Exosomes from Short-Term High-Fat or High-Sucrose Fed Mice Induce Hepatic Steatosis through Different Pathways

Figures

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  Figure 1. High-fat and high-sucrose diets lead to a convergent obese phenotype over time. (A–C): Food (A) and drink (B) intake and calories consumed (C) by male C57BL/6J mice fed either standard chow (CT), a high-fat diet (HFD), or standard chow with drinking water supplemented with 50% sucrose (SAC). (D) Body weight of the same mice during 15 weeks of diet. (E) Magnetic resonance imaging (MRI) analysis of fat distribution of the same mice after 6 (top panels) and 15 weeks (lower panels) of diet. (F–H): After 6 weeks, HFD mice are hypertriglyceridemic (F), glucose intolerant (G), and hyperinsulinemic (H), whereas SAC mice are hyperinsulinemic. (I–K): After 15 weeks, both HFD and SAC mice are hypertriglyceridemic (I), glucose intolerant (J), and hyperinsulinemic (K). (L) After 6 weeks, HFD mice, but not SAC mice, are insulin resistant according to an insulin tolerance test (ITT). (M,N): SAC mice show increased gluconeogenesis (M) and insulin release (N) during a pyruvate tolerance test (PTT), evidencing hepatic insulin resistance. (O–Q): Epididymal white adipose tissue (eWAT) of HFD mice shows adipocyte hypertrophy (O,P) and increased levels of plasma non-esterified fatty acids (NEFA) (Q).…

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  Figure 2. HFD and SAC induce distinct gene expression profiles in eWAT and liver. (A) Comparison of DEGs in HFD/CT and SAC/CT for each tissue analyzed using microarray hybridization. (B) Enrichment analysis showing the log (p value) of pathways associated with upregulated (red) or downregulated genes (green) in the same tissues of HFD and SAC mice. (C–E): The fatty acid biosynthesis pathway is associated with downregulated genes in HFD mice and with upregulated genes in SAC mice (C), including FASN, as determined by mRNA (D) and protein levels (E). (F,G): The glycolysis and gluconeogenesis (GNG) pathway is associated with upregulated genes only in SAC mice (F) including fructose handling genes such as Slc2a5 (G). n = 4/group (A–D,G) and n = 3/group (E). * p < 0.05, and *** p < 0.005 with respect to the CT group. + p < 0.05, and +++ p < 0.005 with respect to the HFD group. Gas = gastrocnemius.

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  Figure 3. SAC mice rely on de novo lipogenesis (DNL) to maintain glucose homeostasis. (A) Oral administration of glucose increased respiratory exchange ratio (RER) in CT but not HFD mice, as measured by indirect calorimetry. SAC mice show significantly higher levels, which are reduced by pretreatment with FASN inhibitor C75. (B) At the end of the calorimetric test shown in (A), only HFD mice show high glucose levels, but after pretreatment with C75, SAC mice also show increased glycemia. (C,D): Oral administration of fructose increase glucose (C) and RER only in SAC mice (D), and the increased RER is blunted by pretreatment with C75. (E,F): At the end of the calorimetric test shown in (D), SAC mice show increased glycemia and triglyceridemia, whereas C75 pretreatment further increases glycemia (E) and decrease TG (F). n = 3/group (A,B,D,F), n = 6/group (C). * p < 0.05, and ** p < 0.01 with respect to the CT group. + p < 0.05 with respect to the HFD group. FTT = fructose tolerance test.

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  Figure 4. Injection of HFD and SAC plasma exosomes reproduce transcriptomic changes observed in donor mice. (A–D): Plasma exosomes from donor mice were characterized by NTA (A), TEM (B), Western blotting of membrane markers CD63 and CD9 (C), and exosome number (D). (E,F): Biodistribution of plasma exosomes from CT, HFD, and SAC mice was followed by transfecting them with a siRNA against EGFP and IV injection into EGFP transgenic mice. Injection of the same siEGFP associated with Invivofectamine was used as a positive control. Loss of fluorescence was determined using confocal microscopy of the tissues of interest (E) and luminometer analysis of tissue lysates (F). (G) Comparison of DEGs in mice injected with HFD plasma exosomes (iHFD) or SAC exosomes (iSAC) with those injected with CT exosomes (iCT) for each tissue analyzed using microarray hybridization. (H) Enrichment analysis showing log (p value) of pathways associated with upregulated (red) or downregulated genes (green) in the same tissues of iHFD and iSAC mice. (I,J): Inflammation is increased in the eWAT of iHFD mice, with decreased prostaglandin synthesis (I) and increased eicosanoid synthesis genes (J). (K,L): The glycolysis and…

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  Figure 5. Injection of HFD and SAC plasma exosomes reproduce the metabolic phenotype observed in donor mice. (A,B): iHFD but not iSAC show glucose intolerance (A) and insulin resistance (B). (C,D): Both experimental groups show increase glucose output during a PTT (C), and lower circulating β-HB levels (D). (E) Only iSAC mice have increased alanine transaminase (ALT) activity. (F) Both experimental groups display hepatic steatosis. (G,H): Only iHFD mice show significant enlargement of adipose depots (G) and elevated circulating NEFA levels (H). (I,J): Oral administration of fructose increased glycemia (I) and circulating TG (J) only in iSAC mice. n = 4/group (A–J). * p < 0.05, ** p < 0.01, and *** p < 0.005 with respect to the iCT group. + p < 0.05, ++ p < 0.01, and +++ p < 0.005 with respect to the iHFD group. NS = not significant.

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  Figure 6. The miRNA profile differs between HFD and SAC plasma and liver exosomes. (A,B): Volcano plots show differential miRNA expression of plasma exosomes from HFD (A) and SAC mice (B) compared with CT mice. (C,D): Comparison of the differentially expressed exosomal miRNAs in HFD/CT and SAC/CT in plasma (C) and liver (D). (E,F): Volcano plots show differential miRNA expression of liver exosomes from HFD (E) and SAC mice (F). n = 3/group (A–C) and n = 4/group (D–F).

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  Figure 7. Circulating miR-22-3p can be used as a biomarker for MAFLD stratification. (A) Heatmap showing positive (blue) or negative (yellow) correlation coefficient values between circulating miRNAs and clinical parameters in a human cohort. (B) Circulating miR-22-3p levels are increased in mice after 15 weeks of a HFD but not a SAC diet. (C) Correlation between circulating miR-22-3p and fatty liver index (FLI) in humans. (D–G): Individuals with FLI > 60 and high miR-22-3p levels (High22) show equivalent body mass index (BMI) (D) but increased fasting plasma insulin (FPI) (E), fasting plasma glucose (FPG) (F), and homeostatic model assessment-insulin resistance (HOMA-IR) (G) than those with FLI > 60, but low miR-22-3p levels (Low22). n = 40 (A,C–G), n = 3/group (B). * p < 0.05 and *** p < 0.005 with respect to CT or FLI < 60 group. + p < 0.05 and ++ p < 0.01 with respect to High22 group. chol = cholesterol; G120 = glucose at 120 min during oral GTT; HDL = high density lipoprotein; LDL = low density lipoprotein; WHR = waist/hip ratio.