Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution

The gut plays a key role in regulating metabolic health. Dietary factors disrupt intestinal physiology and contribute to obesity and diabetes, whereas bariatric procedures such as vertical sleeve gastrectomy (VSG) cause gut adaptations that induce robust metabolic improvements. However, our understanding of these adaptations at the cellular and molecular levels remains limited. In a validated murine model, we leverage single-cell transcriptomics to determine how VSG impacts different cell lineages of the small intestinal epithelium. We define cell type-specific genes and pathways that VSG rescues from high-fat diet perturbation and characterize additional rescue-independent changes brought about by VSG. We show that Paneth cells have increased expression of the gut peptide Reg3g after VSG. We also find that VSG restores pathways pertaining to mitochondrial respiration and cellular metabolism, especially within crypt-based cells. Overall, our study provides unprecedented molecular resolution of VSG’s therapeutic effects on the gut epithelium.

yielded two lists of differentially expressed genes (DEGs) per cluster, which we compared to 154 see if the same genes downregulated by HFD were upregulated by VSG, and vice versa. We 155 grouped these overlapping DEGs into one of two categories, termed "rescue" or "specificity."

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"Rescue" was defined as the fraction of DEGs altered by HFD and changed in the opposite into the rescue category (distinct from VSG-induced DEGs unrelated to dietary perturbation) 159 ( Figure 4A). To compare the extent of "rescue" and "specificity" across different cell types, we 160 first applied filtering criteria to each DEG list (P < 0.05, Padj < 0.20, log2FC > |0.50|). Among all 161 clusters localized to the crypt compartment, 734 genes were differentially expressed after HFD

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Visualizing these proportions in a cluster-specific manner revealed higher overall levels of 165 rescue and specificity in crypts compared to villi, and this pattern was driven predominantly by 166 . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution 6 genes downregulated by HFD and upregulated by VSG ( Figure 4B, Supplemental Figure 2).
Specific clusters that exemplified this trend include crypt-based stem and Paneth cells (as comparable degrees of rescue and specificity between crypt and villus compartments, we found that most genes rescued in EECs were distinct between crypts and villi, with the few shared being predominantly mitochondrial-encoded (Supplemental Figure 3A-B).
Given the critical role of the crypt in maintaining overall intestinal epithelial 173 homeostasis 20 , we further examined the genes rescued in the stem and Paneth clusters 174 (Supplemental Figure 3C). In both cell types, genes related to cellular metabolism, such as Cox7a2 and Gapdh, were rescued to a similar extent ( Figure 4D). Other genes were rescued 176 by VSG to a greater extent in stem versus Paneth cells, such as Gpx4, which encodes an 177 enzyme that mitigates harmful lipid peroxidation 21 , and Prap1, which codes for a protein that 178 protects gut epithelial cells from apoptotic insults 22 . Genes that showed more Paneth-centric 179 rescue effects included Ldha, which encodes a key enzyme in glycolysis leading to the 180 production of lactate 23 , and Reg3g, which codes for an antimicrobial peptide that was recently 181 shown to be required for metabolic improvements induced by VSG or a fiber-enriched diet 14 .

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( Figure 4D). We sought to leverage our single-cell data to follow up on this finding and narrow

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we performed pathway enrichment analysis 24,25 of DEG sets derived from each cell cluster along 192 the crypt-villus axis. We first analyzed VSG-induced DEGs (filtered as previously described) to 193 understand the effects of surgery alone on the epithelium. We then repeated these enrichment 194 analyses with only genes "rescued" by VSG. In villus enterocytes, villus EECs, and some crypt- was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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Nonetheless, to make sure of this, we repeated the differential expression analysis while 233 explicitly accounting for the per-cell proportion of reads mapping to the mitochondrial genome 234 . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023.    supported by glycolytic products (e.g., lactate) from Paneth cells 28,30 . In line with this, we found that HFD reduces while VSG rescues Paneth cell expression of Ldha, which encodes a catalytic 307 subunit of the glycolytic enzyme lactate dehydrogenase 23 . Furthermore, within stem cells we 308 observed more prominent rescue of genes involved in mitigation of oxidative stress (e.g.,

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The authors declare no competing interests.

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In vivo studies 396 Five-week-old male C57BL/6J mice (n=20) were purchased from the Jackson Laboratory 397 (Bar Harbor, ME, USA) and were individually housed in a 12-hour light/dark cycle environment 398 with ad libitum access to water and food. The animal room was maintained at a temperature of 399 25°C with 50%-60% humidity. Following an acclimation period, mice were assigned to receive                            Data depict mean ± SEM analyzed via one-or two-way ANOVA or mixed effects analysis followed by Tukey's post hoc testing. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
. CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution  . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint Working title: Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution (B) Volcano plots of significantly altered stem cell genes across dietary (left) and surgical (right) conditions, filtered by log2 fold change ±0.5 (vertical hashed lines) and Padj < 0.2 (horizontal hashed line). Notably, mitochondrially-encoded genes appear among the most differentially expressed even while accounting for the proportion of genes mapping to the mitochondrial genome as a covariate. (C) Proposed working model as to how chronic consumption of an obesogenic diet and treatment by VSG initiate gut adaptations through cell-type specific changes within the small intestinal epithelium.
. CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted June 4, 2023. ; https://doi.org/10.1101/2023.05.31.543143 doi: bioRxiv preprint