*2.6. Overall Discussion*

Using the 2 × 2 factorial design, we demonstrated that irrespective of the diet, FX increased both O2 consumption and CO2 production, indicating that FX enhanced metabolic rate. This increase in energy expenditure agreed with smaller adipose mass and increases of the PGC-1΅ network gene expressions in iWAT and eWAT. These included Ά3-AR, PGC-1΅, CIDEA, Dio2, PPAR΅ and genes regulating mitochondria biogenesis and fusion (ERR΅, NRF1, NRF2, Mfn1, Mfn2 and OPA1). We speculate that dietary FX might increase the energy expenditure through changes in mitochondria biogenesis, homeostasis and/or activity mediated by the PGC-1΅ network in eWAT and iWAT. 

PGC-1΅ is a master regulator of energy metabolism that orchestrates cellular responses to various types of metabolic stress, such as fasting, cold temperature and physical exertion. The expression of PGC-1΅ is induced by such factors as Ά3-AR and Dio2. It in turn activates the expressions of transcription factors, including PPARs, NRF1/2, ERR΅ and Mfn2. In this study, FX upregulated PGC-1΅ and both of its upstream and downstream genes in iWAT and eWAT, implying that the "PGC-<sup>1</sup>΅ network" is upregulated. As this network is known to regulate energy metabolism, it coincides with our observation that FX increased energy expenditure. 

**Figure 4.** Mitochondrial biogenic gene expressions in epididymal (**A**) and inguinal (**B**) white adipose tissue. Values are means, and error bars are SEM (*<sup>n</sup>* = 4). \* denotes significant effect by either dietary factor at *p* < 0.05 analyzed by two-way ANOVA. HS, HS + F, HF and HF + F: As indicated in Figure 1. The measurement of relative mRNA expression is as indicated in Figure 2. 


**Figure 5.** Mitochondrial homeostatic gene expressions in epididymal (**A**) and inguinal (**B**) white adipose tissue. Values are means, and error bars are SEM (*<sup>n</sup>* = 4). \* denotes significant effect by either dietary factor at *p* <sup>&</sup>lt; 0.05 analyzed by two-way ANOVA. HS, HS + F, HF and HF + F: As indicated in Figure 1. The measurement of relative mRNA expression is as indicated in Figure 2. 

As we only measured the whole body O2 consumption, it is difficult to specify the contribution of different tissues by FX. However, comparison of changes in the gene expression pattern in WATs and BAT prompt us to speculate that WAT might contribute more than BAT in promoting O2 consumption. 

Moreover, our data together with data of previous reports did not support a major role of BAT in the FX enhanced oxygen consumption. These include: (1) FX downregulated Ά3-AR, Mfn2, Fis1, Prdm16 and PPARΈ mRNA levels in the BAT and did not change other thermogenic genes in this study; (2) some studies [31,38] found an anti-obesity effect of FX without BAT enlargement; (3) oxygen consumption did not correlate to BAT mass in our data (Supplementary Information, Figure S1); (4) FX lowered both serum NE (norepinephrine) and BAT Ά3-AR expression, which are key regulators of BAT adaptive thermogenesis. Lowered serum NE might be related to a decreased sympathetic activity or an increased NE degradation. It is not known why and how FX lowered serum NE and downregulated BAT Ά3-AR expression. Although it might not be applicable to FX, there has been a study showing that beta-carotene suppresses exhaustive exerciseinduced plasma levels of adrenocorticotropic hormone, norepinephrine and epinephrine by inhibiting the secretion of corticotropin-releasing hormone [58]. Although NE concentration is very important for the activation of PGC-1alpha networks in whole body, factors other than NE, such as PPARs and thyroid receptor activation, *etc.*, can also lead to the activation of the PGC-1 ΅ network. 

In this study, FX did not significantly increase UCP1 mRNA expression in iWAT, eWAT and BAT ( *p* > 0.05). In contrast, expressions of CIDEA and the PGC-1 ΅ network in eWAT and iWAT were upregulated, although to different extents. These changes were not noted in liver and skeletal muscle. Moreover, differential extents of induction were also observed in other study. For example, mRNA expression levels of adipogenic marker genes (PPAR·, aP2, adiponectin, C/EBP ΅, FATP1, LPL and UCP2) and thermogenic genes and mitochondrial biogenesis genes (UCP1, PGC-1 ΅, NRF1, TFAM, Prdm16, CIDEA and Elovl3) induced by triiodothyronine (T3) were also to different extents [59]. 

## **3. Experimental Section**
