Introduction

Urea is the main nitrogenous end product of mammalian protein catabolism, generated in the liver via the urea-ornithine cycle (G. Stewart, 2011). Facilitative urea transporters (UT’s) are a family of urea selective channel proteins which mediate the movement of urea molecules across cell membranes (G. Stewart, 2011). UT-B is the most widely distributed UT, detected in the human colon (Inoue et al., 2004), rat colon (Inoue et al., 2005), brain (R. A. Fenton et al., 2002), testes (R. A. Fenton et al., 2002), bladder and gastrointestinal tract in mice (N. Lucien et al., 2005) and human urinary (Spector et al., 2007) and gastrointestinal tract (G. Stewart, 2011). A crucial emerging physiological role for UTs is their role in urea nitrogen salvaging (UNS) in the mammalian intestinal tract (G. Steward, 2011). UT-B urea transporters in the epithelial layers mediate urea transport from the bloodstream into the gastrointestinal tract to be catabolised by gastrointestinal bacteria into ammonia and carbon dioxide (G. S. Stewart & Smith, 2005). Bacteria utilise this ammonia as a nitrogen source in the synthesis of amino acids and peptides, which can themselves be reabsorbed by the mammalian host, hence the “salvaging” of urea nitrogen (Fuller & Reeds, 1998). Short chain fatty acids (SCFAs) are a subset of fatty acids produced by anaerobic bacterial populations following fermentation of partially and non-digestible dietary fibres and resistant fibes (Kasubuchi et al., 2015). Most abundant SCFASs acetate, propionate and butyrate are most highly concentrated in the proximal colon at 70-140 mM, decreasing to 20-70 mM in the distal colon (Li et al., 2012). Monocarboxylaye transporters, such as MCT1, mediate the transport of SCFAs such as butyrate (Jackson & Halestrap, 1996). These play a critical role in cellular metabolism, particularly in catalysing the transport of lactate, the end product of glycolysis, in and out of cells based on their metabolic needs (Vijay & Morris, 2014) (Figure 1)

In cancer, the Warburg effect refers to an altered metabolism whereby glucose uptake and fermentation of glucose to lactate is increased (Liberti and Locasale, 2016). Upregulated MCT1 expression has been observed during tumour progression, required for increased lactate transport (Payen et al., 2020).

This study investigated the regulation of MCT1 and UT-B in human colorectal adenocarcinoma cell line DLD-1 in response to SCFA butyrate.

River dolphin aerial survey using the blimp Figure 1 Schematic illustrating the production of short chain fatty acids (SCFAs) and potential pathways through which they influence gut-brain communication.

Materials & Methods

DLD-1 cells were cultured under standard conditions and treated treated with sodium butyrate for 48 hours. Varied concentrations of sodium butyrate were used. Western blotting was employed to measure MCT1 and UT-B protein levels in control vs treated samples.

Densitometry analysis was performed using ImageJ software. Protein ratios (treatment/control) were generated and recorded for statistical analysis.

Results

Effect of 10mM sodium butyrate on MCT1 expression

Effect of 10mM sodium butyrate on MCT1 expression

##             Df Sum Sq Mean Sq F value Pr(>F)
## group        1 0.6842  0.6842   3.706  0.103
## Residuals    6 1.1078  0.1846
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = value ~ group, data = data)
## 
## $group
##             diff       lwr       upr     p adj
## y1-x1 -0.5848769 -1.328333 0.1585794 0.1025548
**Fig.1** Effect of 10mM sodium butyrate on MCT1 expression in human DLD-1 cells treated for 48hrs.

Fig.1 Effect of 10mM sodium butyrate on MCT1 expression in human DLD-1 cells treated for 48hrs.

Effect of 20mM sodium butyrate on MCT1 expression

Effect of 20mM sodium butyrate on MCT1 expression

##             Df  Sum Sq Mean Sq F value Pr(>F)  
## group        1 0.08807 0.08807   7.479  0.034 *
## Residuals    6 0.07065 0.01177                 
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = value ~ group, data = data)
## 
## $group
##             diff        lwr         upr     p adj
## y2-x2 -0.2098412 -0.3975907 -0.02209173 0.0339677
**Fig.1** Effect of 20mM sodium butyrate on MCT1 expression in human DLD-1 cells treated for 48hrs.

Fig.1 Effect of 20mM sodium butyrate on MCT1 expression in human DLD-1 cells treated for 48hrs.

Effect of 10mM sodium butyrate on UT-B expression

Effect of 10mM sodium butyrate on UT-B expression

##             Df Sum Sq Mean Sq F value Pr(>F)
## group        1 0.0967 0.09672   0.838  0.395
## Residuals    6 0.6925 0.11541
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = value ~ group, data = data)
## 
## $group
##             diff        lwr       upr     p adj
## y3-x3 -0.2199095 -0.8077143 0.3678953 0.3952628
**Fig.1** Effect of 10mM sodium butyrate on UT-B expression in human DLD-1 cells treated for 48hrs.

Fig.1 Effect of 10mM sodium butyrate on UT-B expression in human DLD-1 cells treated for 48hrs.

Effect of 20mM sodium butyrate on UT-B expression

Effect of 20mM sodium butyrate on UT-B expression

##             Df Sum Sq Mean Sq F value Pr(>F)
## group        1 0.0587  0.0587   0.125  0.736
## Residuals    6 2.8282  0.4714
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = value ~ group, data = data)
## 
## $group
##             diff        lwr         upr     p adj
## y2-x2 -0.2098412 -0.3975907 -0.02209173 0.0339677
**Fig.1** Effect of 20mM sodium butyrate on UT-B expression in human DLD-1 cells treated for 48hrs.

Fig.1 Effect of 20mM sodium butyrate on UT-B expression in human DLD-1 cells treated for 48hrs.

Discussion

MCT1 expression reduced in DLD-1 cells treated for 48hrs with sodium butyrate at both 10mM and 20mM concentrations (Figure 1 and 2). Anova and Tukey HSD pairwise comparison revealed only 20mM butyrate treatment had a statistically significant effect on MCT1 expression (p value > 0.05, p = 0.0340). No treatment was statistically significant on UT-B expression in DLD-1 cells. In cancer, the Warburg effect refers to the increased uptake of glucose and fermentation of glucose to lactose, required to support tumourogenesis. Increased concentration of SCFA butyrate down regulated MCT1 expression in DLD-1 cells, demonstrating they do indeed act as cancer cells. These cells are therefore optimal for studying MCT1 but not optimal for studying UT-B regulation.

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