Increased gene expression dosage of the triplicated Hsa21 gene orthologs on mouse chromosome 16 (Mmu16) across tissues.

A) Graphical representation of human chromosome 21 (Hsa21) and the syntenic Mmu16 segment that is duplicated in Dp16 mice. B) Global view of the expression of 108 triplicated Hsa21 gene orthologs on Mmu16 in gonadal white adipose tissue (gWAT), inguinal white adipose tissue (iWAT), interscapular brown adipose tissue (BAT), skeletal muscle (gastrocnemius), and hypothalamus. Red denotes transcript that is expressed at ≥1.5-fold the WT level, whereas blue denotes transcript that is expressed at significantly lower level compared to WT control. The Ktrap gene cluster (23 Ktrap genes) located between Cldn8 and Tiam1is not shown. C) Overlap analysis showing differentially expressed Hsa21 gene orthologs that are shared between males and females across six tissues. The criteria for differentially expressed genes (DEGs) is log2(FC) > 0 with padj ≤ 0.05. n = 6 RNA samples per genotype per sex per tissue-type. Chow-fed WT and Dp16 mice were at 27.5 weeks of age at the time of tissue collection.

Sexually dimorphism in body weight, body temperature, food intake, and physical activity in chow-fed Dp16 mice.

A) Body weight of chow-fed male Dp16 and WT mice over time. B) absolute and relative (% of body weight) fat and lean mass in male mice (WT = 15; Dp16 = 12). C) Body weight of chow-fed female Dp16 and WT mice over time. D) absolute and relative (% of body weight) fat and lean mass in female mice (WT = 15; Dp16 = 15). E-F) Food intake, total physical activity level, and energy expenditure of male (E) and female (F) Dp16 and WT mice across the circadian cycle (light and dark) and metabolic states (ad libitum fed, fast, refeed). Sample size for male (WT = 10-12; Dp16 = 11-12) and female (WT = 13-15; Dp16 = 5-6) mice. G-H) fecal frequency, average fecal weight, and fecal energy content (per gram and total) in male (G) and female (H) Dp16 and WT mice. Sample size for male (WT = 6; Dp16 = 6) and female (WT = 6; Dp16 = 6) mice. I-J) Body temperature in the light and dark cycle of male (I) and female (J) Dp16 and WT mice. Sample size for male (WT = 10; Dp16 = 10) and female (WT = 15; Dp16 = 15) mice. All data are presented as mean ± SEM. * P<0.05; *** P<0.001; **** P<0.0001. For body weight over time, data were analyzed by 2-way ANOVA with Sidek post hoc tests.

Glucose intolerance, insulin resistance, and impaired lipid clearance in chow-fed Dp16 mice.

A-B) Overnight fasting insulin, blood glucose, serum triglyceride, cholesterol, non-esterified free fatty acids (NEFA), and β-hydroxybutyrate (ketone) in male (A) and female (B) Dp16 and WT mice. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 15). C-F) Impaired glucose tolerance as determined by the glucose tolerance test (GTT) in male (C) and female (E) Dp16 mice compared to WT controls. Impaired insulin sensitivity as determined by the insulin tolerance test (ITT) in male (D) and female (F) Dp16 compared to WT controls. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 15). G-H) Impaired triglyceride clearance in response to lipid gavage as determined by the lipid tolerance test (LTT) in male (G) and female (H) Dp16 relative to WT controls. Sample size for male mice (WT = 10; Dp16 = 14) and female mice (WT = 15; Dp16 = 15). I-J) Pooled mouse sera from male (I) and female (J) Dp16 and WT mice were fractionated by fast protein liquid chromatography (FPLC), and the triglyceride and cholesterol content of each fraction was quantified. Fractions corresponding to very-low density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL) are indicated. All data are presented as mean ± SEM. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. For all tolerance tests, data were analyzed by 2-way ANOVA with Sidek post hoc tests.

Altered liver and serum metabolome in Dp16 male and female mice.

(A-B) Partial least squares discrimination analysis (PLS-DA) of liver and serum metabolites of Dp16 and WT males and females. N = 6 samples per genotype per sex. (C) Venn diagram of differential metabolites shared between liver and serum in Dp16 male or female mice. (D) Venn diagram of differential liver or serum metabolites shared between Dp16 males and females. (E) KEGG enrichment showing altered metabolic processes in Dp16 female serum. ES, enrichment score; NES, normalized enrichment score.

Selective differential metabolites in the liver and serum of Dp16 male mice

. Metabolites are considered significantly different if fold change (FC) > 1.2 or < 0.833, P-value < 0.05, and the variable importance in projection (VIP) score is > 1. Sample size: WT (n = 6) and Dp16 (n = 6)

Selective differential metabolites in the liver and serum of Dp16 female mice

. Metabolites are considered significantly different if fold change (FC) > 1.2 or < 0.833, P-value < 0.05, and the variable importance in projection (VIP) score is > 1. Sample size: WT (n = 6) and Dp16 (n = 6)

Transcriptomic changes and altered biological pathways across tissues in chow-fed Dp16 male and female mice.

A) Number of differentially expressed genes (DEGs) that up or down regulated across six tissues in male and female Dp16 mice and their WT littermate controls. DEG is defined as any gene with log2(FC) > 0.5 and padj < 0.05. N = 6 per genotype per tissue. gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; BAT, brown adipose tissue. B) Overlap analysis showing DEGs that are shared between males and females, as well as those DEGs found in males or females only, across six tissues. C) Gene ontology highlighting some of the top biological pathways altered across six tissues in male and female Dp16 mice.

Sexually dimorphism in body weight, body temperature, food intake, and physical activity in Dp16 mice in response to a high-fat diet (HFD).

A) Body weight of HFD-fed male Dp16 and WT mice over time. B) absolute and relative (% of body weight) fat and lean mass in male mice (WT = 15; Dp16 = 12). C) Body weight of HFD-fed female Dp16 and WT mice over time. D) absolute and relative (% of body weight) fat and lean mass in female mice (WT = 14; Dp16 = 14). E-F) Food intake, total physical activity level, and energy expenditure of male (E) and female (F) Dp16 and WT mice across the circadian cycle (light and dark) and metabolic states (ad libitum fed, fast, refeed). Sample size for male (WT = 8; Dp16 = 11) and female (WT = 12; Dp16 = 12) mice. G-H) fecal frequency, average fecal weight, and fecal energy content (per gram and total) in male (G) and female (H) Dp16 and WT mice on HFD. Sample size for male (WT = 6; Dp16 = 7) and female (WT = 6; Dp16 = 6) mice. I-J) Body temperature in the light and dark cycle of male (I) and female (J) Dp16 and WT mice on HFD. Sample size for male (WT = 15; Dp16 = 12) and female (WT = 14; Dp16 = 15) mice. All data are presented as mean ± SEM. * P<0.05; *** P<0.001; **** P<0.0001. For body weight over time, data were analyzed by 2-way ANOVA with Sidek post hoc tests.

Exacerbated glucose intolerance and insulin resistance in Dp16 mice fed a high-fat diet (HFD).

A-B) Overnight fasting insulin, blood glucose, serum triglyceride, cholesterol, non-esterified free fatty acids (NEFA), and β-hydroxybutyrate (ketone) in male (A) and female (B) Dp16 and WT mice on HFD. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 14). C-F) Exacerbated glucose intolerance as determined by the glucose tolerance test (GTT) in male (C) and female (E) Dp16 compared to WT controls on HFD. Exacerbated insulin resistance as determined by the insulin tolerance test (ITT) in male (D) and female (F) Dp16 compared to WT controls. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 14). G-H) The rate of triglyceride clearance in response to lipid gavage as determined by the lipid tolerance test (LTT) in male (G) and female (H) Dp16 and WT mice. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 14). I-J) Pooled mouse sera from male (I) and female (J) Dp16 and WT mice were fractionated by fast protein liquid chromatography (FPLC), and the triglyceride and cholesterol content of each fraction was quantified. Fractions corresponding to very-low density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL) are indicated. All data are presented as mean ± SEM. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. For all tolerance tests, data were analyzed by 2-way ANOVA with Sidek post hoc tests.