We thank Johnston and colleagues for their comments and this opportunity to discuss the questions regarding our recent investigation on the role of Toll-like receptor 4 (TLR4) in the development of fructose-induced hepatic steatosis in mice.1 Our answer will address their main points:

First, as pointed out by Johnston and colleagues, we found in an earlier study that chronic intake of different sugars can affect dietary pattern and caloric intake and subsequently weight gain in a specific manner in C57BL/6J mice.2 In these studies, we found that fluid intake of mice varies according to the type of sugar offered in the drinking solution (glucose > sucrose > fructose > water). Contrary to this previous study, mice in the present study were either fed water or fructose solution. In the present study, we determined fluid intake of mice to ascertain that mice had a similar exposure to fructose (data not shown before). Indeed, despite the differences in absolute weight gain, both C3H/HouJ and C3H/HeJ mice consumed on average ∼0.30 mL of 30% fructose solution/day and per gram body weight, amounting to an average fructose intake of ∼0.09 g/day and per gram body weight. In line with the findings of our study, Tsukumo et al.,3 who fed C3H/HeJ mice a high-fat diet for 2-8 months, also reported that the TLR4 mutant mice gained less weight in comparison to wild-type mice, despite no differences in total food intake.

Second, it was also pointed out by Johnston and colleagues that when reanalyzing our data on hepatic triglyceride content, they only found a relative influence of ∼10% after the loss of a functional TLR4. We reanalyzed the data according to the suggestions of Dr. Johnston and his colleagues (data are shown in Fig. 1). When analyses of triglyceride data were performed as per their suggestions, triglyceride levels were 3.3-fold higher in livers of fructose-fed wild-type mice in comparison to wild-type water-fed controls, whereas in livers of TLR4 mutant mice, triglyceride levels were only 2.3-fold higher than in livers of the TLR4 mutant mice fed water, resulting in a difference between fructose-fed wild-type and TLR4 mutant mice of 30%. These results are in accordance with our findings for portal endotxin concentration and MyD88 concentration in the liver, shown in figures 2 and 3 of our article.

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Figure 1. Expression of α-SMA was detected in (A) Kunming mouse liver and (B) CCl4-induced liver by immunohistochemistry. Sections were incubated with antibody against α-SMA (Abcam, Cambridge, MA; diluted 1:50) followed by visualization with the Histostain-Plus Kit (Invitrogen, Carlsbad, CA) after treatment with 3,3-diaminobenzidine and counterstain with hematoxylin (magnification = ×1000).

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  • 1
    Spruss A, Kanuri G, Wagnerberger S, Haub S, Bischoff SC, Bergheim I. Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice. HEPATOLOGY 2009; 50: 1094-1104.
  • 2
    Bergheim I, Weber S, Vos M, Kramer S, Volynets V, Kaserouni S, et al. Antibiotics protect against fructose-induced hepatic lipid accumulation in mice: role of endotoxin. J Hepatol 2008; 48: 983-992.
  • 3
    Tsukumo DM, Carvalho-Filho MA, Carvalheira JB, Prada PO, Hirabara SM, Schenka AA, et al. Loss-of-function mutation in Toll-like receptor 4 prevents diet-induced obesity and insulin resistance. Diabetes 2007; 56: 1986-1998.

Ina Bergheim*, Astrid Spruss*, * Department of Nutritional Medicine and Prevention, University of Hohenheim, Stuttgart, Germany.