The effect of dietary C18:0/C16:0 ratios on body weight and body fat content
At the end of the 6th week, the body weight of mice in HSF group became higher than that in LSF group (Fig. 1a). No significant changes in diet intake were observed among the three groups (data not shown). CT images of body fat distribution showed the richest visceral and subcutaneous fat were in the HSF group, followed by the LSF group, which were much higher than those in NFD group (Fig. 1b). The total body fat ratio and liver fat ratio were also higher in HSF group than those in LSF group (Fig. 1c, d).
The effect of dietary C18:0/C16:0 ratios on energy metabolism
There was no difference in energy expenditure among the 3 groups (Additional file 1: Fig. S1a). Carbohydrate oxidation in HSF and LSF groups was significantly lower, while fat oxidation was much higher, compared with those in NFD group (Fig. S1b, c). Moreover, fat oxidation in LSF group was significantly higher than that in HSF group, indicating a lower oxidation rate of C18:0 than C16:0. There was a trend of decrease in RER in HSF and LSF groups, but it did not reach a statistical significance, compared with that in NFD group (Fig. S1d).
The effect of dietary C18:0/C16:0 ratios on blood biochemical parameters, glucose toleance and inflammatory markers
Mice in both high fat diet groups exhibited decreased glucose tolerance, but the glucose tolerance was impaired much severe in HSF group than that in LSF group (Additional file 2: Fig. S2). Fasting blood glucose, insulin, TC, TG, HDL-c, LDL-c, TNF-α,IL-6 and HOMA-IR in high fat diet groups were significantly higher than those in NFD group, and glucose, insulin, HOMA-IR, TG and IL-6 levels in HSF group were higher than those in LSF group (Additional file 3: Table S1).
The effect of dietary C18:0/C16:0 ratios on fatty acids profile in serum and liver
The levels of serum total fatty acids, saturated fatty acids, unsaturated fatty acids, C16:0, C18:0, C18:1, C18:3 were signifiantly higher in mice fed with high fat diets, and these fatty acids were much higher in HSF group than those in LSF group (Table 1). The levels of C20:2 and C20:4 were also increased in high fat diet groups, but there was no difference between HSF group and LSF group.The changes in liver fatty facids profle were similar with those in serum among groups (Additional file 4: Table S2).
Table 1
Serum fatty acids profile of mice
FAs (µg/ml) | NFD | LSF | HSF |
C14:0 | 5.15 ± 0.97 | 5.55 ± 0.73 | 8.3 ± 2.13 |
C16:0 | 257.4 ± 78.21 | 330.05 ± 58.69* | 377.67 ± 44.33*# |
C16:1 | 37.07 ± 5.08 | 39.68 ± 8.51 | 52.18 ± 10.33* |
C18:0 | 55.39 ± 42.35 | 80.86 ± 29.84* | 120.08 ± 26.37*# |
C18:1 | 159.06 ± 15.99 | 236.41 ± 22.68* | 272.41 ± 23.83*# |
C18:2 | 313.71 ± 54.58 | 256.11 ± 17.52* | 286.88 ± 31.07 |
γ- C18:3 | 13.04 ± 8.41 | 8.42 ± 4.16 | 12.06 ± 5.64 |
C18:3 | 201.41 ± 43.73 | 82.31 ± 15.27* | 114.14 ± 8.37*# |
C20:2 | 35.96 ± 40.21 | 150.16 ± 98.72* | 98.11 ± 46.96* |
C20:4 | 81.72 ± 10.27 | 144.23 ± 37.63* | 148.17 ± 49.17* |
C20:5 | 6.02 ± 1.78 | 2.6 ± 1.04 | 6.66 ± 2.36 |
saturated fatty acid | 317.94 ± 89.10 | 406.24 ± 98.47* | 506.05 ± 69.91*# |
unsaturated fatty acid | 847.98 ± 145.63 | 885.06 ± 165.88 | 990.63 ± 130.54*# |
total free fatty acids | 1128.85 ± 243.39 | 1296.7 ± 129.58* | 1444.79 ± 184.27*# |
All values are presented as mean ± SEM (n = 10). NFD, normal fat diet group; HSF, high stearic acid diet group (C18:0/ C16:0 = 1:2); LSF, low stearic acid diet group (C18:0/ C16:0 = 1:8). * Compared with the NFD group, P༜0.05. # Compared with LSF group, P༜0.05. |
The effect of dietary C18:0/C16:0 ratios on cell ultrastructure in liver and pancreas
Liver mitochondria in NFD group appeared as typical mitochondria, elongated or round in shape, with tubular cristae and a few electron dense granules in the mitochondrial matrix (Additional file 5: Fig. S3a). Mitochondria in high fat diet groups were severely distended compared with NFD group, and mitochondrial distension in HSF group was more serious than that in LSF group (Fig. S3b, c). Some mitochondria in HSF group showed an exvaginated inner mitochondrial membrane. For pancreas, integritied β cell structure was observed in NFD group, with abundant insulin secretion granules in the cytoplasm (Fig. S3d). The amounts of insulin granules decreased significantly in both LSF and HSF groups, and there were obvious vacuoles induced by denatured insulin granules in HSF group (Fig. S3e, f).
Differentially expressed lncRNAs in liver among groups
A total of 34,523 lncRNAs was analyzed. As shown in Heat map and Volcano Plot, there were distinguishable gene expression profilings among groups (Fig. 2). Compared with NFD group, 258 lncRNAs were differentially expressed in LSF group including 152 up-regulated and 106 down-regulated (Fig. 2a, d), while 751 lncRNAs were differentially expressed in HSF group including 364 up-regulated and 387 down-regulated, whcih were much more than those in LSF group (Fig. 2b, e). Among these differentially expressed lncRNAs in comparison with NFD group, there were 148 same lncRNAs in both LSF and HSF groups. There were 216 differentially expressed lncRNAs between LSF and HSF groups, including 116 up-regulated and 100 down-regulated (Fig. 2c, f).
Differentially expressed microRNAs in liver among groups
As shown in Heat map and Volcano Plot, 3544 microRNAs were analyzed and there were distinguishable microRNA expression profiles among groups (Fig. 3). Compared with NFD group, 44 microRNAs were differentially expressed in LSF group including 20 up-regulated and 24 down-regulated (Fig. 3a, d), while 42 microRNAs were differentially expressed in HSF group including 29 up-regulated and 13 down-regulated (Fig. 3b, e). Among these differentially expressed microRNAs in comparison with NFD group, there were 10 same microRNAs in both LSF and HSF groups. There were 32 differentially expressed microRNAs between LSF and HSF groups, including 24 up-regulated and 8 down-regulated (Fig. 3c, f).
Differentially expressed mRNAs in liver among groups
Among 23,047 mRNAs, a total of 302 differentially expressed mRNAs were identified between LSF group and NFD group, including 140 up-regulated and 162 down-regulated (Fig. 4a, c). While 808 differentially expressed mRNAs were identified between the HSF group and NFD group, including 433 up-regulated and 375 down-regulated (Fig. 4b, e). There were much more differentially expressed mRNAs in HSF group than those in LSF when compared with NFD group. And among these differentially expressed mRNAs, there were 150 same mRNAs in both LSF and HSF groups. There were 275 differentially expressed mRNAs between LSF and HSF groups, including 230 up-regulated and 45 down-regulated (Fig. 4c, f).
Function analysis of differentially expressed mRNAs in liver among groups
The GO analysis covered three domains: biological process (Fig. 5), cellular component (Additional file 6: Fig. S4), molecular function (Additional file 7: Figure S5), and we mainly focused on biological processes. Compared with NFD group, differentially up-regulated mRNAs were involved in biological processes including nitric oxide mediated signal transduction, and cellular response to glucose starvation (Fig. 5a, c), and differentially down-regulated mRNAs were involved in positive regulation of fatty acid oxidation, arachidonic acid metabolic process, thioester metablolic process, acyl-CoA metabolic process, long term synaptic depression, and urate metabolic process in both LSF group and HSD group (Fig. 5b, d). Compared with LSF group, differentially up-regulated mRNAs were involved in inclusion body assembly, regulation of gene silencing, mammary gland involution (Fig. 5e), and differentially down-regulated mRNAs were involved in monocyte chemotaxis, antigen processing and presentation of exogenous peptide antigen via MHC class II, and lymphocyte chemotaxis in HSF group (Fig. 5f).
Regarding the up-regulated transcripts, the common pathways involved in both LSF group and HSD group included cytokine-cytokine receptor intervention, and prolactin signaling pathway, compared with NFD group (Fig. 6a, c). Regarding the down-regulated transcripts, the common pathways in LSF group and HSD group included retinol metabolism, fatty acid degradation, peroxisome, steroid hormone biosynthesis, PPAR signaling pathway, and arachidonic acid metabolism (Fig. 6b, d). When compared the difference between LSF group and HSD group, the top 3 score enrichment pathways were chemical carcinogenesis, retinol metabolism, steroid hormone biosynthesis for up-regated mRNAs (Fig. 6e), and steroid hormone biosynthesis, endocytosis and MAPK signaling pathway for down-regated mRNAs (Fig. 6f).