CD8+ T Cells Involved in Metabolic Inflammation in Visceral Adipose Tissue and Liver of Transgenic Pigs.

Anti-inflammatory therapies have the potential to become an effective treatment for obesity-related diseases. However, the huge gap of immune system between human and rodent leads to limitations of drug discovery. This work aims at constructing a transgenic pig model with higher risk of metabolic diseases and outlining the immune responses at the early stage of metaflammation by transcriptomic strategy. We used CRISPR/Cas9 techniques to targeted knock-in three humanized disease risk genes, GIPRdn , hIAPP and PNPLA3I148M . Transgenic effect increased the risk of metabolic disorders. Triple-transgenic pigs with short-term diet intervention showed early symptoms of type 2 diabetes, including glucose intolerance, pancreatic lipid infiltration, islet hypertrophy, hepatic lobular inflammation and adipose tissue inflammation. Molecular pathways related to CD8+ T cell function were significantly activated in the liver and visceral adipose samples from triple-transgenic pigs, including antigen processing and presentation, T-cell receptor signaling, co-stimulation, cytotoxicity, and cytokine and chemokine secretion. The similar pro-inflammatory signaling in liver and visceral adipose tissue indicated that there might be a potential immune crosstalk between the two tissues. Moreover, genes that functionally related to liver antioxidant activity, mitochondrial function and extracellular matrix showed distinct expression between the two groups, indicating metabolic stress in transgenic pigs' liver samples. We confirmed that triple-transgenic pigs had high coincidence with human metabolic diseases, especially in the scope of inflammatory signaling at early stage metaflammation. Taken together, this study provides a valuable large animal model for the clinical study of metaflammation and metabolic diseases.

Genetic characteristics of polycistronic system­mediated randomly­inserted multi­transgenes in miniature pigs and mice.

Multi­transgenic technology is superior to single transgenic technology in biological and medical research. Multi­transgene insertion mediated by a polycistronic system is more effective for the integration of polygenes. The multi­transgene insertion patterns and manners of inheritance are not completely understood. Copy number quantification is one available approach for addressing this issue. The present study determined copy numbers in two multi­transgenic mice (K3 and L3) and two multi­transgenic miniature pigs (Z2 and Z3) using absolute quantitative polymerase chain reaction analysis. For the F0 generation, a given transgene was able to exhibit different copy number integration capacities in different individuals. For the F1 generation, the most notable characteristic was that the copy number proportions were different among pedigrees (P<0.05). The results of the present study demonstrated that transgenes within the same vector exhibited the same integration trend between the F0 and F1 generations. In conclusion, intraspecific consistency and intergenerational copy numbers were compared and the integration capacity of each specific transgene differed in multi­transgenic animals. In particular, the copy number of one transgene may not be used to represent other transgenes in polycistronic vector­mediated multi­transgenic organisms. Consequently, in multi­transgenic experimental animal disease model research or breeding, copy numbers provide an important reference. Therefore, each transgene in multi­transgenic animals must be separately screened to prevent large copy number differences, and inconsistent expression between transgenes and miscellaneous data, in subsequent research.

Systemic overexpression of the 11ß­HSD1 promotes endoplasmic reticulum stress in multiple tissues and the development of metabolic syndrome in mice.

Glucocorticoids are associated with lipid metabolism and their abnormal expression has an important function in the development of metabolic syndrome. The 11ß­hydroxysteroid dehydrogenase type 1 (11ß­HSD1) is a metabolic enzyme of glucocorticoids and may be a potential drug target for the treatment of metabolic syndrome. However, the association between the systemic expression of 11ß­HSD1 and metabolic syndrome remains to be elucidated. The present study used a cytomegalovirus promoter to obtain mice that systemically overexpressed the 11ß­HSD1 gene. The transgenic mice and negative control groups received a high­fat diet at the age of 10 weeks in order to induce metabolic syndrome and this diet was continued for 12 weeks. Several indicators, including body weight, blood glucose, glucose tolerance and insulin resistance, were monitored in vivo. In addition, the protein expression levels of 11ß­HSD1 and DNA damage inducible transcript 3 were detected and the histopathology of important tissues for metabolic syndrome were analyzed. The current findings revealed that the body weights of transgenic mice were significantly higher compared with the control group before and during the periods of high fat diet induction. Transgenic mice also exhibited significantly impaired glucose tolerance, insulin resistance, endoplasmic reticulum stress and increased metabolic syndrome­associated biochemical indicators in the blood and severely impaired liver and kidney functions. The present study successfully established a 11ß­HSD1 systemic overexpression mouse model that exhibited typical characteristics of metabolic syndrome and may be useful for future studies of metabolic syndrome.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Characterization of long non-coding RNA transcriptome in high-energy diet induced nonalcoholic steatohepatitis minipigs.

Today, obesity and nonalcoholic steatohepatitis are a worldwide epidemic, although how these syndromes are regulated with respect to lncRNAs remains largely unknown. Our previous studies have revealed important pathological features and molecular characteristics of nonalcoholic steatohepatitis in the minipig model, and in this study, we analyze the features of lncRNAs and their potential target genes. Minipig samples only from liver were analyzed using next-generation deep sequencing. In total, we obtained 585 million raw reads approximately 70.4 Gb of high quality data. After a strict five-step filtering process, 1,179 lncRNAs were identified, including 89 differentially expressed lncRNAs (P < 0.05) in the experiment group relative to the control group. The cis and trans analysis identified target genes that were enriched for specific GO terms (P < 0.01), including immune processes, chemokine activity, cytokine activity, and G-protein coupled receptor binding, which are closely related to nonalcoholic steatohepatitis. The predicted protein-coding targets of the differentially expressed lncRNAs were further analyzed, such as PPAR, FADS2, DGAT2, ACAA2, CYP2E1, ADH4, and Fos. This study reveals a wealth of candidate lncRNAs involved in NASH and their regulated pathways, which should facilitate further research into the molecular mechanisms of this disorder.

A long-term high-fat, high-sucrose diet in Bama minipigs promotes lipid deposition and amyotrophy by up-regulating the myostatin pathway.

Skeletal muscle is as an important regulator of blood glucose and glycolipid metabolism and is closely related to motor ability. The underlying mechanisms by which dietary ectopic lipids in skeletal muscle prevents muscle growth remain elusive. We utilized miniature Bama swine as a model to mimic human obesity using prolonged dietary induction. After 23 months on a high-fat, high-sucrose diet, metabolic disorders were induced in the animals, which exhibited increased body weight, extensive lipid deposition in the skeletal muscle and amyotrophy. Microarray profiles demonstrated the up-regulation of genes related to fat deposition and muscle growth inhibition. We outline a clear potential pathway that in combination with increased 11ß-hydroxysteroid dehydrogenase type 1, promotes expression of a major inhibitor, myostatin, by converting corticosterone to cortisol, which leads to the growth inhibition of skeletal muscle. This research provides new insights into the treatment of muscle diseases induced by obesity.

Multi­transgenic minipig models exhibiting potential for hepatic insulin resistance and pancreatic apoptosis.

There are currently no multi­transgenic minipig models of diabetes for the regulation of multiple genes involved in its pathogenesis. The foot and mouth disease virus 2A (F2A)­mediated polycistronic system possesses several advantages, and the present study developed a novel multi­transgenic minipig model associated with diabetes using this system. The tissue­specific polycistronic system used in the present study consisted of two expression cassettes, separated by an insulator: (i) 11­ß­hydroxysteroid dehydrogenase 1 (11ß­HSD1), driven by the porcine liver­specific apolipoprotein E promoter; (ii) human islet amyloid polypeptide (hIAPP) and C/EBP homologous protein (CHOP), linked to the furin digested site and F­2A, driven by the porcine pancreas­specific insulin promoter. In the present study, porcine fetal fibroblasts were transfected with this vector. Following somatic cell nuclear transfer using 10 cell clones and the transplantation of 1,459 embryos in total, three Landrace x Yorkshire surrogates became pregnant and delivered three Wuzhishan piglets. Genomic polymerase chain reaction (PCR) demonstrated that the piglets were multi­transgenic. Reverse transcription­quantitative PCR confirmed that 11ß­HSD1 transcription was upregulated in the targeted liver. Similarly, hIAPP and CHOP were expressed at high levels, compared with the control (P<0.05 and P<0.01) in the pancreas, consistent with the western blotting and immunohistochemistry results. The primary results also showed that overexpression of 11ß­HSD1 in the liver increased the liver fat lipid parameters; and the levels of hIAPP and CHOP in the pancreatic islet cells, leading to delayed ß­cell development and apoptosis. This novel tissue­specific polycistronic system offers a promising starting point for efficiently mimicking multigenic metabolic disease.

A Long-Term High-Fat/High-Sucrose Diet Promotes Kidney Lipid Deposition and Causes Apoptosis and Glomerular Hypertrophy in Bama Minipigs.

Metabolic syndrome can induce chronic renal injury in humans. In the present study, Bama minipigs were fed a high-fat/high-sucrose diet (HFHSD) for 23 months, which caused them to develop the pathological characteristics of metabolic syndrome, including obesity, hyperinsulinemia, and hyperlipidemia, and resulted in kidney tissue damage. In the HFHSD group, the ratio of the glomus areas to the glomerulus area and the glomerular density inside the renal cortex both decreased. Lipid deposition in the renal tubules was detected in the HFHSD group, and up-regulated expression levels of SREBP-1, FABP3 and LEPR promoted lipid deposition. The decreased levels of SOD, T-AOC and GSH-PX indicated that the antioxidant capacity of the renal tissues was diminished in the HFHSD group compared with MDA, which increased. The renal tissue in the HFHSD group exhibited clear signs of inflammation as well as significantly elevated expression of key genes associated with inflammation, including tumor necrosis factor-α (TNF-α) and macrophage migration inhibitory factor (MIF), compared with the control group. The tubular epithelial cells in the HFHSD group displayed significantly greater numbers of apoptotic cells, and the expression of proliferating cell nuclear antigen (PCNA) in the renal tubules decreased. Caspase-3 expression increased significantly, and the transcription factor nuclear factor κB (NF-κB) was activated and translocated into the nucleus. In conclusion, long-term HFHSDs cause metabolic syndrome and chronic renal tissue injury in Bama minipigs. These findings provide a foundation for further studies investigating metabolic syndrome and nephropathy.

Hyperinsulinemia shifted energy supply from glucose to ketone bodies in early nonalcoholic steatohepatitis from high-fat high-sucrose diet induced Bama minipigs.

The minipig can serve as a good pharmacological model for human subjects. However, the long-term pathogenesis of high-calorie diet-induced metabolic syndromes, including NASH, has not been well described in minipigs. We examined the development of metabolic syndromes in Bama minipigs that were fed a high-fat, high-sucrose diet (HFHSD) for 23 months, by using histology and serum biochemistry and by profiling the gene expression patterns in the livers of HFHSD pigs compared to controls. The pathology findings revealed microvesicular steatosis, iron overload, arachidonic acid synthesis, lipid peroxidation, reduced antioxidant capacity, increased cellular damage, and inflammation in the liver. RNA-seq analysis revealed that 164 genes were differentially expressed between the livers of the HFHSD and control groups. The pathogenesis of early-stage NASH was characterized by hyperinsulinemia and by de novo synthesis of fatty acids and nascent triglycerides, which were deposited as lipid droplets in hepatocytes. Hyperinsulinemia shifted the energy supply from glucose to ketone bodies, and the high ketone body concentration induced the overexpression of cytochrome P450 2E1 (CYP2E1). The iron overload, CYP2E1 and alcohol dehydrogenase 4 overexpression promoted reactive oxygen species (ROS) production, which resulted in arachidonic and linoleic acid peroxidation and, in turn, led to malondialdehyde production and a cellular response to ROS-mediated DNA damage.

Global Transcriptomic Profiling of Cardiac Hypertrophy and Fatty Heart Induced by Long-Term High-Energy Diet in Bama Miniature Pigs.

A long-term high-energy diet affects human health and leads to obesity and metabolic syndrome in addition to cardiac steatosis and hypertrophy. Ectopic fat accumulation in the heart has been demonstrated to be a risk factor for heart disorders, but the molecular mechanism of heart disease remains largely unknown. Bama miniature pigs were fed a high-fat, high-sucrose diet (HFHSD) for 23 months. These pigs developed symptoms of metabolic syndrome and showed cardiac steatosis and hypertrophy with a greatly increased body weight (2.73-fold, P<0.01), insulin level (4.60-fold, P<0.01), heart weight (1.82-fold, P<0.05) and heart volume (1.60-fold, P<0.05) compared with the control pigs. To understand the molecular mechanisms of cardiac steatosis and hypertrophy, nine pig heart cRNA samples were hybridized to porcine GeneChips. Microarray analyses revealed that 1,022 genes were significantly differentially expressed (P<0.05, ≥1.5-fold change), including 591 up-regulated and 431 down-regulated genes in the HFHSD group relative to the control group. KEGG analysis indicated that the observed heart disorder involved the signal transduction-related MAPK, cytokine, and PPAR signaling pathways, energy metabolism-related fatty acid and oxidative phosphorylation signaling pathways, heart function signaling-related focal adhesion, axon guidance, hypertrophic cardiomyopathy and actin cytoskeleton signaling pathways, inflammation and apoptosis pathways, and others. Quantitative RT-PCR assays identified several important differentially expressed heart-related genes, including STAT3, ACSL4, ATF4, FADD, PPP3CA, CD74, SLA-8, VCL, ACTN2 and FGFR1, which may be targets of further research. This study shows that a long-term, high-energy diet induces obesity, cardiac steatosis, and hypertrophy and provides insights into the molecular mechanisms of hypertrophy and fatty heart to facilitate further research.

Long-term High-fat High-sucrose Diet Promotes Enlarged Islets and ß-Cell Damage by Oxidative Stress in Bama Minipigs.

OBJECTIVES: The effect of a long-term high-fat, high-caloric diet on the dysfunction of pancreas has not been clarified. We investigated the pancreatic histopathology and ß-cell apoptosis in Bama minipigs after 23 months on a high-fat high-sucrose diet (HFHSD). METHODS: Bama minipigs were randomly assigned to control (n = 6) and HFHSD groups (n = 6) for 23 months, and biochemical parameters were measured. Pancreata were subjected to histological analysis, followed by assessment with transmission electron microscopy. Lipid peroxidation was determined by the malondialdehyde concentration and antioxidant enzyme activity. Β-cell apoptosis was measured by an immunohistochemical method. RESULTS: In the HFHSD group, the islets were enlarged, and the pancreatic tissue had observed significant fatty infiltration. Moreover, the feeding program damaged the normal pancreatic tissue structure. The level of lipid peroxidation was increased, and the activities of pancreatic antioxidant enzymes were significantly decreased. The expression levels of caspase-3, Bax, and insulin were significantly increased (P < 0.05), and the expression levels of proliferating cell nuclear antigen and Bcl-2 were decreased (P < 0.05). CONCLUSIONS: The long-term HFHSD promotes pancreatic steatosis and oxidative stress, which increases ß-cell apoptosis as indicated by the activation of caspase-3 through the mitochondrial pathway (Bcl-2/Bax).

Transcriptome analysis on the inflammatory cell infiltration of nonalcoholic steatohepatitis in bama minipigs induced by a long-term high-fat, high-sucrose diet.

Long-term adherence to a high-fat, high-calorie diet influences human health and causes obesity, metabolic syndrome and nonalcoholic steatohepatitis (NASH). Inflammation plays a key role in the development of NASH; however, the mechanism of inflammation induced by over-nutrition remains largely unknown. In this study, we fed Bama minipigs a high-fat, high-sucrose diet (HFHSD) for 23 months. The pigs exhibited characteristics of metabolic syndrome and developed steatohepatitis with greatly increased numbers of inflammatory cells, such as lymphocytes (2.27-fold, P<0.05), Kupffer cells (2.59-fold, P<0.05), eosinophils (1.42-fold, P<0.05) and neutrophils (2.77-fold, P<0.05). High-throughput RNA sequencing (RNA-seq) was performed to explore the systemic transcriptome of the pig liver during inflammation. Approximately 18.2 gigabases of raw sequence data were generated, and over 303 million high-quality reads were assembled into 21,126 unigenes. RNA-seq data analysis showed that 822 genes were differentially expressed in liver (P<0.05) between the HFHSD and control groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the process of inflammation involved the inflammatory signal transduction-related toll-like receptor, MAPK, and PPAR signaling pathways; the cytokine-related chemokine signaling, cytokine-cytokine receptor interaction, and IL2, IL4, IL6, and IL12 signaling pathways; the leukocyte receptor signaling-related T cell, B cell, and natural killer cell signaling pathways; inflammatory cell migration and invasion- related pathways; and other pathways. Moreover, we identified several differentially expressed inflammation-related genes between the two groups, including FOS, JUN, TLR7, MYC, PIK3CD, VAV3, IL2RB and IL4R, that could be potential targets for further investigation. Our study suggested that long-term HFHSD induced obesity and liver inflammation, providing basic insight into the molecular mechanism of this condition and laying the groundwork for further studies on obesity and steatohepatitis.

Molecular characterization of swine leukocyte antigen class I genes by sequence-based and PCR-SSP method in Guizhou minipigs.

The highly polymorphic swine leucocyte antigen (SLA) genes play an important role in swine immune responses to infectious diseases, vaccines and production performance. The pig resource with well defined SLA genes is useful for xenotransplantation and immunological studies. In this study, we have characterized three SLA class I genes (SLA-1, SLA-3, SLA-2) of 22 founder Guizhou minipigs using sequence-based typing method. Thirteen alleles were detected in this population, compared with the SLA allele sequences in GenBank, 11 of 13 SLA class I alleles were novel in Guizhou minipigs. There are four SLA I haplotypes, none of them previously reported in other pigs. Based on these alleles sequences information, we developed a simple method implemented to SLA-typing for unknown offsprings of Guizhou minipigs, relying on designed 13 sequence specific primers that could discriminate each one among which located in each locus using PCR in a SLA typing assay. According the combination methods of sequence-based typing and PCR-SSP, we were able to rapidly conduct SLA typing for Guizhou breeding stock and identify four SLA haplotypes present in the herd. This resource population of SLA-defined Guizhou minipigs will be useful as animal models for xenotransplantation and further immunological research.

Molecular cloning and characterization of the promoter region of the porcine apolipoprotein E gene.

Apolipoprotein E (APOE), a component of lipoproteins plays an important role in the transport and metabolism of cholesterol, and is associated with hyperlipoproteinemia and Alzheimer's disease. In order to further understand the characterization of APOE gene, the promoter of APOE gene of Landrace pigs was analyzed in the present study. The genomic structure and amino acid sequence in pigs were analyzed and found to share high similarity in those of human but low similarity in promoter region. Real-time PCR revealed the APOE gene expression pattern of pigs in diverse tissues. The highest expression level was observed in liver, relatively low expression in other tissues, especially in stomach and muscle. Furthermore, the promoter expressing in Hepa 1-6 was significantly better at driving luciferase expression compared with C2C12 cell. After analysis of porcine APOE gene promoter regions, potential transcription factor binding sites were predicted and two GC signals, a TATA box were indicated. Results of promoter activity analysis indicated that one of potential regulatory elements was located in the region -669 to -259, which was essential for a high expression of the APOE gene. Promoter mutation and deletion analysis further suggested that the C/EBPA binding site within the APOE promoter was responsible for the regulation of APOE transcription. Electrophoretic mobility shift assays also showed the binding site of the transcription factor C/EBPA. This study advances our knowledge of the promoter of the porcine APOE gene.