Gene Expression Profile Analysis of Type 2 Diabetic Mouse Liver

<div><p>Liver plays a key role in glucose metabolism and homeostasis, and impaired hepatic glucose metabolism contributes to the development of type 2 diabetes. However, the precise gene expression profile of diabetic liver and its association with diabetes and related diseases are yet to be further elucidated. In this study, we detected the gene expression profile by high-throughput sequencing in 9-week-old normal and type 2 diabetic db/db mouse liver. Totally 12132 genes were detected, and 2627 genes were significantly changed in diabetic mouse liver. Biological process analysis showed that the upregulated genes in diabetic mouse liver were mainly enriched in metabolic processes. Surprisingly, the downregulated genes in diabetic mouse liver were mainly enriched in immune-related processes, although all the altered genes were still mainly enriched in metabolic processes. Similarly, KEGG pathway analysis showed that metabolic pathways were the major pathways altered in diabetic mouse liver, and downregulated genes were enriched in immune and cancer pathways. Analysis of the key enzyme genes in fatty acid and glucose metabolism showed that some key enzyme genes were significantly increased and none of the detected key enzyme genes were decreased. In addition, FunDo analysis showed that liver cancer and hepatitis were most likely to be associated with diabetes. Taken together, this study provides the digital gene expression profile of diabetic mouse liver, and demonstrates the main diabetes-associated hepatic biological processes, pathways, key enzyme genes in fatty acid and glucose metabolism and potential hepatic diseases.</p> </div

The expression of enzymes directly participated in gluconeogenesis, glycolysis and glycogen metabolism was increased or not significantly changed in diabetic mouse liver.

<p>(A, D) Schematic of gluconeogenesis, glycolysis and glycogen metabolism, and the expression of enzymes directly participated in these processes of diabetic mouse liver. The key enzymes include HK, PFK-1 and PK for glycolysis, G6Pase, FBPase and PEPCK for gluconeogenesis, HK, UDP-GP and GS for glycogen synthesis, and GP for glycogenolysis. Red color represents the upregulated genes in diabetic mouse liver with fold change ≥ 1.5 and FDR < 0.001, gray color indicates no significant change, and white color indicates the genes were not detected. (B, C and E) The mRNA levels of the enzymes directly participated in glycolysis, gluconeogenesis and glycogen metabolism in normal (N) and diabetic (DB) mouse liver. HK, hexokinase; PGI, Phosphoglucoisomerase; PFK-1, phosphofructokinase; ALDB, aldolase; TPI, triose phosphate isomerase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerokinase; PGM, phosphoglyceromutase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; G6Pase, Glucose-6-Phosphatase; FBPase, Fructose 1,6-bisphosphatase; PEPCK, Phosphoenolpyruvate carboxykinase; UDP-GP, Uridine diphosphoglucose pyrophosphorylase; GS, Glycogen Synthase; GP, Glycogen Phosphorylase.</p

Diabetes is correlated with different liver diseases at a transcriptional view.

<p>(A) The map of top 5 liver diseases enriched with the genes altered in 9-week-old db/db mouse liver. 2627 altered genes were assigned to different diseases using the web tool FunDO. The sizes of the disease nodes are proportional to the number of enriched genes. (B) The number of hit genes and <i>P</i>-value of the top 5 enriched liver diseases in (A).</p

The associated genes in the top 5 KEGG pathways significantly altered in diabetic mouse liver.

<p>The associated genes in the top 5 KEGG pathways significantly altered in diabetic mouse liver.</p

Genes and the related biological processes altered in diabetic mouse liver.

<p>(A) The 8551 selected genes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057766#pone.0057766.s001" target="_blank">Figure S1A</a> were separated into two distinct clusters according to the genes upregulated or downregulated in diabetic (DB) mouse liver compared with normal (N) control. Red lines indicate Cluster A including 1933 upregulated genes in diabetic mouse liver. Blue lines indicate Cluster B including 694 downregulated genes . Purple lines indicate the total 2627 altered genes, which include all genes in Cluster A and B. (B) The clustered genes were assigned to different biological processes based on Gene Ontology using the web tool DAVID. The top 10 biological functions and the case genes in each cluster ranked by <i>P</i>-value were listed (case genes ≥ 10).</p

Sequencing and mapping messages of hepatic mRNA profiling of normal and diabetic mice.

<p>(A) The high-quality clean reads from high-throughput sequencing. Total liver RNA from 9-week-old normal (N) and diabetic (DB) db/db mice was used to prepare the high-throughput sequencing library. (B) The proportions of high-quality clean reads unmapped and/or mapped to unique genes, multiple genes and genome. (C) The number of genes detected in normal and diabetic mouse liver. (D) The top 15 abundance change genes downregulated or upregulated in diabetic mouse liver. (E) The top 15 fold change genes downregulated or upregulated in diabetic mouse liver.</p

Identification of mouse and human small RNAs unmapped to reference genome but mapped to rDNA.

<p>(A) Percentage of unique and redundant small RNAs unmapped to reference genome. Mouse neutrophil (GSM304914), young mouse sample (GPL7059) and human liver (GSM531975 and GSM531976) small RNA data obtained from high-throughput sequencing were randomly collected from GEO. (B) Percentage of small RNAs mapped to the 45-kb mouse or 43-kb human rDNA unit. The blue and green histograms present the percentage of unique and redundant reads mapped to rDNA in total reads. The red and purple histograms show the percentage of unique and redundant reads mapped to rDNA among those unmapped to reference genome. (C and D) A continuous tag sequence density estimation by F-Seq showed that srRNAs from mouse (C) and human (D) were mainly enriched in the regions coding 18S, 5.8S and 28S rRNA.</p

Profiling and Identification of Small rDNA-Derived RNAs and Their Potential Biological Functions

<div><p>Small non-coding RNAs constitute a large family of regulatory molecules with diverse functions. Notably, some small non-coding RNAs matched to rDNA have been identified as qiRNAs and small guide RNAs involved in various biological processes. However, a large number of small rDNA-derived RNAs (srRNAs) are usually neglected and yet to be investigated. We systematically investigated srRNAs using small RNA datasets generated by high-throughput sequencing, and found srRNAs are mainly mapped to rRNA coding regions in sense direction. The datasets from immunoprecipitation and high-throughput sequencing demonstrate that srRNAs are co-immunoprecipitated with Argonaute (AGO) proteins. Furthermore, the srRNA expression profile in mouse liver is affected by diabetes. Overexpression or inhibition of srRNAs in cultured cells shows that srRNAs are involved in various signaling pathways. This study presents a global view of srRNAs in total small RNA and AGO protein complex from different species, and demonstrates that srRNAs are correlated with diabetes, and involved in some biological processes. These findings provide new insights into srRNAs and their functions in various physiological and pathological processes.</p> </div

Size and frequency distribution of the srRNAs as well as the top 20 individual srRNAs.

<p>(A) A histogram of size distribution of 18–30 nt unique srRNAs in the indicated mouse and human samples. (B) Cumulative length distribution of srRNAs in the indicated samples. (C) The top 20 abundantly expressed srRNAs in the indicated mouse samples. (D) The top 20 expressed srRNAs in the indicated human samples.</p

The various biological functions of srRNAs.

<p>(A) The effect of the selected srRNA mimics on PEPCK and G6Pase promoter activity. After transfection with the indicated srRNA and luciferase reporter for 72 h, Hepa 1–6 cells were harvested for luciferase assay. In this and all other figures, error bars represent SD. (B) The effect of the selected srRNA inhibitors on PEPCK and G6Pase promoter activity. After transfection with the indicated srRNA mimics and luciferase reporter for 72 h, Hepa 1–6 cells were harvested for luciferase assay. (C) The effect of the selected srRNA inhibitors on PPARγ promoter activity. After transfection with the indicated srRNA inhibitor and PPARγ promoter luciferase reporter for 72 h, Hepa 1–6 cells were harvested for luciferase assay. (D) The effect of the selected srRNA mimics on intracellular ATP levels. After transfection with the indicated srRNA mimics for 72 h, Hepa 1–6 cells were harvested for the measurement of ATP. (E) The effect of the selected srRNA inhibitors on PUMA promoter activity. After transfection with the indicated srRNA inhibitor and PUMA promoter luciferase reporter for 72 h, NIH/3T3 cells were harvested for luciferase assay. (F) The effect of the selected srRNA mimics on ERK pathway including phosohorylation of Erk1/2, p90RSK, Elk-1 and p70S6K. After transfection with the indicated srRNA mimic for 72 h, Hepa 1–6 cells were harvested for western blot. Tubulin was measured as internal control. *p < 0.05, **p < 0.01 versus negative control (NC).</p