MYC mRNA expression throughout the intestine is not associated with body mass index or type 2 diabetes

reflect blink-ered insights into the role of intestinal MYC expression in metabolic disorders. Notably, the genetically and inhibitor– induced reductions in MYC levels in mice scrutinised by Luo et al. supposedly affected MYC throughout the entire intestine and are as such independent of the MYC expression site. Furthermore, we show no difference in MYC mRNA expression between patients with type 2 diabetes and healthy controls, and lastly, we cannot reproduce the association between MYC mRNA levels and BMI as observed by Luo et al. 1 and indicated by Vargas et al. 5 Importantly, our data set was not obtained for these analyses, and thus, the number of subjects might be too low to detect significant correlations; however, no trends seem apparent in any of the plots (Figure 1B– E). Also, differences between the participants in - cluded in our study (Danish subjects with Northern European origin) and the participants examined by Luo et al. 1 (biopsies sampled at a hospital in Shanghai and, thus, presumably a population predomi - nantly of Asian origin) and by Vargas et al. 5 (study conducted in the US on subjects with different racial backgrounds) may explain the discrepant findings. Furthermore, different methodology to deter - mine MYC mRNA expression was used in the different studies, thus, complicating direct comparisons. Lastly, the MYC mRNA level does not necessarily reflect the level of the functional protein, and thus, we cannot exclude a difference in functional MYC between patients with type 2 diabetes and healthy controls. To address this, a full ste-reological analysis is needed. In conclusion, we do not see a correlation between intestinal mucosal MYC mRNA expression and BMI or glycaemic control, and thus, our findings do not support the hypothesis of intestinal MYC as a putative drug target against obesity and metabolic diseases. However, we cannot rule out that the discrepancies between the results presented here and in the literature 1,5 arise due to lack of statistical power in our dataset and/or due to racial and/or methodological differences.


| BACKG ROU N D
In most parts of the world, the prevalence of obesity and other metabolic disorders such as type 2 diabetes is increasing. These conditions typically carry large personal and social consequences.

| Biopsy retrieval and transcriptomics
The study design, the study population, the experimental procedures for biopsy retrieval and storage, as well as the transcriptomic analysis have been previously described. [6][7][8] In brief, 12 patients with type 2 diabetes (treated using diet-counselling alone or in combination with metformin or sulfonylurea) and 12 healthy controls (without first-degree relative with type 1 or type 2 diabetes) matched on age, gender, and BMI were included in the study after obtainment of informed consent. Demographical details are presented in Table 1. During propofol sedation, subjects underwent anterograde and retrograde double-balloon enteroscopies (on two separate days) including the collection of biopsies from every 30 cm throughout the small intestine and from the cecum, the ascending, transverse, descending, and sigmoid large intestine, as well as the rectum. The biopsies were subjected to mRNA extraction, cDNA synthesis, and cDNA sequencing using the NextSeq 500 system (Illumina, San Diego, CA, USA).

| Statistical analyses
To best compare our data with the previous results 1 and to account for possible variation between biopsy sample sites between subjects, we pooled MYC mRNA expression data from several biopsy sample sites into four main categories: proximal small intestine, distal small intestine, proximal large intestine, and distal large intestine. Thus, for each subject, a mean MYC mRNA expression was calculated for each of the four intestinal sections and used for the analyses. To evaluate any differences in MYC expression level, the mean value for each section in the two groups (control and type 2 diabetes) were compared using two-way ANOVA followed by Šídák's multiple comparisons test. Correlations were evaluated with nonparametric Spearman's correlation test. p-values were calculated with 95% confidence intervals and considered statistically significant at p < .05. GraphPad Prism 9.0.0 (GraphPad software, San Diego, CA, USA) was used for the statistical analyses.

| RE SULTS
Inspired by the prospect of intestinal MYC reduction as a putative drug target against metabolic disorders, we investigated MYC mRNA expression in intestinal mucosa biopsies sampled using doubleballoon enteroscopy along the entire intestinal tract of 12 patients with type 2 diabetes and 12 matched healthy controls. 6,7 In these individuals with BMI ranging from 20 to 31 kg/m 2 , the mean MYC mRNA expression varied along the intestine with the lowest level observed at the distal small intestine and the greatest expression levels in the proximal small intestine and in the colon ( Figure 1A).
Intestinal MYC is described as a driver of metabolic diseases. 1 Hence, we investigated whether MYC mRNA expression levels were increased in intestinal biopsies from patients with type 2 diabetes.
We observed no differences between the groups at any of the ex-  Importantly, our data set was not obtained for these analyses, and thus, the number of subjects might be too low to detect significant correlations; however, no trends seem apparent in any of the plots ( Figure 1B-E). Also, differences between the participants in- In conclusion, we do not see a correlation between intestinal mucosal MYC mRNA expression and BMI or glycaemic control, and thus, our findings do not support the hypothesis of intestinal MYC as a putative drug target against obesity and metabolic diseases. However, we cannot rule out that the discrepancies between the results presented here and in the literature 1,5 arise due to lack of statistical power in our dataset and/or due to racial and/or methodological differences.