Hypermetabolism in mice carrying a near complete human chromosome 21

Abstract

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are due to sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca ²⁺ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

Figure 1.. Human chromosome 21 genes are differentially expressed and regulated in mouse adipose tissue, liver, and skeletal muscle.

A) Graphical representation of human chromosome 21 (Hsa21) and the entire long arm (Hsa21q) region carried by a mouse artificial chromosome in the transchromosomic mouse model (TcMAC21). Four deletions that occurred during generation of the transchromosomic mice eliminate 14/213 protein coding genes (PCGs; 7%) and 105/487 predicted or known non-protein coding genes (NPCGs; 22%) (18). B) Global view of transcriptionally expressed and repressed protein and non-protein coding gene regions over the entire Hsa21q across five tissues. Gray box denotes transcript that is not detected. C) Transcriptional activity map showing only Hsa21 genes expressed by at least one tissue. Gray box denotes transcript that is not detected. D) Overlap analysis showing shared expression of human protein coding and non-protein coding genes across five tissues. Of the 235 unique human genes expressed by the MAC21, 54% are protein coding and 46% are non-protein coding genes. PCGs, protein coding genes; NPCGs, non-protein coding genes; B, brown adipose tissue; iW, inguinal white adipose tissue; gW, gonadal white adipose tissue; L, liver; M, skeletal muscle (gastrocnemius); n.d., not detected. n = 5 RNA samples per group per tissue-type. Mice were on high-fat diet for 16 weeks at the time of tissue collection.

Figure 2.. Hypermetabolism in TcMAC21 mice.

A) Body weights of mice fed standard chow. B) Body composition analysis of fat and lean mass (relative to body weight). C) Food intake over a 24 h period. D-E) Energy expenditure (EE) and physical activity level over 24 hr period in chow-fed mice. F) Hematoxylin and eosin (H&E) stained sections of inguinal white adipose tissue (iWAT), and adipocyte cross-sectional area (CSA) quantification. G) Histology of gonadal white adipose tissue (gWAT), and adipocyte CSA quantification. H) Histology of liver tissues with quantification of area covered by lipid droplets per focal plane. I) Fasting serum triglyceride, cholesterol, non-esterified fatty acids (NEFA), β-hydroxybutyrate (ketone) levels. J) fasting blood glucose and insulin levels. K) Insulin resistance index (homeostatic model assessment for insulin resistance (HOMA-IR). L) Glucose tolerance tests. M) Insulin tolerance tests. N) Histology of pancreas and quantification of β-islet CSA. O) Pancreatic insulin and somatostatin (SST) contents (normalized to pancreatic protein input). P) Electron micrographs (EM) of pancreatic β-cells showing dense insulin granules and their surrounding vesicles, and the quantification of insulin granule CSA, insulin vesicle CSA, and the ratio of insulin granule to insulin vesicle. n = 8 euploid and 9 TcMAC21 mice for all graphs from A to Y. n = 8–10 euploid and 5–8 TcMAC21 samples used for pancreatic analysis by H&E and protein quantification, graphs N and P. n = 3 euploid and 3 TcMAC21 used for EM quantification; each data point represents 1,200 insulin granules and 1,200 insulin vesicles quantified across 6 unique locations per mouse, graphs P.

Figure 3.. TcMAC21 mice are resistant to diet-induced obesity and metabolic dysfunction.

A) Body weights over time on a high-fat diet and representative mouse images. B) Body composition analysis of fat and lean mass. C) Histology of inguinal white adipose tissue (iWAT), and quantification of adipocyte cross-sectional area (CSA). D) Histology of gonadal white adipose tissue (gWAT), and quantification of adipocyte CSA. E) Histology of liver tissues, and quantification of area covered by lipid droplets per focal plane. F) Fasting serum triglyceride, cholesterol, non-esterified fatty acids (NEFA), β-hydroxybutyrate (ketone) levels. G) Fasting blood glucose and insulin levels. H) Insulin resistance index (homeostatic model assessment for insulin resistance (HOMA-IR). I) Glucose tolerance tests (GTT). J) Serum insulin levels during GTT. K) Insulin tolerance tests. L) Blood glucose levels after an overnight (16 h) fast and 1, 2, and 3 h of food reintroduction. M) Serum insulin levels after a 16 h fast and 2 h of refeeding. N) Pancreas histology and quantification of β-islet CSA. O-P) Pancreatic insulin and somatostatin (SST) contents (normalized to pancreatic protein input). Q) Electron micrographs (EM) of pancreatic β-cells showing dense insulin granules and their surrounding vesicles, and quantification of insulin granule CSA, insulin vesicle CSA, and the ratio of insulin granule to insulin vesicle. n = 8 euploid and 8–9 TcMAC21 mice for all graphs from A to P. n = 3 euploid and 3 TcMAC21 used for EM quantification; each data point represents 1,200 insulin granules and 1,200 insulin vesicles quantified across 6 unique locations per mouse, graphs Q.

Figure 4.. Hypermetabolism of TcMAC21 mice on HFD is uncoupled from changes in adipose and liver transcriptomes.

A) Food intake in mice fed a high-fat diet (HFD). B) Fecal energy content. C-D) Energy expenditure (EE) and activity level over 24 h period in HFD-fed mice. E) Serum Triiodothyronine (T₃) and Thyroxine (T₄) levels. F) Deep colonic and tail temperature measured over three days in both the light and dark cycle. G) Representative infrared images of mice. H) Body weights of euploid, TcMAC21, and weight-matched (WM) control C57BL/6 mice. I) Interscapular skin temperature of euploid, TcMAC21, and WM control mice. J) Representative histology of brown adipose tissue (BAT). K) Quantification of percent total lipid area coverage per focal plane in BAT of euploid and TcMAC21. L) Expression of mouse genes (by qPCR) known to play major metabolic roles in BAT. M) Differentially expressed mouse genes (DEGs), both protein coding (PCG) and non-protein coding genes (NPCG) in BAT, liver, gonadal white adipose tissue (gWAT), and inguinal white adipose tissue (iWAT). All data is relative to euploid, and presented as Log2(FC). The list of genes shown is all the up and down regulated mouse genes (significant by adjusted p-value cut-off) for all 4 tissues. The red bars indicate up regulated genes and the blue bars indicate down regulated genes. N) General view and summary of transcriptional changes in BAT, Liver, gWAT, and iWAT to highlight the strikingly minimal changes in the mouse transcriptome across the four tissues. Only a combined total of 114 differentially expressed genes (DEGs) across four tissues, with the relative percentage (out of the 114 DEGs) shown for each tissue. Of the 114 DEGs, 105 are protein-coding genes (PCGs; dark yellow bar) and 9 are non-protein coding genes (NPCGs; light yellow bar). Of the 114 DEGs, 46 are upregulated (red bar) and 68 are down regulated (blue bar). In total, only a combined 0.53% change is noted in the transcriptome of all four tissues (out of the 21,752 RNAs detected).

Figure 5.. Sarcolipin overexpression in skeletal muscle drives TcMAC21 hypermetabolism.

A) RNA-sequencing analysis reveals changes in TcMAC21 relative to euploid skeletal muscle (gastrocnemius). Volcano plot of skeletal muscle transcriptome (transcripts from the mouse genome only). The lower dotted line denotes significance at the adjusted P value cut-off (adj. P = 0.05). The vertical dotted lines denote a Log2(FC) of −0.5 or 0.5. B) Gene Ontology analysis of RNA-sequencing results using ClusterProfiler (77). C) Expression of genes (by qPCR) known to be involved in metabolism, fast- or slow-twitch fiber types, futile cycling, thyroid hormone action, and calcium handling in skeletal muscle (gastrocnemius). D) Graphical representation of the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) and its regulator, sarcolipin (SLN). SERCA pump uses the energy derived from ATP hydrolysis to translocate Ca²⁺ from the cytosol back into the sarcoplasmic reticulum (SR), promoting muscle relaxation and restoring intracellular Ca²⁺ level following muscle contraction. SLN binds to SERCA and uncouples its Ca²⁺ transport activity from ATP hydrolysis and heat generation, thus promoting futile SERCA activity. E) qPCR analysis of sarcolipin (Sln) expression in euploid and TcMAC21 mice fed a standard chow, high-fat diet (HFD), and HFD while housed at thermoneutrality (30°C). F) Immunoblot of SLN and GAPDH (loading control) in muscle lysates of Euploid, TcMAC21, and SLN overexpression (OE) transgenic mice. G) Immunoblot quantification of SLN using GAPDH as a loading control. The dotted line marks the OE (SLN overexpression mouse model) level of SLN expression. H-I) Gastrocnemius immunofluorescent labeling of SLN-expressing muscle fibers. J) Muscle fiber cross-sectional area (CSA) quantification from WGA-stained gastrocnemius. K) Immunoblot of OXPHOS complex levels, with GAPDH as a loading control. L) Quantification of OXPHOS complex levels relative to GAPDH. M-N) Gastrocnemius immunofluorescent labeling of Succinate dehydrogenase subunit B (SDHB)-expressing muscle fibers. O-P) Seahorse respirometry analyses of frozen tissue samples. Shown here are the quadricep group average tracings for oxygen consumption rate (OCR) across the experimental time course. Q) OCR of TcMAC21 mitochondrial complex I, II, and IV relative to Euploid, for eleven separate tissues (quadricep, Qd; extensor digitorum longus, EDL; gastrocnemius, Gast; Plantaris, Plnt; Soleus, Sol; Tongue, Tng; Brown adipose tissue, BAT; Liver, Liv; Heart, Hrt; Inguinal white adipose tissue, iWAT; Gonadal white adipose tissue, gWAT) and two dietary conditions (Chow and HFD). DEGs, differentially expressed genes; PCGs, protein coding genes; NPCGs, non-protein coding genes. n = 5 euploid and 4 TcMAC21 for RNA-sequencing experiments. n = 8–9 euploid and 7–8 TcMAC21 for HFD qPCR. n = 7–10 euploid and TcMAC21 for Chow qPCR. n = 4–5 euploid and TcMAC21 for HFD + thermoneutrality qPCR. n = 6 euploid and 6 TcMAC21 for all immunoblots. n = 7 Euploid and 8 TcMAC21 for gastrocnemius CSA quantification. n = 6 euploid and 4 TcMAC21 for all mitochondrial respiration assays; each biological replicate represents the average of three technical replicates.

Figure 6.. Potential regulators of sarcolipin expression in skeletal muscle.

A.) Body weights at thermoneutrality (30°C) for 8 weeks. B) Body composition analysis of fat and lean mass. C) Fasting insulin levels. D) Energy expenditure in the dark and light cycles. E) Food intake. F) Volcano plot of skeletal muscle transcriptome (transcripts from the mouse genome only). The lower dotted line denotes significance at the P value cut-off (P = 0.05), and the upper dotted line denotes significance at the adjusted P value cut-off (adj. P = 0.05). The vertical dotted lines denote a Log 2 (Fold Change) of −0.5 or 0.5. Flanking the Volcano plot are the top down- and up-regulated mouse genes. G) Comparison of differentially expressed genes (DEGs) shared and not shared by TcMAC21 mice housed at 22°C vs. 30°C. Heat map showing all significantly up- or down-regulated shared genes. H) Direct comparison of TcMAC21 mice housed at 22°C and 30°C for most stably expressed mouse genes. Data filtered first by genes with Log2(FC) within ±0.5 (least change), then by lowest significance, and finally compared to the shared DEGs found in G. Table shows DEGs with least variation in expression at 22°C and 30°C. I) Overlap analysis of Hsa21-derived human transcripts expressed in TcMAC21 mice (skeletal muscle) housed at 22°C and 30°C. J) Graph showing the most stably expressed Hsa21-derived human genes in the TcMAC21 gastrocnemius. Top axis refers to the difference in Log2(FC) between human genes in TcMAC21 mice housed at 22°C and 30°C. Bottom axis shows the Log2(FC) value of a particular gene at 22°C and 30°C. The gene list shows all the human genes expressed by both groups with the least amount of change between the two temperatures.