Tcf7l2 in hepatocytes regulates de novo lipogenesis in diet-induced non-alcoholic fatty liver disease in mice

Aims/hypothesis Non-alcoholic fatty liver disease (NAFLD) associated with type 2 diabetes may more easily progress towards severe forms of non-alcoholic steatohepatitis (NASH) and cirrhosis. Although the Wnt effector transcription factor 7-like 2 (TCF7L2) is closely associated with type 2 diabetes risk, the role of TCF7L2 in NAFLD development remains unclear. Here, we investigated how changes in TCF7L2 expression in the liver affects hepatic lipid metabolism based on the major risk factors of NAFLD development. Methods Tcf7l2 was selectively ablated in the liver of C57BL/6N mice by inducing the albumin (Alb) promoter to recombine Tcf7l2 alleles floxed at exon 5 (liver-specific Tcf7l2-knockout [KO] mice: Alb-Cre;Tcf7l2f/f). Alb-Cre;Tcf7l2f/f and their wild-type (Tcf7l2f/f) littermates were fed a high-fat diet (HFD) or a high-carbohydrate diet (HCD) for 22 weeks to reproduce NAFLD/NASH. Mice were refed a standard chow diet or an HCD to stimulate de novo lipogenesis (DNL) or fed an HFD to provide exogenous fatty acids. We analysed glucose and insulin sensitivity, metabolic respiration, mRNA expression profiles, hepatic triglyceride (TG), hepatic DNL, selected hepatic metabolites, selected plasma metabolites and liver histology. Results Alb-Cre;Tcf7l2f/f essentially exhibited increased lipogenic genes, but there were no changes in hepatic lipid content in mice fed a normal chow diet. However, following 22 weeks of diet-induced NAFLD/NASH conditions, liver steatosis was exacerbated owing to preferential metabolism of carbohydrate over fat. Indeed, hepatic Tcf7l2 deficiency enhanced liver lipid content in a manner that was dependent on the duration and amount of exposure to carbohydrates, owing to cell-autonomous increases in hepatic DNL. Mechanistically, TCF7L2 regulated the transcriptional activity of Mlxipl (also known as ChREBP) by modulating O-GlcNAcylation and protein content of carbohydrate response element binding protein (ChREBP), and targeted Srebf1 (also called SREBP1) via miRNA (miR)-33-5p in hepatocytes. Eventually, restoring TCF7L2 expression at the physiological level in the liver of Alb-Cre;Tcf7l2f/f mice alleviated liver steatosis without altering body composition under both acute and chronic HCD conditions. Conclusions/interpretation In mice, loss of hepatic Tcf7l2 contributes to liver steatosis by inducing preferential metabolism of carbohydrates via DNL activation. Therefore, TCF7L2 could be a promising regulator of the NAFLD associated with high-carbohydrate diets and diabetes since TCF7L2 deficiency may lead to development of NAFLD by promoting utilisation of excess glucose pools through activating DNL. Data availability RNA-sequencing data have been deposited into the NCBI GEO under the accession number GSE162449 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162449). Graphical abstract Supplementary Information The online version of this article (10.1007/s00125-023-05878-8) contains peer-reviewed but unedited supplementary material..

According to genome-wide association studies in humans, Tcf7l2 is one of the strongest risk factors associated with type 2 diabetes [9,10]. However, it remains unclear whether the genetic alteration of Tcf7l2 affects its expression. Therefore, Tcf7l2-deficient in vitro and in vivo models are being used to track the association between SNPs in Tcf7l2 and Tcf7l2 expression, and to gain insights into diabetes-related molecular pathways [1,11]. Many studies have shown that alterations in Tcf7l2 expression are metabolically associated with type 2 diabetes [1,11].
Tcf7l2 has also been proposed as a genetic factor that can explain the association between type 2 diabetes and nonalcoholic fatty liver disease (NAFLD), as genetic alterations in Tcf7l2 can make individuals susceptible to NAFLD [12]. Recently, the coexistence of NAFLD and type 2 diabetes has emerged as an important issue because individuals with NAFLD plus type 2 diabetes have a poor metabolic profile and a higher risk of worsening with more advanced forms of disease, such as non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC) [13][14][15]. However, the association of TCF7L2 with NAFLD remains unclear and little is known about the underlying mechanisms and function of TCF7L2 in the pathogenesis of NAFLD.
NAFLD is characterised by excessive hepatic triglyceride (TG) accumulation, which is primarily caused by excessive exogenous fatty acid uptake and increased endogenous fatty acid synthesis (de novo lipogenesis [DNL]) in the liver [16][17][18][19]. In this study, to determine the role of hepatic Tcf7l2 deficiency in NAFLD, we generated liver-specific Tcf7l2 knockout (KO) mice (Alb-Cre;Tcf7l2 f/f ). Based on the major risk factors for NAFLD development [18], mice lacking hepatic Tcf7l2 were given a high-fat diet (HFD) to provide fatty acids, or a refeeding/high-carbohydrate diet (HCD) to stimulate DNL. We thoroughly explored the underlying mechanisms and metabolic phenotype of liver TG accumulation owing to hepatic Tcf7l2 deficiency in in vivo and in vitro systems.

Methods
For detailed methods, please refer to the electronic supplementary material (ESM).
Human liver tissue samples Human liver tissue samples in ESM Fig. 1a were collected from 32 individuals who underwent hepatectomy or cholecystectomy at the universityaffiliated Severance Hospital, Yonsei University College of Medicine, Republic of Korea, between September 2014 and October 2019, as previously described [20]. Liver histology was assessed by an experienced pathologist and, according to diagnostic criteria [21], the samples were put into the following groups: (1) normal (n=13); (2) simple steatosis (NAFLD; n=10); and (3) NASH (n=9). All individuals gave informed consent and the study protocol was approved by the Institutional Review Board at Severance Hospital (IRB No 4-2014-0674).
Animal experiments Seven-week-old male C57BL/6N mice were purchased from ORIENT BIO (ORIENT BIO, Republic of Korea). Hepatocyte-selective Tcf7l2 ablation in C57BL/6N mice (liver-specific Tcf7l2 KO mice: Alb-Cre;Tcf7l2 f/f ) was obtained by crossing mice carrying Cre recombinase driven by the albumin (Alb) promoter with mice carrying Tcf7l2 alleles floxed at exon 5. Six-week-old male Alb-Cre;Tcf7l2 f/f and their wild-type littermates (Tcf7l2 f/f mice; developed in-house from Tcf7l2 +/− mice [Tcf7l2 tm2a(EUCOMM)Wtsi ; the EUCOMM Consortium; www.mousephenotype.org/data/genes/MGI: 1202879, accessed 8 September 2022]) were given either an HFD (catalogue no. D12492; Research Diet, USA) or HCD (catalogue no. TD.98090; Envigo, USA) for the indicated periods. For GTTs and ITTs, mice were fasted for 16 h or 6 h, respectively, and injected intraperitoneally with 1.5 g/kg of glucose or 1 U/kg of insulin. Blood glucose levels were measured in samples taken from the tail vein (LifeScan; One-Touch, USA).VO 2 , carbon dioxide production (VCO 2 ), respiratory exchange ratio (RER;VCO 2 /VO 2 ) and energy expenditure were measured in mice at 10 weeks using the Oxylet Pro system (Panlab, Spain). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience & Biotechnology (KRIBB-AEC-21051) and were performed in accordance with the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Quantitative PCR Total RNA from human liver tissues, mouse tissues or mouse primary hepatocytes was extracted using the easy-spin Total RNA Extraction Kit (catalogue no. 17221; iNtRON Biotechnology, Republic of Korea). Quantitative PCR (qPCR) was performed using the SYBR green PCR kit in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, USA). All data were normalised to the expression of ribosomal L32. Primer sequences are listed in ESM Table 1. miRNA (miR) expression was analysed as previously described [22] and the miR primer sequences are listed in ESM Table 2.
Western blotting Western blot analysis of extracts from mouse tissues and whole cells was performed as previously described [23]. Primary and secondary antibodies were used according to manufacturer's instructions and are listed in ESM Table 3.
Histological analysis Liver tissue sections were stained with Oil Red O (catalogue no. O0625; Sigma-Aldrich) and H&E, as previously described [24]. Liver fibrosis was detected with Sirius red staining (catalogue no. ab150681; Abcam, USA) according to the manufacturer's instructions. NAFLD Activity Score (NAS) and fibrosis stage were calculated as the sum of the scores for steatosis (0-3), lobular inflammation (0-3), hepatocyte ballooning (0-2), as assessed by H&E staining, and fibrosis (0-4), assessed by Sirius red staining [21,25]. Histological samples were anonymised and assigned a number before the slides were analysed by an experienced pathologist.
Fatty acid uptake assay Mouse primary hepatocytes isolated from Tcf7l2 f/f or C57BL/6N mice were transfected with the indicated plasmid DNA vectors using Lipofectamine 3000 transfection reagent (catalogue no. L3000015; Invitrogen, USA). Fatty acid uptake in mouse primary hepatocytes was measured with the Fatty Acid Uptake Assay Kit (catalogue no. K408-100; Biovision, USA) according to the manufacturer's instructions. Fluorescent fatty acid probes (green) were detected by a fluorescence microscope (DM IL LED FLUO; Leica Microsystems, Germany) and fluorescence intensity was quantified using Image J software (v 1.50i).
Glucose uptake assay Mouse primary hepatocytes isolated from Tcf7l2 f/f mice were infected with adenoviruses expressing gfp only and Cre and treated with 1 nmol/l insulin for 30 min. 2-Deoxyglucose (2-DG) levels in mouse primary hepatocytes were measured using the 2-DG Uptake Measurement Kit (catalogue no. CSR-OKP-PMG-K01; Cosmo Bio, Japan) according to the manufacturer's instructions.
DNL measurements For in vitro DNL analysis, mouse primary hepatocytes were treated with 37 kBq of 14 C-labelled glucose (catalogue no. NEC042; PerkinElmer, USA) and 10 nmol/l insulin for 48 h. Lipids were extracted from cells with chloroform and 14 C radioactivity was measured using the Tri-Carb 2910 TR liquid scintillation analyser (PerkinElmer). For in vivo DNL analysis, mice were fasted for 24 h, then refed an HCD for 12 h. Subsequently, they were intraperitoneally injected with 14 C-labelled sodium acetate (555 kBq/mouse; catalogue no. NEC553; PerkinElmer), or 3% (vol./vol.) ethanol in saline (136.9 mmol/l NaCl) as a control, and euthanised 1 h post injection. Lipids were extracted using the Folch method [7] and incorporation rates of [1-14 C]acetic acid into lipids were measured using the Tri-Carb 2910 TR liquid scintillation analyser.
Luciferase assay Luciferase assays were performed to determine the effects of TCF7L2 on the sterol regulatory element binding protein 1 (SREBP1)c, ChREBP/max-like protein X (MLX) transcriptional activities and the miR-33-5p promoter activity in HEPG2 cells (catalogue no. HB-8065; ATCC, USA) and Srebf1 3′UTR promoter activity in HEK293T cells (catalogue no. CRL-3216, ATCC). HEPG2 and HEK293T cells were tested for mycoplasma using the BioMycoX Mycoplasma PCR Detection Kit (catalogue no. D-50; CellSafe, Republic of Korea) and were found to be mycoplasma negative. Promoter activity was measured using a luciferase reporter assay system (catalogue no. E1910; Promega, USA) and normalised to β-galactosidase activity.
Immunoprecipitation and wheat germ agglutinin purification Total lysates from liver tissues were centrifuged at 16,000 × g for 10 min and the supernatant was extracted. For immunoprecipitation, 3 mg of protein lysates were incubated with carbohydrate response element binding protein (ChREBP) antibody and 30 μl of protein G plus A agarose beads (catalogue no. IP05; Millipore, USA) was added to each sample. For wheat germ agglutinin (WGA) precipitation, 3 mg of protein lysates were incubated with 30 μl of WGA agarose beads (catalogue no. AL-1023; Vector Laboratories), after which beads were eluted in 2× sample buffer without βmercaptoethanol.
Chromatin immunoprecipitation Cross-linking, nuclear isolation and chromatin immunoprecipitation (ChIP) assays were performed on AML12 cell line samples and mouse primary hepatocyte samples, using previously reported methods [27]. The precipitated DNA fragments were analysed by PCR.
Statistical analysis Statistical differences between two experimental groups were evaluated by the two-tailed unpaired Student's t test. One-way ANOVA with Tukey's multiple comparison test was performed using GraphPad Prism 8.0.1 (GraphPad Software, USA) when comparing three or more groups, as reported in the figure legends. Data are shown as mean±SD or mean±SEM. A p value <0.05 was considered statistically significant.

Results
Loss of hepatic TCF7L2 exerts lipogenic potential We found that Tcf7l2 expression was significantly reduced in the liver of individuals with NAFLD and NASH (ESM Fig. 1a). Therefore, to gain new insights into the role of TCF7L2 in NAFLD development, we generated liver-specific Tcf7l2 KO mice (Alb-Cre;Tcf7l2 f/f ; Fig. 1a,b and ESM Fig. 1b). Essentially, Alb-Cre;Tcf7l2 f/f mice exhibited increased fasting glucose levels without changes in body weight or plasma insulin and IGF-1 levels ( Fig. 1c-e and ESM Fig. 1c). They also displayed impaired glucose and insulin tolerance (Fig. 1f,h and ESM Fig. 1d). However, there were no changes in insulin concentration patterns during GTT (Fig. 1g) or in islet glucagon-positive alpha cell and insulin-positive beta cell areas (ESM Fig. 1e,f). Additionally, hepatic Tcf7l2 deficiency did not lead to changes in hepatic insulin signalling, RER,VO 2 ,VCO 2 and energy expenditure, as compared with wild-type controls (Fig. 1i,j and ESM Fig. 1g). Although there were no significant changes in liver TG content (Fig. 1k), hepatic Tcf7l2 depletion significantly enhanced hepatic expression of lipogenic genes, without altering genes involved in β-oxidation and lipolysis (Fig. 1l). protein levels is also shown. eWAT, epididymal white adipose tissue; WT, wild-type. (c-e) 10-week-old Tcf7l2 f/f (n=9) and Alb-Cre;Tcf7l2 f/f (n=6) mice fed an NCD were assessed for body weight (c), blood glucose levels (d) and plasma insulin levels (e). (f) GTT (1.5 g/kg body weight) in 10-week-old Tcf7l2 f/f (n=9) and Alb-Cre;Tcf7l2 f/f (n=6) mice fed an NCD, with glucose AUC is presented. *p<0.05, **p<0.01, Alb-Cre;Tcf7l2 f/f vs wild-type mice at the same time point, analysed by unpaired Student's t test. (g) Plasma insulin concentration during GTT in 10-week-old Tcf7l2 f/f (n=9) and Alb-Cre;Tcf7l2 f/f (n=6) mice fed an NCD, with insulin AUC presented. (h) ITT (1 U/kg body weight) in 10-week-old Tcf7l2 f/f (n=9) and Alb-Cre;Tcf7l2 f/f (n=6) mice fed an NCD, with glucose AUC is presented. *p<0.05, **p<0.01, ***p<0.001, Alb-Cre;Tcf7l2 f/f vs wildtype mice at the same time point, analysed by unpaired Student's t test.
(i) Ten-week-old Tcf7l2 f/f (n=12) and Alb-Cre;Tcf7l2 f/f (n=12) mice were fasted for 6 h and then injected intraperitoneally with insulin (10 U/kg body weight) or saline for 10 min. Representative western blot showing the effects of hepatic Tcf7l2 depletion on the hepatic insulin signalling pathway. The p-Akt/Akt and phosphorylated glycogen synthase kinase-3 β (p-GSK3β/GSK3β ratios are presented. (j) RER in 10-week-old Tcf7l2 f/f (n=12) and Alb-Cre;Tcf7l2 f/f (n=11) mice fed an NCD. RER was measured hourly in each metabolic chamber using the Oxylet Pro system. Average RER values for light (09:00-21:00 hours) and dark (21:00-09:00 hours) phases in a 12 h/12 h light/dark cycle are presented. (k, l) Ten-week-old Tcf7l2 f/f (n=6) and Alb-Cre;Tcf7l2 f/f (n=5) mice were fasted for 6 h, after which hepatic TG levels (k) and gene expression (l) were analysed. (l) Quantitative PCR (qPCR) analysis showing mRNA expression of genes involved in lipid metabolism in the liver of mice. The key in (b) also applies to (c-i) (k) and (l). Data in (b), (i) and (l) are presented as mean±SD; data in (c-h) (j) and (k) are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, analysed by t test Additionally, there were no changes in Igf1 and Igf1R mRNA expression (ESM Fig. 1h).
Hepatic Tcf7l2 deficiency contributes to NAFLD development by inducing preferential metabolism of carbohydrates In rodents, studies have shown that feeding with an HFD or HCD for 22 weeks leads to the development of NAFLD, including non-alcoholic fatty liver (NAFL; simple steatosis) and NASH [28][29][30]. As with individuals with NAFLD, Tcf7l2 expression was markedly decreased to below-normal levels in the liver of C57BL/6N mice fed an HFD or HCD for 22 weeks ( Fig. 2a,b). To assess the role of TCF7L2 in the development of NAFLD, Alb-Cre;Tcf7l2 f/f mice were fed an HFD or HCD for 22 weeks (Fig. 2c). Although hepatic TCF7L2 expression was decreased in the liver of wild-type Tcf7l2 f/f mice by both HFD and HCD feeding for 22 weeks, hepatic TCF7L2 expression in wild-type Tcf7l2 f/f mice was clearly higher compared with that in Alb-Cre;Tcf7l2 f/f mice (ESM Fig. 2a). Under HCD conditions, hepatic Tcf7l2 deficiency promoted lipid droplet formation and TG accumulation in the liver, resulting in elevated alanine aminotransferase (ALT) levels ( Fig. 2c-e). In contrast, these differences were not observed when mice were fed an HFD ( Fig. 2c-e). Consistently, genes responsible for lipogenesis and inflammation in NAFLD/NASH were found to be significantly increased in the liver of Alb-Cre;Tcf7l2 f/f mice, as compared with wild-type mice, following HCD feeding, whilst elevation of these genes was less consistently observed following HFD feeding (ESM Fig. 2b,c). However, there were no significant changes in islet size or islet glucagon-positive alpha cell and insulin-positive beta cell areas in mice fed an HFD or HCD (ESM Fig. 2d-g). Representative liver images of Alb-Cre;Tcf7l2 f/f mice fed an HCD for 22 weeks are shown in Fig.  2f. In response to 22 weeks of HCD, hepatic Tcf7l2 deficiency caused impairment of glucose and insulin tolerance (Fig. 2g,h). This impeded hepatic insulin signalling, as confirmed by the reduced level of Akt phosphorylation on Ser473 in the liver of Alb-Cre;Tcf7l2 f/f mice vs wild-type mice (Fig. 2i). However, it did not affect Akt phosphorylation levels in epididymal white adipose tissue and skeletal muscle (ESM Fig. 2h).
TCF7L2 does not affect HFD-induced intrahepatic TG accumulation Compared with an HCD, an HFD did not have as marked an effect on NAFLD development in hepatic Tcf7l2-deficient mice. To investigate whether this could have been owing to saturation of liver lipid content under an HFD diet, we used Alb-Cre;Tcf7l2 f/f mice fed an HFD for 4 or 12 weeks. Hepatic Tcf7l2 depletion exacerbated HFD-induced glucose intolerance and insulin intolerance without causing changes in body weight (ESM Fig. 3a-c). However, it did not alter hepatic TG content or plasma TG and NEFA levels (ESM Fig. 3d-g). To specifically address why hepatic Tcf7l2 did not affect HFD-induced TG content, we analysed the expression of genes involved in various lipid-related metabolic pathways (ESM Fig. 3h). Notably, hepatic Tcf7l2 deficiency increased the expression of several lipogenic genes (Srebf1c [encoded by the Srebf1 gene], Fasn and Acaca), whereas it decreased the expression of several fatty acid transporter genes (Cd36, Slc27a1, Slc27a4 and Fabp3; ESM Fig. 3h). In contrast, adenovirus-mediated expression of hepatic Tcf7l2 inversely regulated many of these genes (Srebf1c, Fasn, Cd36, Slc27a4 and Fabp3) without altering HFD-induced hepatic TG content (ESM Fig. 3i,j). Further, in a cell-autonomous manner, Tcf7l2 KO via Cre deletion of floxed sequences in primary hepatocytes impeded insulin-induced fatty acid uptake, as confirmed by use of a fluorescent fatty acid probe (ESM Fig. 4a), whereas forced expression of Tcf7l2 in C57BL/6N primary mouse hepatocytes promoted it (ESM Fig. 4h). It is well known that HFD suppresses DNL [31]. Accordingly, fatty acids can inhibit insulinstimulated glucose uptake [32,33]. Therefore, we hypothesised that hepatic Tcf7l2 deficiency did not affect HFD-induced liver lipid content, despite there being a decrease in fatty acid uptake, because lipogenesis is balanced by a relative increase in glucosesensing in response to reduced fatty acid uptake. Indeed, Tcf7l2 KO in primary hepatocytes impaired fatty acid-induced inhibition of glucose uptake (ESM Fig. 4b). Expression of L-type pyruvate kinase (L-PK; encoded by the pklr gene) and Pklr, a glucosesensing marker, increased in the liver of Alb-Cre;Tcf7l2 f/f mice vs wild-type mice, even under HFD conditions (ESM Fig. 4c,d).
Fatty acid β-oxidation, another lipid-associated metabolic pathway, was also altered by hepatic Tcf7l2 modulation under HFD conditions (ESM Fig. 3h,j). The AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC)/carnitine palmitoyltransferase 1α (CPT1α) axis is a key pathway for regulating fatty acid oxidation [34]. However, neither KO nor overexpression of Tcf7l2 functionally affected fatty acid βoxidation, as determined by changes in OCR following treatment with fatty acid and/or etomoxir, an irreversible inhibitor of CPT1α (ESM Fig. 4e,i). Further, Tcf7l2 KO or overexpression did not affect AMPK phosphorylation levels following showing relative protein levels of TCF7L2, ACC and FAS in the liver of mice fed an HCD (n=4) or HFD (n=7) for 22 weeks compared with a normal chow diet (NCD; n=3). *p<0.05, **p<0.01, 22 weeks vs 0 weeks, analysed by unpaired Student's t test. (c-e) Six-week-old Tcf7l2 f/f and Alb-Cre;Tcf7l2 f/f mice were fed an NCD for 22 weeks (n=4 for both genotypes; control groups), or were given an HCD (n=7 for both genotypes) or an HFD for 22 weeks (Tcf7l2 f/f , n=7; Alb-Cre;Tcf7l2 f/f , n=5) to induce NAFLD progression. Subsequently, frozen liver sections were stained using Oil Red O and H&E (representative images are shown; ×20 magnification; scale bars, 200 μm; c), and hepatic TG levels (d) and plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (e) were measured. (f) Six-week-old Tcf7l2 f/f (n=9) and Alb-Cre;Tcf7l2 f/f mice (n=11) were fed an HCD for 22 weeks. Subsequently, intra-abdominal liver images were taken (representative images shown). (g, h) Six-week-old Tcf7l2 f/f (n=9) and Alb-Cre;Tcf7l2 f/f mice (n=11) were fed an HCD for 22 weeks. Following this, a GTT (1.5 g/kg body weight; g) and ITT (1 U/kg body weight; h) were conducted. Glucose AUCs are also presented for both GTT and ITT. *p<0.05, **p<0.01, ***p<0.001, Alb-Cre;Tcf7l2 f/f vs wild-type mice at same time point, analysed by unpaired Student's t test. (i) Six-week-old Tcf7l2 f/f (n=8) and Alb-Cre;Tcf7l2 f/f (n=9) mice were fed an HCD for 22 weeks. Mice were fasted for 6 h and then injected intraperitoneally with insulin (10 U/kg body weight) or saline for 10 min. Presented is a representative western blot showing the effects of hepatic Tcf7l2 deficiency on the hepatic insulin signalling pathway. The p-Akt/Akt ratio is also shown. HSP90, heat shock protein 90. Key in (d) also applies to (e-h). Data in (b) and (i) are presented as mean±SD; data in (d) (e), (g) and (h) are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, analysed by t test treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a selective activator of AMPK, under HFD conditions (ESM Fig. 4f,g,j,k).
Hepatic TCF7L2 regulates hepatic DNL in a cell-autonomous fashion HFD provides excessive exogenous fatty acids, whereas HCD and refeeding mechanistically stimulate endogenous fatty acid synthesis from glucose by postprandial blood glucose and glucose-responsive insulin action, which are increased following carbohydrates consumption. Therefore, we hypothesised that the difference in effects of TCF7L2 on HCD-and HFD-induced fatty liver might originate from differential responses to exogenous fatty acid and glucose/ insulin pools. Hepatic TCF7L2 expression decreased in primary hepatocytes treated with palmitic acid, the most common saturated fatty acid in the human body, and in the liver of HFD-fed mice (Fig. 3a). Conversely, hepatic TCF7L2 expression significantly increased following in vitro treatment with glucose/insulin and following in vivo refeeding ( Fig. 3b and ESM Fig. 5a-c). To further investigate the relationship between TCF7L2 expression and carbohydrate sensing, we measured hepatic TCF7L2 expression at the same time as measuring the expression of lipogenic factors over varying HCD exposure time. Hepatic TCF7L2 expression increased after 2 and 4 weeks of HCD feeding vs baseline but was downregulated after 8 weeks of HCD feeding ( Fig. 3c and  ESM Fig. 5d,e). However, expression of the lipogenic factors ACC (encoded by Acaca) and fatty acid synthase (FAS; encoded by Fasn), and hepatic TG levels increased with HCD feeding (Fig. 3c,d and ESM Fig. 5d,e). Eventually, as shown in Fig. 2a, hepatic TCF7L2 expression decreased to below-normal levels after 22 weeks of HCD feeding, at which point excessive intrahepatic fatty acid was expected.
Hence, we hypothesised that although excessive increases in intrahepatic fatty acid as a result of long-term HCD feeding can eventually reduce hepatic TCF7L2 expression and activity, hepatic TCF7L2 restrains endogenous fatty acid synthesis to maintain lipid homeostasis (Fig. 3e). Therefore, we investigated whether TCF7L2 could regulate hepatic DNL in a cellautonomous fashion. Adenovirus-mediated Tcf7l2 expression suppressed the expression of glucose-/insulin-stimulated DNL genes involved in glycolysis (Gck and Pklr) and lipogenesis (Acaca and Fasn) and inhibited DNL in primary hepatocytes, as compared with control cells infected with Ad-gfp (Fig.  3f,g). Conversely, Tcf7l2 f/f primary hepatocytes infected with adenovirus expressing Cre (Ad-Cre) exhibited increased DNL genes and promoted DNL, as compared with cells infected with Ad-gfp (Fig. 3h,i). Consistent with previous reports, Tcf7l2 deficiency did not significantly affect hepatic insulin signalling pathway in a cell-autonomous manner (ESM Fig.  5f). Further, to investigate Tcf7l2 functional knockdown whilst avoiding potential redundancy of other TCF protein members, we generated a TCF7L2 dominant-negative (DN) mutant (TCF7L2DN) that lacks the N-terminal β-catenin interaction domain. TCF7L2DN inhibited the suppressive effects of TCF7L2 on DNL-associated genes and DNL in primary hepatocytes (Fig. 3j,k).  Fig. 5a and ESM Fig. 5c. (c, d) C57BL/6N mice were fed an HCD for 2, 4 and 8 weeks (n=5 per group; for this experiment, at '0 weeks of HCD feeding', n=5 mice that had been fed an NCD for 8 weeks were used). Representative western blot showing TCF7L2, ACC and FAS protein levels is shown alongside the quantified relative protein levels (TCF7L2/ heat shock protein 90 [HSP90], ACC/HSP90 and FAS/HSP90 ratios) (c; further quantification data, including details of statistical significance, are presented in ESM Fig. 5d). Hepatic TG levels are also presented (d). Hepatic Tcf7l2 depletion increases hepatic DNL in a time-and amount-dependent manner following carbohydrate loading To further assess the physiological function of hepatic Tcf7l2 deficiency in hepatic DNL, Alb-Cre;Tcf7l2 f/f mice were fasted and then refed. Compared with wild-type mice, this promoted the expression of DNL genes involved in glycolysis (Slc2a2, Gck and Pklr) and lipogenesis (Acly, Acaca and Fasn) following refeeding in a time-dependent manner (Fig. 4a). These genes are shown (in red) in Fig. 4b, which depicts the DNL process. However, there were no changes in the expression of genes involved in lipolysis and β-oxidation upon refeeding (ESM Fig. 6). Importantly, loss of hepatic Tcf7l2 led to increased liver TG content after 24 h of refeeding, but not levels of hepatic glycogen and β-OH, which are glucose products that are dispersed into and metabolised by other pathways during the DNL process (Fig. 4c-e). Additionally, there were no changes in plasma TG and NEFA levels (Fig. 4f,g). These data suggest that the activation of the glycolytic/lipogenic pathway for DNL is the primary factor that affects fat deposition in the liver of Alb-Cre;Tcf7l2 f/f mice.
Dietary carbohydrates are the primary stimulus for hepatic DNL. To further elucidate the carbohydrate dependency of Tcf7l2 deficiency-induced hepatic TG accumulation, we compared normal chow refeeding with high-carbohydrate refeeding (HCR) in Alb-Cre;Tcf7l2 f/f mice. Hepatic Tcf7l2 deficiency promoted expression of glycolytic and lipogenic factors and hepatic TG accumulation, which was more evident under HCR conditions compared with normal chow refeeding conditions (Fig. 4h-j). A high-carbohydrate intake markedly enhanced hepatic DNL induced by hepatic Tcf7l2 depletion (Fig. 4k). These data suggest that hepatic Tcf7l2 deficiency can aggravate liver steatosis and that the extent to which this occurs is dependent on carbohydrate intake, owing to increased hepatic DNL with carbohydrate loading.
SREBP1c and ChREBP are core factors associated with lipid metabolism in hepatic Tcf7l2-deficient mice To investigate the major pathways and key instigators upregulated in the liver of Alb-Cre;Tcf7l2 f/f mice, we analysed mRNAsequencing data from liver samples from refed mice. In total, 526 genes were upregulated (log 2 fold-change cut-off >1.5) and were analysed using GSEA (www.gsea-msigdb.org/gsea/ msigdb/mouse/annotate.jsp, accessed on 1 November 2021), based on canonical pathways gene sets derived from the Reactome pathway database (www.gsea-msigdb.org/gsea/ msigdb/mouse/genesets.jsp?collection=CP:REACTOME, accessed on 1 November 2021). The lipid metabolism-related gene set was most significantly upregulated (its significance can be more intuitively and integratedly presented as a radial line graph; see Fig. 5a and ESM Table 6). Specifically, the SREBP-and ChREBP-related pathways were identified as major signalling pathways (Fig. 5a). Careful investigation of mRNA-sequencing data showed that specific target genes and commonly shared lipogenic target genes of SREBP1c and ChREBP increased in the liver of Alb-Cre;Tcf7l2 f/f mice ( Fig. 5b and ESM Table 7). However, despite a reduction in Srebf2 mRNA expression in the liver of Alb-Cre;Tcf7l2 f/f vs wild-type mice, the expression of its target cholesterogenic genes was not altered (ESM Fig. 7a,b and ESM Table 8). SREBP1c and ChREBP are the main transcription factors for DNL [35] that regulate target gene transcription by binding to the sterol regulatory element (SRE) and carbohydrate response element (ChoRE) within their target gene promoters, respectively [36]. In the HEPG2 cell line, transfection with TCF7L2 inhibited ChREBP/MLX-induced activation of Pklr promoters containing ChoRE as well as ChREBP/MLXinduced activation of the ChoRE promoter itself (Fig. 5c). Furthermore, TCF7L2 inhibited SREBP1c-induced activation of an Fasn promoter region containing the SRE only as well as SREBP1c-induced activation of the SRE promoter itself (Fig.  5d). Liver X receptors (LXRs) are nuclear receptors that can  The radial line graph relates to the data for the Reactome gene set. In addition, data for the major pathways relating to the most significantly altered gene set ('metabolism of lipids'; see ESM Table 6 for complete gene set) are presented. Values in brackets are overlap/input numbers. Rho, Ras homologous; SREBF, sterol regulatory element-binding transcription factor (also known as sterol regulatory element binding protein directly regulate both SREBP1c and ChREBP [35]. However, neither KO nor overexpression of Tcf7l2 in primary hepatocytes affected lipogenic gene expression induced by the LXR agonist T0901317 (ESM Fig. 7c,d). These data suggest that TCF7L2 regulates ChREBP and SREBP1c transcriptional activities in an LXR-independent manner.
TCF7L2 promotes ChREBP transcriptional activity by modulating O-GlcNAcylation and ChREBP protein stability Among the two ChREBP isoforms (ChREBPα and ChREBPβ; both encoded by the Mlxipl gene) [37], the 100 kDa protein band corresponding to ChREBPα [35] was found to be significantly increased in the refed state and after 2, 4 and 8 weeks of HCD, but decreased after 22 weeks of chronic HCD (Fig. 6a,b). However, hepatic Tcf7l2 deficiency resulted in increased protein expression of ChREBPα and its target L-PK (encoded by the pklr gene), not only under acute HCR conditions but also following 22 weeks of chronic HCD (Fig. 6c,d). However, there were no changes in mRNA levels and promoter activity of Mlxipl(α) (Fig.  6e,f). Therefore, we investigated whether TCF7L2 regulates ChREBPα protein levels to modulate ChREBPα transcriptional activity. As shown in Fig. 6g, TCF7L2 inhibited ChREBPα/MLX-induced Mlxipl(β) promoter activity. In a set of experiments using the same concentration ratio of Flag-tagged ChREBPα:HA-tagged TCF7L2 used in the promoter activity assay, ChREBPα protein levels appeared to gradually decrease in a TCF7L2 expression-leveldependent manner, without changes in Mlxipl(α) mRNA expression (Fig. 6h). Further, the TCF7L2-mediated reduction in ChREBPα protein expression was restored by treatment with the proteasome inhibitor MG132 (Fig. 6i). Conversely, ChREBP ubiquitination was found to be significantly decreased in the liver of hepatic Tcf7l2-deficient mice under HCR conditions ( Fig. 6j and ESM Fig. 8a). O-GlcNAcylation of ChREBP is induced by high glucose levels, blocking its ubiquitination to increase glucose utilisation and lipogenic gene transcription [38]. Loss of hepatic Tcf7l2 significantly increased O-GlcNAcylation of ChREBP under HCR conditions, whereas hepatic Tcf7l2 restoration reduced it to normal levels (Fig. 6k,l and ESM  Fig. 8b,c). Consistently, CRISPR/Cas9-mediated Tcf7l2 KO in the AML12 cell line significantly promoted ChREBPα protein expression and the expression and promoter activity of its target gene Pklr in a cellautonomous manner, under high-glucose conditions (ESM Fig. 9). Indeed, the high-glucose-stimulated promoter activity of the ChREBP target Pklr in Tcf7l2 KO cell lines was suppressed by the O-GlcNAc transferase (OGT) inhibitor OSMI-1 (Fig. 6m). These data suggest that TCF7L2 regulates ChREBP transcriptional activity via O-GlcNAcylation (Fig. 6n).
Restoration of hepatic Tcf7l2 alleviates hepatic steatosis induced by acute and chronic HCD To further investigate the physiological role of hepatic Tcf7l2 in high-carbohydrateinduced fatty liver, we restored hepatic Tcf7l2 expression in Alb-Cre;Tcf7l2 f/f mice under acute and chronic HCD conditions. The restoration of hepatic Tcf7l2 expression in mice subjected to HCR reduced the expression of glycolytic and lipogenic factors to normal levels (Fig. 8a,b). This resulted in the restoration of hepatic DNL and TG levels (Fig. 8c,d). Under chronic HCD conditions, hepatic Tcf7l2 restoration did not cause changes in body weight, adiposity or body composition, and it did not affect the weights of the liver, epididymal white adipose tissue, inguinal white adipose tissue or brown adipose tissues (Fig. 8e-i). Plasma TG and NEFA levels also remained unchanged (ESM Fig. 11a-c). However, similar to the model of acute HCD, hepatic Tcf7l2 restoration significantly recovered protein levels of key lipogenic enzymes (ACC and FAS) and mRNA levels of glycolytic Gck and lipogenic Fasn, and restored chronic HCD-induced hepatic lipid droplet formation and hepatic TG levels as shown by Oil Red O staining of liver sections (Fig. 8j-l and ESM Fig.  11d,e). Indeed, adenovirus-mediated expression of hepatic Tcf7l2 in wild-type C57BL/6N mice fed an HCD for 22 weeks decreased chronic HCD-induced hepatic lipid droplets and TG content, concomitant with reductions in DNL-associated genes, without changes in body weight (ESM Fig. 11f-j).
Alb-Cre;Tcf7l2 f/f mice fed an HCD develop a NASH phenotype To further emphasise the pathological role of hepatic Tcf7l2 deficiency in the liver, we explored whether the absence of hepatic Tcf7l2 exacerbated NAFLD into more severe forms. Mice lacking hepatic Tcf7l2 displayed simple liver steatosis at 16 weeks of HCD feeding and progressive fatty liver at 34 weeks of a long-term HCD (ESM Fig. 12a). In detail, Alb-Cre;Tcf7l2 f/f mice fed an HCD for 34 weeks exhibited steatosis and lobular inflammation (F4/80-positive area) in the liver (ESM Fig. 12b,c). Although the data are not shown, we observed some hepatocellular ballooning with Mallory-Denk bodies in the liver. Alb-Cre;Tcf7l2 f/f mice had elevated NAS, which is the sum of scores for steatosis, lobular inflammation and ballooning (ESM Fig. 12d). Additionally, liver fibrosis, as determined by the proportion of the Sirius redstained area, was significantly increased upon hepatic Tcf7l2 deficiency (ESM Fig. 12e). Inflammation and fibrosis in the liver of Alb-Cre;Tcf7l2 f/f mice was further confirmed by analysis of mRNA and protein levels (ESM Fig. 12f,g).

Discussion
This study aimed to explore how changes in TCF7L2 expression in the liver affect NAFLD development. We found that  ; n=4), 24 h fasted (n=4) or 24 h refed (n=4) conditions. (b) C57BL/6N mice were fed a an HCD for 2, 4 and 8 weeks (n=5 per group; for this experiment, at '0 weeks of HCD feeding', n=5 mice that had been fed a normal chow diet [NCD] for 8 weeks were used) or they were fed an HCD for 22 weeks (n=4; for this experiment, at '0 weeks of HCD feeding', n=3 mice that had been fed an NCD for 22 weeks were used). Representative western blot showing ChREBP protein levels in mouse livers is shown alongside the ChREBP/heat shock protein 90 (HSP90) ratio. **p<0.01, ***p<0.001, analysed by one-way ANOVA with Tukey's post hoc test. (c) Ten-week-old Tcf7l2 f/f (n=8) and Alb-Cre;Tcf7l2 f/f (n=6) mice were subjected to HCR for 24 h. Representative western blots of protein levels of TCF7L2, ChREBP and L-PK in the liver are shown. ChREBP/HSP90 and L-PK/HSP90 ratios are also shown. (d) Six-week-old Tcf7l2 f/f (n=7) and Alb-Cre;Tcf7l2 f/f (n=7) mice were fed an HCD for 22 weeks. Representative western blots of protein levels of TCF7L2, ChREBP and L-PK in the liver are shown. ChREBP/HSP90 and L-PK/HSP90 ratios are also shown. (e) Ten-week-old Tcf7l2 f/f (n=8) and Alb-Cre;Tcf7l2 f/f (n=6) mice were subjected to HCR for 24 h or 6-week-old Tcf7l2 expression was reduced in the liver of both human and diet-induced mouse models of NAFLD/NASH. To gain new insights into the relationship between reduced hepatic Tcf7l2 expression and NAFLD development, we generated a mouse model with hepatocyte-specific Tcf7l2 deficiency. Several previous studies have attempted to elucidate the role of TCF7L2 in hepatic lipid metabolism using mouse models [42][43][44]; however, contrasting results have been obtained. Whole-body Tcf7l2 deficiency contributed to a reduction in hepatic lipid content, whereas Tcf7l2DN caused an increase in hepatic lipid content [38][39][40]. Despite advanced tools for in vitro-derived 3D cell culture systems, including liver organoids that can replace animal models, there are still several limitations to reproducing the in vivo process of dietinduced NAFLD, which is influenced by varying and complex factors. Therefore, we used a mouse model capable of inducing NAFLD in vivo.
Hepatic Tcf7l2 deficiency exacerbated liver steatosis via carbohydrate dependency owing to cell-autonomous increases in DNL. During the DNL process, glucose 6-phosphate can be synthesised by or broken down into glycogen [45], and acetyl-CoA can be converted into or back from ketone bodies [44,46]. However, hepatic glycogen and β-OH levels did not change in the Alb-Cre;Tcf7l2 f/f mice used in our study, as compared with wild-type mice, indicating that the glycolytic/lipogenic pathway may be the key signalling pathway for liver TG accumulation upon Tcf7l2 deletion without interference from other signalling pathways. As a strategy to avoid the potentially redundant or compensatory effects of other TCF members following Tcf7l2 deficiency [8,44,47], we performed Tcf7l2DN-mediated functional knockdown studies on hepatic DNL. Adenovirus-mediated Tcf7l2DN expression promoted hepatic DNL in a cell-autonomous manner, similar to that shown in Tcf7l2 f/f -derived primary hepatocytes infected with adenovirus expressing Cre.
Excessive exogenous fatty acid uptake is one of the leading contributors to intrahepatic TG accumulation in the pathogenesis of NAFLD. However, hepatic Tcf7l2 deficiency did not increase HFD-induced fat accumulation in the liver. Fatty acids typically inhibit glucose uptake [32,33]. In the fatty acid/glucose axis, hepatic Tcf7l2 deficiency alleviated inhibition of glucose uptake by fatty acids, resulting in relatively increased glucose uptake compared with controls. Further, this promoted glucose-sensing under HFD conditions, as confirmed by the expression of pklr, a glucose-sensing marker. This might contribute to balanced basal induction of hepatic lipogenesis under HFD conditions. This might be the reason why hepatic Tcf7l2 KO did not alter hepatic lipid content despite the partially decreased fatty acid uptake observed under HFD conditions. Importantly, these data suggest that excessive fatty acid uptake is not the mechanism driving NAFLD following hepatic Tcf7l2 loss.
Hepatic Tcf7l2 expression increased under conditions that stimulate DNL by refeeding with both a standard chow diet or an HCD. Increased hepatic Tcf7l2 expression suppressed hepatic DNL. However, excessively increased intrahepatic fatty acid owing to chronic HCD feeding eventually decreased hepatic TCF7L2 expression and activity. Therefore, the early induction of hepatic TCF7L2 expression by dietary carbohydrates may play an important role in maintaining hepatic lipid homeostasis, although reduced hepatic TCF7L2 expression may indicate a transition to a pathological state.
Conclusion Although this study did not model the Tcf7l2 SNP associated with the risk of type 2 diabetes, based on its potential to utilise the excess glucose pool more efficiently, we suggest that TCF7L2 may be a promising regulator of NAFLD associated with dietary carbohydrates and diabetes. Pathologically, hepatic Tcf7l2 deficiency-induced fatty liver progressed to NASH, a severe form of NAFLD, demonstrating the potent effect of TCF7L2 on hepatic DNL. Therefore, Alb-Cre;Tcf7l2 f/f mice could be a useful mouse model to investigate NAFLD development and progression under HCD conditions.
Data availability RNA-sequencing data have been deposited into the NCBI GEO under the accession number GSE162449 (www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE162449). Data that support the findings of this study are available from the lead contact upon reasonable request.
Funding This work was supported by grants from the KRIBB (KGM1052112 to K-JO; KGM5392212 to WKK) and the National Research Foundation of Korea (2016R1C1B2010257 to K-JO; 2020R1A2C2102308 to K-JO; 2020R1A2C2007111 to K-HB).
Authors' relationships and activities The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.
Contribution statement K-JO, K-HB and HailK supervised the experiments. K-JO, K-HB and DSL designed the experiments. DSL, THA and HyunmiK contributed to the experimental preparation and data analysis. K-JO generated liver-specific KO mice. S-HK and C-HL helped generate mice. JKS, JWP, GK and SYO contributed to the evaluation of liver histology and body composition monitoring. S-HK, EJ, T-SH and JSK helped to generate the plasmid construct. HJC, DHH and Y-HL contributed to the preparation and analysis of human liver samples. D-SK and JJ helped with bioinformatics analysis. WKK, E-WL, B-SH and SCL contributed to data interpretation. KH and Y-HL interpreted the physiological meaning of the study. S-HK provided critical reagents, samples and comments. K-JO organised the data and wrote the manuscript. All authors made substantial contributions to editing and revising the manuscript and have approved the final version of the manuscript. K-JO is the guarantor of this work and is responsible for the integrity of the work as a whole.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are Fig. 8 Effects of restoring hepatic Tcf7l2 expression on liver steatosis induced by acute and chronic HCD feeding in Alb-Cre;Tcf7l2 f/f mice. (ac) Eight-week-old Tcf7l2 f/f (n=6) and Alb-Cre;Tcf7l2 f/f (n=8) mice infected with Ad-gfp were fasted (F) for 24 h. Subsequently, Tcf7l2 f/f (n=5) and Alb-Cre;Tcf7l2 f/f mice infected with Ad-gfp (n=8) or Ad-Tcf7l2 (n=8) were subjected to HCR for 24 h. A schematic diagram showing the experimental design for generation of the acute highcarbohydrate loading mouse model is shown alongside a representative western blot showing TCF7L2, SREBP1 (full length [FL] and nuclear [N] forms), ChREBP, ACC and FAS protein levels in mouse liver (a). Quantitative PCR (qPCR) analysis showing expression levels of Tcf7l2 and glycolytic (Gck and Pklr) and lipogenic (Acaca, Fasn and Scd1) genes in mouse liver (b). Hepatic TG levels in mice (c). (d) DNL rate in primary hepatocytes isolated from Tcf7l2 f/f and Alb-Cre;Tcf7l2 f/f mice. Cells were infected with Ad-gfp or Ad-Tcf7l2 and treated with [ 14 C]glucose and 10 nmol/l insulin or 3% (vol./vol.) ethanol in saline (control [Con]) for 48 h. (e-i) Six-week-old Tcf7l2 f/f (n=7) and Alb-Cre;Tcf7l2 f/f (n=15) mice were fed an HCD for 22 weeks. At 21 weeks of HCD, Tcf7l2 f/f mice were infected with Ad-gfp (n=7) and Alb-Cre;Tcf7l2 f/f mice were infected with Ad-gfp (n=7) or Ad-Tcf7l2 (n=8). A schematic diagram showing the experimental design for generation of the chronic high-carbohydrate loading mouse model is shown alongside representative images of livers, epididymal white adipose tissues (eWAT), inguinal white adipose tissues (iWAT) and brown adipose tissues (BAT) from mice (e). Body, fat and lean weights (f). Percentages of fat mass, lean mass and free body fluid (g). Liver weight (h). Weights of eWAT, iWAT and BAT (i). (j-l) Six-week-old Tcf7l2 f/f (n=7) and Alb-Cre;Tcf7l2 f/f (n=8) mice were fed an HCD for 22 weeks. At 21 weeks of HCD, Tcf7l2 f/f mice were infected with Ad-gfp (n=7) and Alb-Cre;Tcf7l2 f/f mice were infected with Ad-gfp (n=4) or Ad-Tcf7l2 (n=4). A representative western blot showing TCF7L2, ACC and FAS protein levels in mouse liver (j; quantification data are presented in ESM  Fig. 11d). Representative H&E and Oil Red O staining of liver sections (×20 magnification; scale bars, 200 μm; k). Hepatic TG levels (l). HSP90, heat shock protein 90. Key in (b) also applies to (c), (d), (h), (i) and (l). Data in (b-d), (f-i) and (l) are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, assessed by one-way ANOVA with Tukey's post hoc test included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.