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5.2. B. longum APC1472 administration improves glucose tolerance, circulating levels of leptin and corticosterone in obese mice Effects of HFD feeding ( F (5, 155) = 3.321, p = 0.007) and B. longum APC1472 supplementation ( F (5, 155) = 4.792, p < 0.001) were observed, as well as an interaction effect between these two fac- tors and time ( F (5, 155) = 3.307, p = 0.007) in the glucose toler- ance test. Supplementation with B. longum APC1472 normalized glucose levels after 15 mins of glucose administration in HFD-fed obese mice ( p = 0.006) and significantly decreased glucose after 90 ( p =0.019) and 120 min ( p = 0.018) respectively ( Fig. 3A ) as determined by 2 way ANOVAs at each individual timepoint. Moreover, HFD feeding ( F (1, 33) = 29.761, p <0.001), B. longum APC1472 ( F (1, 33) = 4.425, p = 0.043) and interaction effects between these two factors ( F (1, 33) = 5.337, p = 0.027) were also observed when analysing the area under the curve (AUC) for glucose levels ( Fig. 3B ), with B. longum APC1472 administration significantly reducing AUC in HFD-fed mice ( p = 0.003) as determined by post-hoc comparison ( Fig. 3B ). In ad- dition, both a HFD feeding (F (1, 33) = 9.167, p = 0.005) and a B. longum APC1472 effect ( F (1, 33) = 4.796, p = 0.036) were observed for non-fasting insulin levels ( Fig. 3C) . Interestingly, B. longum APC1472 reduced non-fasting insulin levels in LFD-fed mice ( p =0.054) but not in HFD-fed mice ( Fig. 3C) . However, for fasting glucose lev- els, only a HFD feeding effect was observed (F (1, 32) = 29.153, p < 0.001) ( Fig. 3D ). Moreover, both a HFD feeding (F (1, 31) = 30.926, p < 0.001) and a B. longum APC1472 effect ( F (1,
31) = 17.917, p < 0.001) were observed for epididymal insulin recep- tor substrate 1 ( IRS-1 ) expression ( Fig. 3E ). Post-hoc comparisons de- termined that B. longum APC1472 significantly reduced IRS -1 expres- sion in both LFD ( p = 0.002) and HFD-fed mice ( p =0.011) ( Fig. 3E ). Both a HFD feeding ( F (1, 33) = 38.023, p < 0.001) and a B. longum APC1472 ( F (1, 33) = 5.340, p = 0.027) effect as well as an interac- tion effect (F (1, 33) = 4.237, p = 0.048) were observed for fasting leptin levels ( Fig. 3F) . The effect of HFD on leptin levels was attenu- ated by B. longum APC1472 treatment ( p = 0.004). Finally, we found a significant B. longum APC1472 treatment effect ( F (1, 32) = 7.774, p = 0.009) for plasma corticosterone levels ( Fig. 3G ). Administration of B. longum APC1472 significantly decreased plasma corticosterone lev- els in HFD-fed mice ( p =0.011) ( Fig. 3G ), which may have contributed to its overall impact on glucose homeostasis . 5.3. B. longum APC1472 induces changes of hypothalamic neuropeptide expression in mice Analysis of the gene expression levels of hypothalamic neuropep- tides involved in appetite modulation revealed a significant HFD effect on the gene expression of the orexigenic peptide agouti-related protein ( AgRP ) ( F (1, 33) = 10.412, p = 0.003) but a non-significant reduction in neuropeptide Y ( NPY ) expression ( Figure S3 ). Interestingly, both a B. longum APC1472 effect ( F (1, 33) = 7.820, p = 0.009) and an inter- action effect ( F (1, 33) = 5.881, p = 0.021) were observed for cocaine- and amphetamine-regulated transcript ( CART ) expression ( Figure S3 ). Indeed, B. longum APC1472 administration significantly reduced CART
Fig. 3. Bifidobacterium longum APC1472 improved glucose tolerance, leptin plasma levels and stress-induced corticosterone circulating levels in high-fat diet-induced obesity in mice. (A and B) Glucose tolerance test (GTT) glucose curve and area under the curve (AUC) after 1 g/kg glucose challenge, (C and D) non-fasting and fasting insulin plasma levels, (E) fasting leptin plasma levels, (F) epididymal fat insulin receptor substrate (IRS) − 1 mRNA expression and (G) fasting-induced corticosterone plasma in control mice treated with drinking water containing sterile PBS (2% v/v) and glycerol (0.5% v/v) and fed a control low-fat diet (LFD) ( n = 10 in A, B, C, E, F and G) or a high-fat diet (HFD) ( n =9inA,B,C,D,EandG; n =8 in F) and in mice treated with B. longum APC1472 in drinking water (2 × 10 8 CFU/mL) and fed a LFD ( n =9inA,B,C,D,EandF; n =8inG)oraHFD( n =9inA,B,C,D,EandF; n =8 in G) for 15 (A, B,C) or 16 weeks (D, E, F and G). Data are shown as mean ± SEM. Data are significant different ( p <0.05) accordingly to Repeated Measures ANOVA (A) or two-way ANOVA followed by LSD post-hoc test (B, C, D, E, F and G). * indicates significant diet treatment effect (* p <0.05, ** p <0.01, *** p <0.001) and # indicates significant B. longum APC1472 treatment effect ( # p <0.05, ## p <0.01).
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