Table 1.
Diet formulation.
Fig 1.
Supplementation with kale did not attenuate HFD diet induced weight gain and insulin resistance.
Mice were fed isocaloric diets for 12 weeks and data was subjected to an ANOVA. (A). The calculated calorie consumption per day was not different among all groups (P = ns; n = 9). (B). The HFD induced greater body weight gain compared to LFD. Supplementing the HFD with kale from beginning of the study did not lower weight gain (P = ns; n = 9). Supplementing the HFD diet with kale after 6 weeks on HFD did not reduce weight gain compared to HFD group (P = ns; n = 9). (C). Weekly body weight gain was increased in the HFD group. Weight gain in both groups fed with kale was not different from that of the HFD group (P = ns; n = 9). Fasting glucose and fasting insulin were determined by glucometer and ELISA test respectively. (D). At week 0 (baseline), all groups had similar fasting glucose. By week 6 the HFD diet induced higher fasting glucose and kale had no effect. By week 12, the HFD fed group and HFKV groups had the same levels of fasting glucose (P>0.05; n = 8–9). (E). At baseline, all groups had similar fasting Insulin. By week 6 the HFD diet induced higher fasting glucose in the two groups and group 3 fed with kale had significantly lower insulin levels compared to the HF group. By week 12, the HFD fed group and the two groups fed kale had similar levels of fasting insulin (P>0.05; n = 8–9). (F). At baseline, all groups had similar HOMA-IR values. By week 6, the HFD diet induced higher HOMA-IR and group 3 fed with kale had significantly lower HOMA-IR (P<0.05; n = 8–9). By week 12, the HFD fed group and groups fed HFD supplemented with kale had similar levels of HOMA-IR (P>0.05; n = 8–9). ‘a’ Different letters indicate significant differences after ANOVA at P<0.05.
Fig 2.
Supplementation with kale vegetable did not lower fat accumulation but reduced inflammatory markers in adipose tissue.
(A and B). The weight of the epididymal fat pad and combined weight of perirenal and retroperitoneal fat pads were higher in HFD compared to LFD fed group (P<0.05; n = 9). Supplementing the HFD diet with kale from beginning of the study or after 6 weeks of HFD feeding did not lower fat pad weights compared to the HFD diet (P>0.05; n = 9) after ANOVA. (C). Cell size of epididymal fat on H and E stained sections was determined by measuring cross-sectional width of the cells at 40X magnification and standardizing LFD group size to 100. HFD diet increased cell size. The cell size of HFD and HFKV was not different (P>0.05; n = 9) after ANOVA. (D-F). H and E stained sections of the epididymal fat pad showed that LFD group had smaller adipocytes in size with no markers of inflammation visible. The HFD sections showed larger adipocytes and presence of crown like structures the markers of inflammation (shown by arrow). The HFKV sections showed large adipocytes but no crown like structures. ‘a’ Different letters indicate significant differences after ANOVA at P<0.05.
Fig 3.
Supplementation with kale lowered liver weight and liver triglycerides but not liver lipid droplet number.
(A). The weight of the liver was higher in HFD compared to LFD fed group (P<0.05; n = 9). Supplementation of the HFD diet with kale from beginning of the study or after 6 weeks of HFD feeding lowered liver weight to levels not different from those of the LFD (P<0.05; n = 8–9 after ANOVA). (B). Amount of liver triglycerides was higher in HFD compared to LFD fed group (P<0.05; n = 9). Supplementation of the HFD diet with kale from beginning of the study or after 6 weeks of HFD feeding lowered liver triglycerides to levels similar to those of the LFD (P<0.05; n = 8–9 after ANOVA). (C). Quantification of fat droplet number in liver sections at 40X magnification (P<0.05; n = 8–9). (D-F). ORO stained liver slides from HFD fed mice showed highly increased lipid droplets compared to the LFD group. The lipid droplet number in HFKV group was not different from that of the HFD group. ‘a’ Different letters indicate significant differences after ANOVA at P<0.05.
Fig 4.
Supplementation with kale lowered serum TAGs, LDL cholesterol, endotoxemia and inflammation markers.
TAGs were analyzed by a colorimetric kit. LDL and HDL cholesterol were quantified by Elisa kits. All data was subjected to after ANOVA. (A). TAGS in colon fecal samples were higher in HFD compared to LFD fed group (P<0.05; n = 9). TAG levels in HFKV samples were higher than those in HFD and LFD (P<0.05; n = 9). (B). TAG levels in serum were higher in HFD compared to LFD fed group (P<0.05; n = 9). TAG levels in HFKV samples were lower than those in HFD (P<0.05; n = 9). (C and D). LDL cholesterol levels in HFKV samples were lower than those in HFD and LFD (P<0.05; n = 9). HDL cholesterol levels were not different between the 3 diet groups (P = ns; n = 9). LPS, MCP-1 and IL-10 levels in serum were quantified using mouse ELISA kits. All data was subjected to ANOVA. (E) The HFD increased levels of LPS compared to LFD (P<0.05; n = 9). The HFKV diet attenuated the increase in LPS to levels below those of both the HFD diet and LFD (P<0.05; n = 9). (F) The HFD diet increased levels of MCP-1(Ccl2) compared to LFD (P<0.05). The HFKV diet attenuated the increase in MCP-1 to levels below those of both the HFD diet and LFD diet groups (P<0.05; n = 9). (G) The HFD diet increased the levels of IL-10 compared to LFD diet (P<0.05). The HFKV diet further increased IL-10 to levels higher than those of the HFD diet group (P<0.05; n = 9). ‘a’ Different letters indicate significant differences after ANOVA and Tukeys test of multiple comparisons at P<0.05.
Fig 5.
Supplementation with kale reduced adipose tissue markers of inflammation.
Quantitative PCR and western blotting showed that: (A). The HFD increased gene expression of F4/80 compared to LFD (P<0.05). The HFKV diet decreased expression of F4/80 to levels below those of both the HFD diet and LFD groups (P<0.05). (B). The HFD diet increased gene expression of CD11c compared to LFD diet (P<0.05). The HFKV diet attenuated the increase in CD11c to levels below those of both the HFD and LFD groups (P<0.05). (C). The HFD diet increased the protein expression of F4/80 compared to LFD. The HFKV diet decreased F4/80 to levels not detectable by western blotting (P<0.05). Bar chart represents quantification of the gel blot in Image-J. (D). The HFD diet increased the expression of CD11c compared to LFD. The HFKV diet decreased expression of CD11c to levels not detectable by western blotting (P<0.05). The bar chart represents quantification of the gel blot in Image-J. The test proteins and β-actin were determined on the same membranes but at different exposure times. Full membranes are shown in S1 Fig. ‘a’ Different letters indicate significant differences after ANOVA at P<0.05.
Fig 6.
Fold change in gene expression of inflammatory cytokines, chemokines and receptors in adipose tissue.
cDNA was synthesized using the RT2 first strand kit and using the RT2 Profiler TM PCR Array, the differential expression of the chemokines, cytokines and receptors was determined. (A). The HFD diet increased ten chemokines and chemokine receptors by over 3.0-fold and the HFKV diet reduced these. The HFD reduced 3 cytokines and the HFKV diet increased them. All genes shown were changed by over 3.0-fold and were statistically different at P<0.05 after paired T tests. (B). The layout of the PCR array plate. (C-E). Heat maps displaying changes between the HFD and LFD, HFD and HFKV and HFKV and LFD respectively.
Fig 7.
Gene expression and protein expression in adipose tissue and skeletal muscle.
Gene expression was determined by qPCR and Cq values for each gene and that of β-actin as reference gene were used to calculate gene expression by calculating 2-ΔΔCT. The bar charts next to the western blots shows quantification of the western blot band in image J and after ANOVA. (A). Genes involved in fatty acid oxidation (B). Genes involved in adipogenesis. (C). Markers of adipose tissue lipogenesis. (D). Protein expression of Adiponectin. HFD decreased adiponectin expression and HFKV had no effect (P = ns). (E). Protein expression of PPARα. HFD decreased PPARα expression and HFKV had no effect. (F). Genes involved in fatty acid oxidation in skeletal muscle. HFKV lowered the expression of FIAF and increased that of PPARα (P<0.05). (G). Protein expression in skeletal muscle. The bar chars represent quantification of the protein standardized by expression of β-Actin. Expression of PPARα was higher in HFKV diet compared to the LFD and HFD (P<0.05). The test proteins and β-actin were determined on different membranes. Full blots are shown in the S2 Fig. ‘a’ Different letters indicate significant differences at P<0.05.