5f2e6578e81139fe6aa1e8438c82c575 289946.pdf 599579aec90c04f818a131a1ce175381ca6402cc 289946.pdf b77a35484fa2719d1cc296a29bbedff516c755743e2a46309d426cb8d3b872e7 289946.pdf Title: Intergenerational Inheritance of Hepatic Steatosis in a Mouse Model of Childhood Obesity: Potential Involvement of Germ-Line microRNAs Subject: Childhood obesity increases the risk of developing metabolic syndrome later in life. Moreover, metabolic dysfunction may be inherited into the following generation through non-genomic mechanisms, with epigenetics as a plausible candidate. The pathways involved in the development of metabolic dysfunction across generations in the context of childhood obesity remain largely unexplored. We have developed a mouse model of early adiposity by reducing litter size at birth (small litter group, SL: 4 pups/dam; control group, C: 8 pups/dam). Mice raised in small litters (SL) developed obesity, insulin resistance and hepatic steatosis with aging. Strikingly, the offspring of SL males (SL-F1) also developed hepatic steatosis. Paternal transmission of an environmentally induced phenotype strongly suggests epigenetic inheritance. We analyzed the hepatic transcriptome in C-F1 and SL-F1 mice to identify pathways involved in the development of hepatic steatosis. We found that the circadian rhythm and lipid metabolic process were the ontologies with highest significance in the liver of SL-F1 mice. We explored whether DNA methylation and small non-coding RNAs might be involved in mediating intergenerational effects. Sperm DNA methylation was largely altered in SL mice. However, these changes did not correlate with the hepatic transcriptome. Next, we analyzed small non-coding RNA content in the testes of mice from the parental generation. Two miRNAs (miR-457 and miR-201) appeared differentially expressed in the testes of SL-F0 mice. They are known to be expressed in mature spermatozoa, but not in oocytes nor early embryos, and they may regulate the transcription of lipogenic genes, but not clock genes, in hepatocytes. Hence, they are strong candidates to mediate the inheritance of adult hepatic steatosis in our murine model. In conclusion, litter size reduction leads to intergenerational effects through non-genomic mechanisms. In our model, DNA methylation does not seem to play a role on the circadian rhythm nor lipid genes. However, at least two paternal miRNAs might influence the expression of a few lipid-related genes in the first-generation offspring, F1. Keywords: circadian rhythm; intergenerational epigenetic inheritance; childhood obesity; litter size reduction; DNA methylation; small non-coding RNAs Author: Francesc Ribas-Aulinas, Sílvia Ribo, Eduard Casas, Marta Mourin-Fernandez, Marta Ramon-Krauel, Ruben Diaz, Carles Lerin, Susana G. Kalko, Tanya Vavouri and Josep C. Jimenez-Chillaron Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Mar 1 07:42:03 2023 CET ModDate: Wed Mar 1 09:01:40 2023 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2379108 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WLENKM+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 IBTYZX+VnURWPalladioL Type 1 Custom yes yes no 15 0 EWQNMW+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 YNJWVQ+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 EDRNWR+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 HGMNRX+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 46 0 FFDGWE+CMSY10 Type 1 Builtin yes yes no 72 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 80 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 84 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 88 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 92 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 118 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 122 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 126 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 130 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 147 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 151 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 155 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 168 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 172 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 176 0 BCDJEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 180 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 193 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 197 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 201 0 LWLIVD+EURM10 Type 1 Builtin yes yes no 226 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-03-15 02:41:56 CET RepresentationInformation: 289946.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-03-14 14:50:31 CET Size: 2379108 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 550 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Animal Care and Experimental Design Destination: subsection.2.1 Item: Title: TAG and Cholesterol Determination Destination: subsection.2.2 Item: Title: Tissue Culture and Incubations with AZA Destination: subsection.2.3 Item: Title: Sperm Isolation Destination: subsection.2.4 Item: Title: DNA and RNA Extraction Destination: subsection.2.5 Item: Title: Affymetrix Microarrays Destination: subsection.2.6 Item: Title: Agilent DNA Methylation Microarrays Destination: subsection.2.7 Item: Title: Small RNA Sequencing and Analysis Destination: subsection.2.8 Item: Title: Real-Time Quantitative PCR (qPCR) Destination: subsection.2.9 Item: Title: Statistical Analysis Destination: subsection.2.10 Item: Title: Data and Resource Availability Destination: subsection.2.11 Item: Title: Results Destination: section.3 Children: Item: Title: Paternal Overnutrition Induced Hepatic Steatosis in the First-Generation Offspring Destination: subsection.3.1 Item: Title: Litter Size Reduction Altered the Expression of Genes Involved in the Circadian Rhythm and Lipid Metabolism Destination: subsection.3.2 Item: Title: DNA Methylation Did Not Correlate with the Expression of Clock and Lipid-Related Genes Destination: subsection.3.3 Item: Title: Two miRNAs Were Differentially Expressed in the Testes of SL-F0 Mice and Might Be Linked to the Hepatic Lipid Metabolism in the Offspring Destination: subsection.3.4 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Intergenerational Inheritance of Hepatic Steatosis in a Mouse Model of Childhood Obesity: Potential Involvement of Germ-Line microRNAs Author: Francesc Ribas-Aulinas, Sílvia Ribo, Eduard Casas, Marta Mourin-Fernandez, Marta Ramon-Krauel, Ruben Diaz, Carles Lerin, Susana G. Kalko, Tanya Vavouri and Josep C. Jimenez-Chillaron Subject: Childhood obesity increases the risk of developing metabolic syndrome later in life. Moreover, metabolic dysfunction may be inherited into the following generation through non-genomic mechanisms, with epigenetics as a plausible candidate. The pathways involved in the development of metabolic dysfunction across generations in the context of childhood obesity remain largely unexplored. We have developed a mouse model of early adiposity by reducing litter size at birth (small litter group, SL: 4 pups/dam; control group, C: 8 pups/dam). Mice raised in small litters (SL) developed obesity, insulin resistance and hepatic steatosis with aging. Strikingly, the offspring of SL males (SL-F1) also developed hepatic steatosis. Paternal transmission of an environmentally induced phenotype strongly suggests epigenetic inheritance. We analyzed the hepatic transcriptome in C-F1 and SL-F1 mice to identify pathways involved in the development of hepatic steatosis. We found that the circadian rhythm and lipid metabolic process were the ontologies with highest significance in the liver of SL-F1 mice. We explored whether DNA methylation and small non-coding RNAs might be involved in mediating intergenerational effects. Sperm DNA methylation was largely altered in SL mice. However, these changes did not correlate with the hepatic transcriptome. Next, we analyzed small non-coding RNA content in the testes of mice from the parental generation. Two miRNAs (miR-457 and miR-201) appeared differentially expressed in the testes of SL-F0 mice. They are known to be expressed in mature spermatozoa, but not in oocytes nor early embryos, and they may regulate the transcription of lipogenic genes, but not clock genes, in hepatocytes. Hence, they are strong candidates to mediate the inheritance of adult hepatic steatosis in our murine model. In conclusion, litter size reduction leads to intergenerational effects through non-genomic mechanisms. In our model, DNA methylation does not seem to play a role on the circadian rhythm nor lipid genes. However, at least two paternal miRNAs might influence the expression of a few lipid-related genes in the first-generation offspring, F1. Keywords: circadian rhythm; intergenerational epigenetic inheritance; childhood obesity; litter size reduction; DNA methylation; small non-coding RNAs Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Mar 01 07:42:03 CET 2023 ModDate: Wed Mar 01 09:01:40 CET 2023 ID: 0x1886ba3f19a93631ec6cc63b5ca48978, 0x1886ba3f19a93631ec6cc63b5ca48978 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LWLIVD+EURM10 FontSubset: true FirstChar: 12 LastChar: 12 FontDescriptor: FontName: LWLIVD+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: EWQNMW+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 135 FontDescriptor: FontName: EWQNMW+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YNJWVQ+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 248 FontDescriptor: FontName: YNJWVQ+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 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