid contents, and relative size of viscera and total fat of three groups of rats
Variables NC group HFD group LFBE Food intake (g/d) 27.66 ± 1.83a 28.84 ± 1.98a 28.12 ± 1.48a Fat mass (g) 15.51 ± 1.75c 59.10 ± 6.78a 29.86 ± 3.56b Epididymal fat mass (g) 6.05 ± 0.79c 18.52 ± 2.15a 12.48 ± 1.44b Abdominal fat mass (g) 8.70 ± 0.92c 39.61 ± 4.46a 26.82 ± 2.06b Serum TG (mmol/L) 1.06 ± 0.32c 3.31 ± 0.89a 1.96 ± 0.25b TC (mmol/L) 1.30 ± 0.11b 1.68 ± 0.14a 1.29 ± 0.20b HDL-C (mmol/L) 1.12 ± 0.15a 0.87 ± 0.14b 1.02 ± 0.14ab LDL-C (mmol/L) 0.27 ± 0.05a 0.14 ± 0.06b 0.19 ± 0.06ab Insulin (mIU/L) 17.66 ± 0.82b 21.17 ± 0.89a 18.30 ± 0.67b Liver TG (mmol/gprot) 0.90 ± 0.08c 2.26 ± 0.32a 1.49 ± 0.14b TC (mmol/gprot) 0.42 ± 0.09b 0.67 ± 0.02a 0.42 ± 0.07b HDL-C (mmol/gprot) 1.65 ± 0.06a 0.85 ± 0.04b 1.52 ± 0.07a LDL-C (mmol/gprot) 0.44 ± 0.09c 0.92 ± 0.03a 0.65 ± 0.05b Relative size (g/100 g body weight) Liver 2.49 ± 0.09c 3.02 ± 0.22a 2.68 ± 0.07b Spleen 0.16 ± 0.01a 0.14 ± 0.01a 0.15 ± 0.02a Kidney 0.50 ± 0.03a 0.45 ± 0.02a 0.47 ± 0.04a Pancreas 0.10 ± 0.02a 0.08 ± 0.01a 0.09 ± 0.02a Body fat 2.83 ± 0.48c 8.52 ± 1.44a 6.87 ± 1.05b Epididymal fat 1.12 ± 0.35c 2.60 ± 0.41a 2.10 ± 0.32b Abdominal fat 1.61 ± 0.28c 5.57 ± 0.87a 4.22 ± 0.65b Note. NC: group consisted of normal rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with control diet; HFD: group consisted of diet-induced obese rats treated with distilled water (4 mL/kg body weight) through gavage administration and fed with HFD; LFBE: group consisted of diet-induced obesity rats treated with LFBE (1 g/kg body weight) through gavage administration and fed with HFD. Values are means ± SEM. n = 8. Different superscript letters indicate a significant difference (P < 0.05). Table 2. Effect of LFBE and RBE on glucose consumption and relative TG contents after 24 h treatment of HepG2 cells
Concentration (μg/mL) Glucose consumption (mmol/L) TG contents (%) Control 2.64 ± 0.24** 100.00 ± 1.41** Model 1.26 ± 0.25 135.97 ± 3.25 LFBE 200 2.02 ± 0.81** 104.05 ± 2.15** 100 2.51 ± 0.36** 106.21 ± 2.31** 50 2.18 ± 0.17** 112.63 ± 3.57** RBE 200 1.42 ± 0.26 129.45 ± 4.29 100 1.34 ± 0.22 130.58 ± 3.25 50 1.23 ± 0.13 132.85 ± 2.43 Note. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, compared to model. RBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; Control: normal cells without PA treatment; Model: HepG2 cells treated with PA. Table 3. Effect of miR-212 and miR-34a on glucose consumption and relative TG contents after 24 h treatment of HepG2 cells
Concentration (nmol/L) Glucose consumption (mmol/L) TG contents (%) Control 3.05 ± 0.22** 100.00 ± 1.41** Model 1.68 ± 0.15 135.97 ± 3.25 miR-212 mimic 1.71 ± 0.23 147.89 ± 4.14 miR-212 inhibitor 2.95 ± 0.14** 107.14 ± 2.88** miR-34a mimic 1.52 ± 0.13 153.45 ± 4.89 miR-34a inhibitor 2.88 ± 0.24** 102.85 ± 1.73** Note. Data are presented as mean ± SD (n = 3). **P < 0.01, compared to model. Control: normal cells without PA treatment; Model: HepG2 cells treated with PA. -
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