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- Materials and methods
- Conflict of interest statement
Maturity onset diabetes of the young (MODY) represents a spectrum of disorders characterized by autosomal dominant inheritance, early onset (usually before the age of 25 years) and β-cell dysfunction. The exact prevalence is unknown but MODY has been estimated to account for 1–2% of individuals with type 2 diabetes (T2D) . Heterozygous mutations of the transcription factor 1 (TCF1) gene, encoding hepatocyte nuclear factor 1α (HNF1α), cause MODY3 . More than 200 different mutations in the TCF1 gene have been reported [3, 4]. The clinical expression of MODY3 is highly variable which may, at least partially, be explained by the type and location of the mutation .
Bile acid (BA) synthesis serves as the major elimination route of excess cholesterol, to maintain cholesterol homoeostasis. In the liver, cholesterol is converted to 7α-hydroxycholesterol by cholesterol 7α-hydroxylase [also known as cytochrome P450 7A1 (CYP7A1)], the rate-limiting enzyme of the classic pathway of bile acid (BA) synthesis, which is then converted to 7α-hydroxy-4-cholesten-3-one (C4). Cholesterol is also oxidized by CYP27A1 and CYP7B1 in an alternative pathway that exists in all tissues. In humans, the classic pathway is mainly responsible for BA synthesis. It has been reported that serum levels of C4 reflect BA synthesis and correlate with the enzymatic activity of CYP7A1 [5-8]. Hence, determination of serum C4 levels allows investigation of BA synthesis in vivo without collection of liver biopsies.
Farnesoid X receptor (FXR) is regarded as the BA nuclear receptor as it regulates the expression of several key genes involved in BA synthesis, metabolism and transport [9, 10]. In the liver, FXR activation upregulates the expression of small heterodimeric partner (SHP), which then forms a heterodimer with liver-related homologue 1 (LRH-1) leading to repression of CYP7A1 [11, 12]. In the intestine, FXR activation induces the synthesis of fibroblast growth factor 19 (FGF19). Secreted FGF19 binds to its cognate receptor (FGFR4) which activates intracellular pathways that target the expression of CYP7A1 [13, 14].
BAs actively reabsorbed in the terminal ileum by the apical sodium-dependent bile salt transporter (ASBT/SLC10A2) contribute substantially to levels in the enterohepatic circulation. Mice lacking Asbt have increased faecal BA excretion and increased Cyp7a1 expression . In humans, mutations in the ASBT gene lead to BA malabsorption , and patients with obstructive cholestasis have lower intestinal ASBT expression .
The role of HNF1α in the regulation of cholesterol homoeostasis has been investigated in Tcf1−/− mice . These mice were reported to have defective BA transport, increased BA and cholesterol synthesis, and impaired HDL metabolism. By contrast, patients with MODY3 have been reported to have similar HDL cholesterol and apolipoprotein A1 levels as healthy control subjects , but whether these patients have altered BA and cholesterol synthesis remains unknown. The aim of this study was to investigate whether patients with MODY3 have altered BA synthesis, cholesterol synthesis and intestinal cholesterol absorption, and to investigate the effects of TCF1 mutations on some of the key promoters in BA metabolism.
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- Materials and methods
- Conflict of interest statement
In this study, we used serum biomarkers to investigate possible alterations in cholesterol homoeostasis in patients with MODY3, compared with healthy control subjects. We found that BA synthesis was increased, whereas markers of cholesterol synthesis and intestinal cholesterol absorption were not different in patients with MODY3 compared with control subjects. Promoter studies in human hepatoma (HuH7) and intestinal (Caco2) cells showed that the mutations in our patients with MODY3 affect promoters involved in BA metabolism.
Cholesterol homoeostasis is maintained by balancing endogenous cholesterol synthesis and intestinal cholesterol absorption with excretion of biliary cholesterol and BAs. Dyslipidaemia, characterized by a high plasma triglyceride concentration, low HDL cholesterol and an increased level of small dense LDL particles, is very common in individuals with T2D . Additionally, increased cholesterol synthesis has been reported in those with T2D . Thus, monogenic forms of diabetes may also lead to unfavourable effects on lipid metabolism. However, MODY1 patients with a specific mutation (K99fsdelAA) in the gene encoding HNF4α have lower plasma triglyceride levels compared with control subjects , and we have previously shown that such patients have lower levels of very low-density lipoprotein and LDL esterified cholesterol . In the same study, we also found that patients with MODY3 have similar lipoprotein profiles as healthy control subjects . However, there was a trend towards lower LDL cholesterol levels, although – probably due to the small study sample size – this did not reach statistical significance. Unfortunately, in the present study, we were not able to analyse lipoprotein profiles as samples that have not undergone freeze-thawing are required (but were not available) for accurate measurements. Thus, whether the higher BA synthesis in patients with MODY3, without significant changes in cholesterol synthesis and cholesterol absorption, affects LDL cholesterol levels need to be further investigated. However, these results suggest that the dyslipidaemic phenotype reported in T2D is not present in individuals with MODY3.
The observed lipidaemic phenotype in patients with MODY3 is in contrast to mice lacking Tcf1  in which increased cholesterol synthesis and impaired HDL metabolism were reported. However, there are major species-related differences between mice and humans with respect to cholesterol metabolism. For example, plasma cholesterol is mainly transported in LDL in humans and in HDL in mice, humans have much lower levels of cholesterol synthesis than mice , and the feed-forward regulation of Cyp7a1 by cholesterol (mediated by liver X receptor α) in mice is absent in humans . Moreover, most patients with MODY3 have heterozygous mutations , whereas Tcf1+/− mice have been reported to be similar to wild-type mice . Thus, the effects in Tcf1−/− mice are expected to be more severe and may not be clinically relevant.
For ethical reasons, we were not able to investigate the molecular mechanisms responsible for the increased BA synthesis in patients with MODY3 in vivo. In an attempt to gain some mechanistic insights, each mutation was introduced into an expression vector for HNF1α and used for transient cotransfection of human hepatoma (HuH7) and intestinal (Caco2) cells along with promoters for CYP7A1, FXR, SHP, FGF19 and ASBT. The human HNF1α protein consists of 631 amino acids (aa) subdivided into three functional regions: an amino-terminal dimerization domain (aa 1–32), a DNA-binding motif (aa 203–279) and a carboxyl-terminal transactivation domain (aa 281–631) . Dimerization is necessary for interaction with specific cis-acting elements in promoters of target genes, as deletion of the dimerization domain prevents DNA binding. Thus, it has been suggested that the transactivation domain may be more tolerant of minor structural changes than the DNA-binding motif .
P379A is a missense mutation located in the transactivation domain, and it has previously been reported that the transactivation potential is decreased compared with HNF1α . However, we found that this mutation increased the activation of the CYP7A1 promoter compared with HNF1α (Fig. 1), whereas no other major differences were observed for the FXR (Figs 3 and 5), SHP (Figs 4 and 6), FGF19 (Fig. 7) or ASBT (Fig. 8) promoters. The discrepancy between these findings and those of the previous study  may be due to the different cell lines used [i.e. mouse pancreatic (Min6) and African green monkey kidney (Cos7) cells, compared with our human hepatoma and intestinal cells]. Other possible reasons for the inconsistent results include species differences and the use of different promoters and expression vectors.
P291fsinsC, the most common mutation in HNF1α, is a frameshift mutation leading to synthesis of a 315-aa protein lacking most of the transactivation domain . Consistent with this, we found that P291fsinsC reduced or abolished the activation of the CYP7A1 and FXR promoters in HuH7 cells as well as of the FXR and ASBT promoters in Caco2 cells. The reduced activation of FXR was followed by reduced repression, at the higher concentration (1.0 μg), of the SHP promoter in HuH7 cells (Fig. 4) but not in Caco2 cells.
R131Q is a missense mutation that is not defective in the DNA-binding motif but leads to decreased transactivation potential compared with HNF1α . In line with this, we found that R131Q reduced the activation of CYP7A1 (Fig. 1) and FXR (Fig. 3) promoters in HuH7 cells and of the ASBT promoter in Caco2 cells (Fig. 8). However, similar activation of the FXR (Fig. 5) and SHP (Fig. 6) or increased activation of the FGF19 (Fig. 7) promoters were observed in Caco2 cells. Thus, it seems that the effects of this mutation are also dependent on the promoter and the cell line that are used.
L107I is a missense mutation that has been reported to result in loss of function of the protein and impaired transcriptional activity due to impaired DNA binding . In the present study, the transfection experiments showed reduced activation of the CYP7A1 (Fig. 1) and FXR (Fig. 3) promoters in HuH7 cells and of the ASBT promoter in Caco2 cells (Fig. 8). By contrast, no major differences (FXR and SHP, Figs 5 and 6, respectively) or increased (FGF19, Fig. 7) activation were observed in Caco2 cells. Again, it appears that differences in promoters and/or cell lines may affect the results.
T260M is a missense mutation within the DNA-binding motif [3, 47]. We showed that this mutation abolished the activation of the CYP7A1 (Fig. 1), FXR (Fig. 3) and SHP (Fig. 4) promoters in HuH7 cells as well as of the ASBT promoter in Caco2 cells (Fig. 8). It is interesting that our results in Caco2 cells showed decreased activation of the FXR promoter (Fig. 5) at the higher concentration (1.0 μg), but similar effects on SHP (Fig. 6) and FGF19 (Fig. 7) promoters.
FXR is expressed at high levels in the human liver and intestine and may inhibit the expression of CYP7A1 by indirect mechanisms and not via direct binding to CYP7A1 . These indirect mechanisms may be initiated by the FXR/SHP/LRH-1 pathway in the liver [11, 12] or the FXR/FGF19/FGFR4 pathway in the intestine [13, 14]. We used two human cell lines in this study, one hepatic and one intestinal, to investigate whether mutations in the gene encoding HNF1α may affect the expression of CYP7A1 via direct or indirect mechanisms. Only one of the MODY3 mutations (P379A) increased the CYP7A1 promoter activity compared with HNF1α, suggesting direct effects on CYP7A1 expression by this mutation. The other four mutations reduced the FXR promoter activity and then reduced repression of SHP, suggesting indirect effects of these mutations on the expression of CYP7A1. Although two of these mutations (T260M and P291fsinsC) significantly reduced FXR promoter activity in Caco2 cells, this was not followed by reduced SHP or FGF19 promoter activity in these cells, suggesting that the hepatic FXR/SHP/LRH-1 pathway may be responsible for the effects on CYP7A1 expression in subjects carrying these four mutations. This is also supported by the significant correlation between serum C4 levels and FXR and SHP promoter activities in HuH7 cells.
The mutations found in the patients with MODY3 in this study were within different domains of the HNF1α protein. However, whether the mutation was within the DNA-binding motif (i.e. T260M) or within the transactivation domain (i.e. P379A or P291fsinsC) did not affect the severity of the effect. Furthermore, the P379A and P291fsinsC mutations did not produce similar effects on the promoters used in this study. Instead, P379A seemed to have direct effects on the CYP7A1 promoter, which may, at least partially, contribute to the increased BA synthesis in individuals with this mutation. For the other four mutations (including P291fsinsC), reduced hepatic FXR activation followed by reduced repression of SHP may contribute to the higher degree of BA synthesis in individuals with MODY3. Thus, different MODY3 mutations may alter different targets; however, all these mutations lead to increased BA synthesis compared with controls.