Model-identification of diabetes-related sarcopenia in db/db mouse
Fifteen-week-old db/db mice showed a phenotype of severe obesity (Fig. 1a, b) with obvious hyperglycaemia (>25 mmol/L) (Fig. 1c) compared to normal control db/m. Morphological changes in GAS were observed by haematoxylin and eosin (H&E), Oil-Red O, and Masson’s trichrome staining compared to db/m mice (Fig. 1d–f). The average cross-sectional area (CSA) of muscle fibres was markedly reduced (Fig. 1g), and lipid droplet infiltration and increased fibrosis were observed in GAS of db/db mice, indicating evident pathological changes of diabetes-induced sarcopenia. The forelimb grip strength of db/db mice was significantly decreased (Fig. 1h), and the muscle mass and size of lower limbs were decreased (Fig. 1i) compared with db/m mice. These results indicate that muscle strength and mass were significantly decreased in db/db mice. Decreased bone mineral density (BMD), lower lean mass, and increased fat mass were observed by dual‐energy X‐ray absorptiometry (DEXA, Hologic Discovery A, Hologic Inc) analysis in the db/db group (Fig. 1j–l). Serum biochemical parameters were indicative of the systematic metabolic disorder. Triglycerides, glucose, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were increased significantly in db/db mice compared to db/m control (Supplementary Table 1). Morphological abnormalities of mitochondria, swelling of the endoplasmic reticulum, reduced muscular glycogen, and sarcomere damage were observed by transmission electron microscopy (TEM) in GAS of db/db compared with the db/m group (Fig. 1m, n). Our data showed that muscular weakness and loss with subcellular structural dysfunction are evident in db/db mice.
Identification of dif-mRNAs in GAS of db/db mice
To identify new molecular mechanisms during diabetes-accelerated sarcopenia, total RNA from GAS of db/db and db/m mice was used for whole transcriptome sequencing. Raw sequencing data were filtered, and clean reads were mapped to the reference genome (reference species: Mus_musculus. Reference Genome Version: GCF_000001635.26_GRCm38.p6). Filtered clean reads were spliced into putative transcripts (Supplementary Table 2). An absolute log2 FC value of 0.585 was used as the standard to confirm the dif-mRNAs (false discovery rate, FDR < 0.05). Based on that standard, 607 upregulated mRNAs and 1332 downregulated mRNAs were identified in db/db vs. db/m (Supplementary Data 1–2). The heatmap and a volcano plot (Fig. 2a, b) showed differential mRNA expression between db/db vs. db/m (FDR < 0.05). The top 20 dysregulated mRNAs are shown in Supplementary Table 3. The top5 up-regulated mRNAs are Ttll7, Sorbs2, Sh3rf2, Prelp, and Ppp1r3c, while the top5 down-regulated genes include Xirp1, Vldlr, Vgll2, Ugp2, and Ucp3 in Supplementary Table 3.
Functional prediction of differentially expressed mRNAs (dif-mRNAs)
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify potential biological functions and implications. Dif-mRNAs were exhibited in three modules of the GO pathway enrichment analysis in GAS of the db/db group. Upregulated mRNAs were mainly enriched in the extracellular matrix (ECM) organization (GO:0030198), negative regulation of the Wnt pathway (GO:0030178), retinoic acid catabolic process (GO:0034653), inflammatory response (GO:0006954), and collagen fibril organization (GO:0030199) in the biological process category of GO terms (Fig. 2c). Downregulated mRNAs were primarily enriched in terms such as immune system process (GO:0002376), and skeletal muscle contraction (GO:0006936) (Fig. 2d). The top 10 GO terms associated with dif-mRNAs of db/db mice are shown in Supplementary Table 4–5. For dif-mRNAs, enriched transcription factors (TFs), including Nr4a1, Mafa, Jun (also known as AP-1), ATF5, Trp63, and Tbx1, were differentially expressed in the db/db group (Supplementary Table 6). These data showed that differential TFs might be essential for the progression of diabetes-induced sarcopenia.
Then, 607 upregulated mRNAs and 1332 downregulated mRNAs were enriched in dysregulated pathways by KEGG analysis. Our data showed that 607 upregulated genes were enriched in 204 signalling pathways, which were mainly enriched in the pathway of neuroactive ligand-receptor interaction (PATH: map04080), cAMP signaling (PATH: map04024), ECM-receptor interaction (PATH: map04512), regulation lipolysis in adipocytes (PATH: map04923) and protein digestion and absorption (PATH: map04974) (Fig. 2e). KEGG enrichment showed that 1332 downregulated genes were enriched in 249 significant pathways, including the pathway of muscle contraction (PATH: map04260), cell adhesion molecules (PATH: map04514), TNF signaling (PATH: map04668), arginine and proline metabolism (PATH: map00330), and glutathione metabolism (PATH: map00480) (Fig. 2f). The top 10 KEGG pathways associated with dysregulated mRNAs of diabetes-related sarcopenia are shown in Supplementary Table 7–8, indicating that these significant changes in pathways may contribute to the development of diabetes-induced sarcopenia.
Based on shared biological function, location, or expression of genes, Gene set enrichment analysis (GSEA) has a broad scope of enrichment analysis and discovers potential mechanisms in disease. The dif-mRNAs were associated with significant pathways in GSEA, as shown in Supplementary Table 9 (p-value < 0.05). The GSEA results showed that the top 2 upregulated mRNAs were closely related to neuroactive ligand-receptor interactions (PATH: map04080) and the renin secretion pathway (PATH: map04924) (Supplementary Fig. 1a, b). In contrast, the top 2 downregulated mRNAs were highly correlated with the pathway of oxidative phosphorylation (PATH: map00190), arginine and proline metabolism (PATH: map00330) (Supplementary Fig. 1c, d).
To investigate deeper interactions in significant pathways, 370 dif-mRNAs enriched into 10 mainly significant pathways of KEGG were subjected to a visual pathway-act-network analysis (Supplementary Fig. 2a). A pathway-act network was constructed with 233 dif-mRNAs enriched with 10 significant pathways of GSEA (Supplementary Fig. 2b). These data showed that significant metabolic pathway regulation might be involved in the development of diabetes-induced sarcopenia.
Identification of differentially expressed lncRNAs (dif-lncRNAs) in GAS of db/db vs. db/m
According to the screening criteria, 306 upregulated lncRNAs and 554 downregulated lncRNAs were identified in db/db vs. db/m (Supplementary Data 3–4). All dif-lncRNAs were widely found on all chromosomes, including the X and Y sex chromosomes (Fig. 3a). These dif-lncRNAs were divided into four categories based on different genic loci: exonic sense, intronic sense, antisense, and intergenic (Fig. 3b). Heatmap and volcano plot were constructed (Fig. 3c, d). We also identified the top 20 dysregulated lncRNAs in db/db mice (Supplementary Table 10).
Co-expression network construction and Real-Time Quantitative PCR (RT-qPCR) validation
Based on the co-expression relationship of lncRNA-mRNA, we generated diabetes-induced sarcopenia‐specific lncRNA‐mRNA co-expression networks. Through using correlation analysis of the expression, 24 lncRNA (log2 FC > 1, q-value < 0.05 and expression >2) and 860 mRNAs (Pearson correlation coefficient, PCC > 0.95, p-value < 0.05) were used to construct a highly correlated co-expression network. Several core dif-lncRNAs in the co-expression network were validated by RT-qPCR in db/db and db/m mice. The results showed that 1700047G03Rik and Gm31814 were upregulated, while Gm20743, Gm35438, Gm36131, and A330074k22rik were downregulated in GAS of the db/db group compared with db/m mice (Fig. 4a–f). Accumulated evidence showed that the elevated serum PA level in T2DM is involved in the development of insulin resistance and implicated in skeletal muscle inflammation, oxidative stress, and mitochondrial dysfunction by causing lipotoxicity, which leads to skeletal muscle loss21,22. In our results, PA-treated C2C12 myotubes decreased the diameter of and inhibited the differentiation level of myotubes using fusion index analysis (Fig. 5a, b). Atrogin-1, MuRF-1, and Mstn, which serve as muscle atrophy markers, were higher relative to NC (Fig. 5c–e). These results suggested that PA efficiently induced muscle atrophy in C2C12 myotubes. The expression of these six dif-lncRNAs was further confirmed in a PA-induced muscle atrophy C2C12 cell model by RT-qPCR (Fig. 6a–f). The results found that the expression of most lncRNAs was consistent with animal models and RNA-seq data except for Gm38141 (Fig. 6e), which may be due to the differences in tissue and cellular components. Overall, the RT-qPCR validation of dysregulated lncRNAs of co-expression network in diabetes-associated sarcopenia mouse models and PA-treated C2C12 cells provide us with further indications to characterize their functions in the development of diabetes-associated sarcopenia. In addition, we found that 3 core dif-lncRNAs named Gm20743 (log2FC = −1.57, q-value < 0.001), Gm35438 (log2FC = −1.90, q-value < 0.001) and 1700047G03Rik (log2FC = 6.34, q-value < 0.001) had the highest number of interactions in the co-expression network and may contribute to the development of diabetes-related sarcopenia (Figs. 7a and 8a).
The potential function of candidate lncRNAs
We performed functional prediction of the co-expressed dif-mRNAs highly correlated with the three candidate lncRNAs by KEGG analysis, respectively. Functional analysis revealed that Gm20743 may be involved in mitochondrial functions like oxidative phosphorylation and glutathione pathway (Fig. 7b). Through RT-qPCR verification, the key genes (like ATP1B1, Gclc, Gclm, and GPX4) in the co-expression network which contributed to oxidative phosphorylation and glutathione pathway were downregulated in vivo (Fig. 4g–j) and in vitro (Fig. 6g–j) consistent with the RNA-seq results. These results suggested that Gm20743 may be involved in the regulation of mitochondrial function and redox homeostasis in skeletal muscle. KEGG analysis found that the dif-mRNAs co-expressed with Gm35438 are mainly enriched in muscle contraction and adrenergic signaling pathways (Fig. 7c). The co-expressed contractile proteins Tnnc1 and TPM3 in these differentially enriched pathways were downregulated in db/db mice (Fig. 4k, l) and PA-treated C2C12 cells (Fig. 6k, l) compared to their control groups, and these results suggested that Gm35438 may be related to skeletal muscle contraction and strength. Through co-expression network and bioinformatic analysis, we found the dif-mRNAs co-expressed with 1700047G03Rik (the top upregulated lncRNA in Supplementary Table 10) were mainly enriched in the neuroactive ligand-receptor interaction and glycolipid metabolism pathway (Fig. 8b). The RT-qPCR validation found that co-expressed key catalytic enzymes ACSL3 and DGAT2 in the glycolipid metabolism pathway were upregulated in both animal (Fig. 4m, n) and cell models of diabetic sarcopenia (Fig. 6m, n). It is suggested that 1700047G03Rik may be involved in the intramuscular lipid deposition of diabetes-related sarcopenia.
The role of Gm20743 in the development of diabetic sarcopenia
In this study, we specifically focused on the role of lncRNA Gm20743 on mitochondrial function and redox homeostasis in skeletal muscle cells. The key reasons are (1) First, based on the criteria |log2 FC | > 0.585, FDR < 0.05, and expression >2, we ranked lncRNAs in terms of FDR and found that lncRNA Gm20743 ranked in the top 20 differentially expressed lncRNAs. (2) Secondly, through a systematic literature review, many studies found that mitochondrial dysfunction and oxidative stress play an important role in sarcopenia; (3) Moreover, our previous study found that insulin resistance, mitochondrial dysfunction, and oxidative stress were involved in the development of muscle atrophy in PA-treated C2C12 cells23,24; (4) In addition, through functional prediction of dif-mRNAs and lncRNA-mRNA co-expression network. These results showed that the down-regulated mitochondrial oxidative phosphorylation, citrate cycle, and glutathione metabolism were the most significantly enriched pathways based on GSEA (Supplementary Figure 1c, e, f). The KEGG analysis also revealed that the genes highly correlated with Gm20743 were mainly enriched in down-regulated oxidative phosphorylation and glutathione metabolism pathways (Fig. 7b). The above findings suggested that Gm20743 may play an important role in regulating mitochondrial dysfunction and oxidative stress in diabetic sarcopenia.
To verify the role of Gm20743 in diabetes-related sarcopenia, through fluorescence in suit hybridization (FISH), our results found that the distribution of Gm20743 was found in both the cytoplasm and nucleus of skeletal muscle C2C12 cells (Fig. 9a). Gm20743 was knocked down in C2C12 cells by siRNA transfection. The expression of Gm20743 was significantly downregulated by Gm20743si-1, Gm20743si-2, and Gm20743si-3 in C2C12 cells (Fig. 9b), and Gm20743 was overexpressed in C2C12 cells by lentiviral transfection and the expression of Lv-Gm20743 was verified by RT-qPCR (Fig. 9c). After knocking down Gm20743, we found that the extent of knockdown of Gm20743si-1, and Gm20743si-2 was lower in C2C12 cells. To avoid off-target situations, we used Gm20743si-1 and Gm20743si-2 to knock down Gm20743 in C2C12 cells, respectively. Through the detection of Mito-Sox and 2’, 7’-dichlorofluorescein-diacetate (DCFH-DA), our results found that knockdown Gm20743 increased mitochondrial reactive oxygen species (ROS) (Fig. 9d, e) and intracellular ROS (Fig. 9f, g) in C2C12 cells with Gm20743si-1 and Gm20743si-2, while overexpression of Gm20743 significantly reduced mitochondrial ROS (Fig. 9h, i) and intracellular ROS (Fig. 9j, k) in PA-induced C2C12 cells. These results suggest that Gm20743 can regulate mitochondrial function and redox homeostasis in C2C12 cells. Previous studies found that mitochondrial dysfunction and oxidative stress prevent the proliferation and differentiation in skeletal muscle myoblasts25,26,27. To further assess whether Gm20743 is involved in the cell proliferation and myotube differentiation in skeletal muscle myoblasts, the detection of ethynyl-2′-deoxyuridine (EdU) and myosin heavy chain (MyHC) staining was determined. In our results, we found that knockdown Gm20743 decreased the number of EdU-positive cells in C2C12 myoblasts compared to the siNC group (Fig. 10a, b), and overexpressed Gm20743 increased the number of EdU-positive cells in PA-induced C2C12 myoblasts (Fig. 10c, d), suggesting Gm20743 improves cell proliferation in C2C12 cells. In addition, we observed that knockdown Gm20743 significantly decreased the number and diameter of myotube in C2C12 cells compared to the siNC group (Fig. 10e, f), while overexpressed Gm20743 significantly increased the number and diameter of myotube in PA-induced C2C12 myoblasts (Fig. 10g, h), compared to Lv-vector control. Together, these results indicate that Gm20743 may promote cell proliferation and myotube differentiation by preventing mitochondrial dysfunction and oxidative stress in skeletal muscle cells.
Wise (formerly TransferWise) is the cheaper, easier way to send money abroad. It helps people move money quickly and easily between bank accounts in different countries. Convert 60+ currencies with ridiculously low fees - on average 7x cheaper than a bank. No hidden fees, no markup on the exchange rate, ever.
How to access the offer?
1- Click here
2- Select “Register''
3- Enter your email address, create a password, and select your country of residence
4- Fill out the required personal information, and the free first transfer offer will be applied automatically.
Benefits of the Multi-Currency Account:
- Free to create online
- Hold 50+ currencies
- Get multiple local bank details in one account (including EU, UK, US)
- Convert currency at the real exchange rate, even on weekends
- Spend whilst travelling on the Wise debit card without high conversion fees
Wise International Transfers:
- $1.5 billion saved by customers every year
- Send money to over 60 target currencies
- Lower fees for larger transfers
- No hidden fees. No bad exchange rates. No surprises.
- Send your money with a bank transfer, or a debit or credit card