Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (8), e106159
eCollection

The Development of Diet-Induced Obesity and Glucose Intolerance in C57BL/6 Mice on a High-Fat Diet Consists of Distinct Phases

Affiliations

The Development of Diet-Induced Obesity and Glucose Intolerance in C57BL/6 Mice on a High-Fat Diet Consists of Distinct Phases

Lynda M Williams et al. PLoS One.

Abstract

High-fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12-16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1
A. Body weight of the HF fed mice differed significantly from those of the LF fed mice from 3 days of feeding (P<0.01) and (P<0.001) onwards. B. The increase in body weight seen in A is mostly fat mass which was increased in the HF mice from 3 days onwards (P<0.001). C. Mean food intake of mice receiving either a LF or HF diet (grams per day). The intake of the HF fed mice was decreased at 3 days on diet (P<0.01) and (P<0.001) thereafter (n = 6–8). D. PF animals showed a decrease in body weight after 3 days on diet compared to LF (P<0.05 and HF fed animals (P<0.001). E. PF animals had a similar body weight to LF fed mice after 1 week on diet. Both LF and PF mice had lower body weight than the HF mice (P<0.001). F. Body fat measured by MRI after 3 days of diet was increased in PF (P<0.05) and HF mice (P<0.001) compared to LF mice despite the lower body weight seen in PF mice in C. G. Body fat after 1 week on diet was higher in both PF (P<0.001) and HF fed mice (P<0.001) compared to LF and was also higher in HF (P<0.001) compared to PF mice. H. Lean body mass after 3 days on diet was similar in all mice LF, PF and HF indicating that all weight changes were due to changes in adiposity. I. Lean mass after 1 week on diet was only increased in HF fed mice compared to LF and PF (P<0.001) (n = 6–8).
Figure 2
Figure 2
A, B &C. Liver lipid measured by image analysis of oil red O in arbitrary units (AU). A. staining increased linearly with time on diet in HF fed mice from 1 week (P<0.001) onwards. Representative images of oil red O stained sections for HF and LF fed mice after 8 weeks on diet. Mag X 100. B. Liver lipid in PF mice was reduced compared to both LF and HF mice after 3 days (P<0.01). There was no difference in liver lipid between LF and HF fed mice. C. After 1 week on diet there was no difference in liver lipid between LF and PF mice but HF mice had more lipid than LF (P<0.001) and PF (P<0.01) mice (n = 6–8). D & E. Liver (TAG), measured by Kone analysis. D. LF and HF levels are not significantly different while PF mice show a lower level of TAG compared to LF fed mice (P<0.05) after 3 days on diet. E. LF and HF levels are not significantly different while PF mice show a lower level of TAG compared to LF fed mice (P<0.05) after 1 week on diet.
Figure 3
Figure 3
A. Plasma triacylglycerol (TAG) was significantly lower in HF fed mice at both 3 days and 1 week on diet (P<0.001), but did not differ at 16 weeks on diet. B. Plasma non esterified fatty acids (NEFA)/free fatty acids were unchanged by a HF diet at all times tested. C. Liver TAG levels were unchanged in the HF fed mice at 3 days and 1 week and significantly higher after 16 weeks on HF diet (P<0.05). D. Liver NEFA levels were unchanged at 3 days and 1 week on HF diet but were significantly lower after 16 weeks on HF diet (P<0.001) E. Plasma low-density lipoprotein (LDL) cholesterol was elevated in HF fed mice after 3 days (P<0.01) and 16 weeks (P<0.001) on diet, but was not different from LF values after 1 week. F. Plasma high-density lipoprotein (HDL) cholesterol followed the same pattern as LDL cholesterol and was higher in HF fed mice at 3 days (P<0.01) and 16 weeks (P<0.001) on diet, but was not different from LF after 1 week of diet (n = 6–8).
Figure 4
Figure 4
A–D. Intraperitoneal glucose tolerance tests (IPGTT) in LF fed mice •, HF fed mice • and PF fed mice ▪, fed the HF diet restricted to the caloric intake of the LF fed mice, after 3 days, 1, 12 and 16 weeks after the start of diet. IPGTT was carried out as a non-recovery procedure as the effect of fasting and glucose administration can alter gene expression for some time afterwards. (n = 6–8). E. Comparison of the % difference in total AUC between LF and HF mice over time using a two-way ANOVA showed that when time points 3 days and 16 weeks were included that diet and time had a significant effect on glucose tolerance (F1,58 = 94.56, P<0.01) and (F4,58 = 11.22, P<0.01) respectively and that there was an interaction between diet and time (F4,58 = 2.83, P<0.05). However, when these time points were omitted there was no effect of time or diet indicating that there was no change in AUC between 1 and 12 weeks. One way ANOVA of the difference between LF and HF total AUC revealed that is was higher at 3 days (P<0.01) and 16 weeks (P<0.01) compared to the other time points tested and that there was no change in the % difference in total AUC between 1, 4, 8 and 12 weeks on diet. F. A comparison of total AUC in LF, PF and HF fed mice after 3 days on diet shows the PF (P<0.05) and HF (P<0.01) mice have higher total AUC compared to LF mice but that the total AUC for PF and HF was not significantly different (NS). G. A comparison of total AUC in LF, PF and HF fed mice after 1 week on diet shows a different pattern with no difference in total AUC between LF and PF mice (NS). The total AUC for HF mice is significantly higher than PF (P<0.01) and LF mice (P<0.05) (n = 6–8).
Figure 5
Figure 5
A. Plasma leptin levels were significantly higher in HF fed mice after 3 days on diet (P<0.05), did not differ after 1 week on diet (NS) and increased greatly after 16 weeks on HF diet (P<0.001). B. Plasma insulin levels were not different in HF mice compared to LF fed mice after 3 days or 1 week on diet (NS) but were increased after 16 weeks (P<0.05). C. Fasted glucose levels approached statistical significance for HF vs. LF after 3 days and 1 week on diet (P = 0.08 and P = 0.052 respectively) but was not different at 16 weeks. Fasted glucose in PF mice was significantly lower than LF mice at both 3 days and 1 week of feeding (P<0.05) (n = 6–8).
Figure 6
Figure 6. Representative 2D Coomassie stained gel of mouse plasma after 3 days on the HF diet. Bio-Rad, 11 cm, immobilized pH gradient (IPG) strips (pH 3–10) were used for the separation of plasma proteins in the first dimension.
After the first dimension the IPG strip was applied to the top of a precast Criterion XT Bis-Tris 3–12% IPG+ 1 well gel cassette and 5 µl of All Blue Precision Protein Standards (Bio-Rad) were loaded in the reference well. The gels were fixed and stained with Coomassie Blue. Numbered spots indicate those with significantly different average normalised volumes (P<0.05) (n = 5) in HF compared to LF mice. Proteins were identified by LC/MS/MS. Spots inside dotted lines have been identified as the same protein. See Table 1 for protein identification.
Figure 7
Figure 7
A. Plasma protein levels of haptoglobin (P<0.05), alpha-1-antichymotrypsin (aACT) (P<0.01) and ApoA-IV (P<0.001) were all significantly higher in HF compared to LF after 3 days on the diet. B. After 1 week on the diet plasma protein levels of ApoA-IV were significantly greater in HF fed mice compared with LF (P<0.01). There was no significant difference in haptoglobin or aACT protein levels between mice on HF vs. LF diets after 1 week. C. After 16 weeks on the diet there was significantly greater level of haptoglobin protein in plasma from HF compared to LF (P<0.01), there was no significant difference in the levels of ApoA-IV or aACT in HF compared to LF (n = 6–8). D. Alpha-1-antichymotrypsin (aACT) plasma protein levels were significantly greater in HF compared to LF (P<0.01) and PF (P<0.001) at 3 days. There were no significant differences in aACT plasma protein levels after 1 week. E. ApoA-IV plasma protein was significantly increased in HF compared to LF at 3 days (P<0.001). At 1 week ApoA-IV plasma protein levels were higher in HF compared to both LF (P<0.001) and PF (P<0.05). The amount of ApoA-IV protein was also greater in plasma from PF mice compared to LF (P<0.01) after 1 week. F. Levels of haptoglobin protein in plasma from HF mice were significantly greater at 3 days compared to LF (P<0.05). There was no significant difference in plasma protein levels of haptoglobin between the different diets after 1 week (n = 6–8).
Figure 8
Figure 8
A. Plasma levels of IL-6 were increased after 3 days on a HF diet (P<0.05) but were unchanged after 1 week and 16 weeks on diet. B. Plasma levels of TNFα were not significantly different at any of the time points tested but individual measurements were variable. C. As with TNFα plasma levels of IL -1β were not significantly different at any of the time points tested and individual measurements were variable particularly in HF fed mice after 3 days on diet (n = 6–8).
Figure 9
Figure 9
A. SerpinA3N gene expression in the liver was significantly up-regulated in a HF diet at all times tested 3 days (P<0.01), 1 week (P<0.001) and 16 weeks (P<0.01). B. Haptoglobin gene expression was unchanged in HF fed mice liver after 3 days on diet but was increased after 1 week (P<0.01) and 16 weeks (P<0.01). C. SAA gene expression was significantly higher on a HF diet after 3 days (P<0.01) and 1 week (P<0.001) but was not different after 16 weeks. D. ApoA-IV gene expression was significantly down-regulated in the liver of HF fed mice after 3 days (P<0.05) and 1 week (P<0.01) on diet but was unchanged after 16 weeks on diet. E. In comparison ApoA-IV gene expression was significantly up-regulated in the ileum of HF fed mice after 3 days, 1 week and 16 weeks on diet (P<0.05). F. SerpinA3N gene expression in the liver was significantly down-regulated with time on a LF diet between 3 days and 16 weeks (P<0.001). Units are fold expression changes between experimental groups relative to B2M and calculated from the ΔΔC t values (n = 6–8).
Figure 10
Figure 10
A. The area of F4/80 staining in the liver measured as arbitrary units (AU) was significantly increased in HF fed mice at all the time points tested (P<0.01) at 3 days and (P<0.001) at all other time points. Values appeared to peak at 4 weeks on the HF diet. Representative images of F4/80 staining (brown) for HF and LF fed mice after 1 week on diet. Mag X 200. B. MRC-1 gene expression was significantly decreased after 3 days on a HF diet (P<0.001) but was unchanged after 1 week and 16 weeks. None of the other genes tested Il-1β, IL-6, TNFα, CXCL1, F4/80, MRC-1, ARG and ITgax showed any change with time on a HF diet. IL-10 expression was undetectable (data not shown) (n = 6-8). C. IL-1β gene expression in the liver is up-regulated between 3 days and 16 weeks on a LF diet (P<0.05). Units are fold expression changes between experimental groups relative to B2M and calculated from the ΔΔC t values (n = 6–8).
Figure 11
Figure 11
A. IL-1β gene expression in muscle was unchanged after 3 days of HF diet but was up-regulated at 12 week (P<0.001) and 16 weeks (P<0.01) B. IL-6 gene expression was not detected (ND) after 3 days on a HF diet but was up-regulated after 12 week (P<0.01) and 16 weeks (P<0.001) C. TNFα gene expression was unaffected by a HF diet at 3 days and 12 weeks but was up-regulated at 16 weeks (P<0.001) Units are fold expression changes between experimental groups relative to GAPDH and calculated from the ΔΔC t values. D. Oil red O staining showed an increase in muscle lipid after 12 weeks on HF diet. Mag X 100 (n = 6–8). E IL-1β gene expression in muscle is strongly up-regulated between 3 days and 1 week (P<0.001) and between 3 days and 16 weeks (P<0.001) on a LF diet. F. TNFα gene expression in muscle is up-regulated over time on a LF diet between 3 days and 1 week (P<0.001) and between 3 days and 16 weeks (P<0.05). Units are fold expression changes between experimental groups relative to GAPDH and calculated from the ΔΔC t values (n = 6–8).
Figure 12
Figure 12
A. The area of F4/80 staining in the WAT measured as arbitrary units (AU) was increased in HF fed mice at 12 and 16 weeks compared to the LF diet and almost reached significance at 16 weeks (P = 0.058). B. IL-1β gene expression in white adipose tissue (WAT) was slightly up-regulated after 3 days of HF diet but was also below the level considered to be significant (P = 0.06) and was unchanged at 1 week and up-regulated at 16 weeks (P<0.05). C. IL-6 gene expression was up-regulated after 3 days on a HF diet (P<0.05), unchanged after 1 week and marginally up-regulated at 16 weeks (P = 0.054). D. TNFα gene expression was unchanged after 3 days and 1 week on HF diet but was up-regulated at 16 weeks (P<0.001). E. CXCL-1 gene expression was unchanged at 3 days and 1 week on a HF diet but was up-regulated after 16 weeks (P<0.001). F. F4/80 gene expression was unchanged after 3 days and 1 week on a HF diet and up-regulated 16 weeks (P<0.01) G. MRC-1 gene expression was unchanged after 3 days and 1 week of HF diet but was up-regulated 16 weeks (P<0.01) H. IL-10 gene expression was unchanged after 3 days and 1 week on a HF diet and was up-regulated at 16 weeks (P<0.01) I. ARG gene expression was unchanged after 3 days and 1 week of HF diet but was up-regulated 16 weeks (P<0.01). J ARG gene expression in WAT is unchanged between 3 days and 1 week and up-regulated between 3 days and 16 weeks (P<0.05) on a LF diet. K. IL-6 gene expression in WAT is unchanged between 3 days and 1 week and up-regulated between 3 days and 16 weeks on a LF diet (P<0.01). L. IL-1β gene expression in WAT is unchanged between 3 days and 1 week and up-regulated between 3 days and 16 weeks (P<0.01) on a LF diet. Units are fold expression changes between experimental groups relative to B2M and calculated from the ΔΔC t values (n = 6–8).

Similar articles

See all similar articles

Cited by 39 PubMed Central articles

See all "Cited by" articles

References

    1. Xu H, Barnes GT, Yang Q, Tan G, Yang D, et al. (2003) Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 112: 1821–1830. - PMC - PubMed
    1. Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444: 860–867. - PubMed
    1. Davis JE, Gabler NK, Walker-Daniels J, Spurlock ME (2008) Tlr-4 deficiency selectively protects against obesity induced by diets high in saturated fat. Obesity (Silver Spring) 16: 1248–1255. - PubMed
    1. Caricilli AM, Nascimento PH, Pauli JR, Tsukumo DM, Velloso LA, et al. (2008) Inhibition of toll-like receptor 2 expression improves insulin sensitivity and signaling in muscle and white adipose tissue of mice fed a high-fat diet. J Endocrinol 199: 399–406. - PubMed
    1. Kleinridders A, Schenten D, Konner AC, Belgardt BF, Mauer J, et al. (2009) MyD88 signaling in the CNS is required for development of fatty acid-induced leptin resistance and diet-induced obesity. Cell Metab 10: 249–259. - PMC - PubMed

Publication types

Grant support

LMW, FMC, JED, CG, GH, ACM, PN, AJF, NH, WDR, SMH were funded by the Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS). AT and KC were funded by the German Ministry of Research and Education (Ref. No: 0315087), HTN and I-LS were funded by The Research Council of Norway (project no. 196112/H10. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Feedback