Altered gene expression and repressed markers of autophagy in skeletal muscle of insulin resistant patients with type 2 diabetes

Abstract

This case-control study was designed to investigate the gene expression profile in skeletal muscle from severely insulin resistant patients with long-standing type 2 diabetes (T2D), and to determine associated signaling pathways. Gene expression profiles were examined by whole transcriptome, strand-specific RNA-sequencing and associated signaling was determined by western blot. We identified 117 differentially expressed gene transcripts. Ingenuity Pathway Analysis related these differences to abnormal muscle morphology and mitochondrial dysfunction. Despite a ~5-fold difference in plasma insulin, we did not observe any difference in phosphorylation of AKT or AS160, although other insulin-sensitive cascades, as mTOR/4EBP1, had retained their sensitivity. Autophagy-related gene (ATG14, RB1CC1/FIP200, GABARAPL1, SQSTM1/p62, and WIPI1) and protein (LC3BII, SQSTM1/p62 and ATG5) expression were decreased in skeletal muscle from the patients, and this was associated with a trend to increased phosphorylation of the insulin-sensitive regulatory transcription factor FOXO3a. These data show that gene expression is highly altered and related to mitochondrial dysfunction and abnormal morphology in skeletal muscle from severely insulin resistant patients with T2D, and that this is associated with decreased expression of autophagy-related genes and proteins. We speculate that prolonged treatment with high doses of insulin may suppress autophagy thereby generating a vicious cycle maintaining insulin resistance.

Figure 1. Heat map of the 117 genes differentially expressed genes between controls and type 2 diabetic subjects.

Fold change in gene expression is color coded: red: expression higher than the median of all samples; blue: expression lower than the median of all samples; yellow: median expression. Supervised hierarchical clustering was performed vertically in samples and horizontally in genes. As illustrated by the dendrogram, the analysis identified two distinct clusters separating healthy subjects from patients with type 2 diabetes. The length of the lines indicates the degree of separation between the clusters.

Figure 2. Autophagy-related protein expression is repressed in skeletal muscle from T2D patients.

The ratio of LC3BII to LC3BI was equal in the two groups (A). Separate analysis of LC3BII and LC3BI showed that LC3BII was decreased in the diabetics (B), but the difference in LC3BI did not reach statistical significance (C). p62 and ATG5 were decreased in the patients (D,E). GABARAP was decreased in the patients, but the difference did not reach statistical significance (F). Values are means ± SEM. *Indicate difference in the mean values based on Student’s t-test. Representative western bots are shown below the graphs. Based on the applied molecular standards, approximated molecular weights are indicated on the right.

Figure 3. Autophagic signaling through ULK1 and Foxo3a are repressed in skeletal muscle from T2D patients.

AKT phosphorylation at Ser⁴⁷³ was equal in the two groups (A). mTOR phosphorylation at Ser²⁴⁴⁸ was elevated in the patients, but the difference did not reach statistical significance (B). ULK1 phosphorylation at Ser⁷⁵⁷ was equal in the two groups (C), while ULK1 phosphorylation at Ser⁵⁵⁵ was decreased in patients with T2D (D). AMPKα phosphorylation at Thr¹⁷² was equal in the two groups (E). FOXO3a phosphorylation at Ser^321/318 was increased in the patients, but the difference did not reach statistical significance (F). Values are means ± SEM. *Indicate difference in the mean values based on Student’s t-test. Representative western bots are shown below the graphs. Based on the applied molecular standards, approximated molecular weights are indicated on the right.

Figure 4. Signaling to protein synthesis is stimulated in skeletal muscle from T2D patients.

4EBP1 phosphorylation at Thr^37/46 was elevated, in the patients as demonstrated by decreased non-p-4EBP1 (A). S6rp phosphorylation at Ser^235/236 was equal in the two groups (B). Values are means ± SEM. *Indicate difference in the mean values based on Student’s t-test. Representative western bots are shown below the graphs. Based on the applied molecular standards, approximated molecular weights are indicated on the right.

Figure 5. Expression of mitochondrial protein is repressed in skeletal muscle from T2D patients.

Cyt-C, SDHA, VDAC, and COX-IV were suppressed in the patients (A–D), while the difference in PDHα1 did not reach statistical significance (E). Values are means ± SEM. *Indicate difference in the mean values based on Student’s t-test. Representative western bots are shown below the graphs. Based on the applied molecular standards, approximated molecular weights are indicated on the right.