Succinate induces skeletal muscle fiber remodeling via SUNCR1 signaling.

The conversion of skeletal muscle fiber from fast twitch to slow-twitch is important for sustained and tonic contractile events, maintenance of energy homeostasis, and the alleviation of fatigue. Skeletal muscle remodeling is effectively induced by endurance or aerobic exercise, which also generates several tricarboxylic acid (TCA) cycle intermediates, including succinate. However, whether succinate regulates muscle fiber-type transitions remains unclear. Here, we found that dietary succinate supplementation increased endurance exercise ability, myosin heavy chain I expression, aerobic enzyme activity, oxygen consumption, and mitochondrial biogenesis in mouse skeletal muscle. By contrast, succinate decreased lactate dehydrogenase activity, lactate production, and myosin heavy chain IIb expression. Further, by using pharmacological or genetic loss-of-function models generated by phospholipase Cß antagonists, SUNCR1 global knockout, or SUNCR1 gastrocnemius-specific knockdown, we found that the effects of succinate on skeletal muscle fiber-type remodeling are mediated by SUNCR1 and its downstream calcium/NFAT signaling pathway. In summary, our results demonstrate succinate induces transition of skeletal muscle fiber via SUNCR1 signaling pathway. These findings suggest the potential beneficial use of succinate-based compounds in both athletic and sedentary populations.

Effect of Moringa oleifera supplementation on productive performance, colostrum composition and serum biochemical indexes of sow.

Moringa oleifera has been considered as a potential functional feed or food, since it contains multiple components beneficial to animal and human. However, little is known about the effects of Moringa oleifera supplementation on productive performances in sows. In the current study, the results showed that dietary Moringa oleifera significantly decreased the farrowing length and the number of stillborn (p < .05), while had an increasing trend in the number of live-born (0.05 < p < .10). Furthermore, 8% Moringa oleifera supplementation significantly elevated protein levels in the colostrum (p < .05); 4% Moringa oleifera lowed serum urea nitrogen of sows after 90 days of gestation (p < .05) and significantly decreased serum glucose on 10 days of lactation (p < .05). Both groups showed significant elevation in serum T-AOC activity (p < .05). The serum malondialdehyde (MDA) of sows declined significantly in 4% Moringa oleifera addition group (p < .05). 8% Moringa oleifera meal significantly elevated serum CAT activity after 60 days of gestation (p < .05), while decreased the serum MDA level and increased the serum GSH-Px activity of sows at 10 days of lactation (p < .05). Of piglets, both two dosages of Moringa oleifera supplementation essentially reduced the serum urea nitrogen (p < .05), and 4% Moringa oleifera meal increased serum total protein (p < .05). In addition, piglets that received 8% Moringa oleifera had the highest serum CAT and SOD activities among all groups (p < .05). The present study indicated that Moringa oleifera supplementation could enhance the reproduction performances, elevate protein levels in the colostrum and improve the serum antioxidant indices in both sows and piglets.

Plant MIR156 regulates intestinal growth in mammals by targeting the Wnt/ß-catenin pathway.

MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. Recent studies have shown that miRNAs might regulate gene expression among different species in a cross-kingdom manner. However, the specific roles of plant miRNAs in animals remain poorly understood and somewhat. Herein, we found that plant MIR156 regulates proliferation of intestinal cells both in vitro and in vivo. Continuous administration of a high plant miRNA diet or synthetic MIR156 elevated MIR156 levels and inhibited the Wnt/ß-catenin signaling pathway in mouse intestine. Bioinformatics predictions and luciferase reporter assays indicated that MIR156 targets Wnt10b. In vitro, MIR156 suppressed proliferation by downregulating the Wnt10b protein and upregulating ß-catenin phosphorylation in the porcine jejunum epithelial (IPEC-J2) cell line. Lithium chloride and an MIR156 inhibitor relieved this inhibition. This research is the first to demonstrate that plant MIR156 inhibits intestinal cell proliferation by targeting Wnt10b. More importantly, plant miRNAs may represent a new class of bioactive molecules that act as epigenetic regulators in humans and other animals.

ß-Glucanase specific expression in the intestine of transgenic pigs.

Producing heterologous enzymes in the animal digestive tract to improve feed utilization rate is a new research strategy by transgenic technology. In this study, transgenic pigs specifically expressing ß-glucanase gene in the intestine were successfully produced by somatic cell nuclear transfer technology in order to improve digestibility of dietary ß-glucan and absorption of nutrients. The ß-glucanase activity in the intestinal juice of 4 transgenic pigs was found to be 8.59 ± 2.49 U/mL. The feeding trial results showed that the crude protein digestion of 4 transgenic pigs was significantly increased compared with that of the non-transgenic pigs. In order to investigate the inheritance of the transgene, 7 G1 transgenic pigs were successfully obtained. The ß-glucanase activity in the intestinal juice of 7 G1 transgenic pigs was found to be 2.35 ± 0.72 U/mL. The feeding trial results showed the crude protein digestion and crude fat digestion were significantly higher in 7 G1 transgenic pigs than in non-transgenic pigs. Taken together, our study demonstrated that the foreign ß-glucanase expressing in the intestine of the transgenic pigs could reduce the anti-nutritional effect of ß-glucans in feed. In addition, ß-glucanase gene could be inherited to the offsprings and maintain its physiological function. It is a promising approach to improve feed utilization by producing transgenic animals.

Exploration of long noncoding RNA in bovine milk exosomes and their stability during digestion in vitro.

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell-cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.

miRNAome, mRNAome and degradome analysis of Tibetan minipigs anterior pituitary.

Tibetan minipig is an important animal model for human diseases. The anterior pituitary is the master gland responsible for growth, reproduction, and metabolism and is regulated by thousands of miRNAs/mRNAs molecules. However, little is known about miRNAs and their relationships with mRNAs in Tibetan minipig anterior pituitary. Using microarray and mRNA-Sequencing, we identified 203 miRNAs and 12,040 mRNA transcripts from the anterior pituitary of Tibetan minipigs. These miRNAs were corresponding to 194 hairpin precursors, 25 miRNA clusters and 24 miRNA families. In addition, 64 intragenic miRNAs were annotated. Using three bioinformatic algorithms (TargetScan, miRanda and RNAhybrid), 359,184 possible miRNA-mRNA interactions were predicted, and an integrated network of miRNAs and pituitary-specific mRNA transcripts was established. To validate the predicted results, the degradome sequencing was employed to confirm miRNA-mRNA interactions, totally, 30 miRNA-mRNA pairs were identified. The present study provided a general overview of miRNA and mRNA annotation in Tibetan minipig anterior pituitary and established a miRNA-mRNA interactions database at the whole genome scale, which helps shed light on the molecular mechanisms in the anterior pituitary of pigs even other mammals.

miR-146b Inhibits Glucose Consumption by Targeting IRS1 Gene in Porcine Primary Adipocytes.

Adipose tissue plays an important role in energy metabolism. Adipose dysfunction is closely related to obesity and type II diabetes. Glucose uptake is the key step for fat synthesis in adipocyte. miRNAs have been proven to play a crucial role in adipocyte differentiation, adipogenesis and glucose homeostasis. In this paper, we firstly reported that miR-146b decreased glucose consumption by up-regulating miR-146b in a porcine primary adipocyte model, while the inhibitor of endogenous miR-146b rescued the reduction. Then, miR-146b was predicated to target IRS1 by bioinformatics analysis, and a dual-luciferase reporter assay validated this predication. Western blot analyses indicated both IRS1 and glucose transporter type 4 (GLUT4) were down-regulated by miR-146b overexpression. Our study demonstrated that miR-146b regulated glucose homeostasis in porcine primary pre-adipocyte by targeting IRS1, and provided new understandings on regulations of lipogenesis by miRNAs.

miR-361-3p regulates FSH by targeting FSHB in a porcine anterior pituitary cell model.

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.

Improvement of anti-nutritional effect resulting from ß-glucanase specific expression in the parotid gland of transgenic pigs.

ß-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although ß-glucanase supplementation in diet can help to eliminate the adverse effects, enzyme stability is substantially modified during the feed manufacturing process. To determine whether the expression of endogenous ß-glucanase gene (GLU) in vivo can improve digestibility of dietary ß-glucan and absorption of nutrients, we successfully produced transgenic pigs via nuclear transfer which express the GLU from Paenibacillus polymyxa CP7 in the parotid gland. In three live transgenic founders, ß-glucanase activities in the saliva were 3.2, 0.07 and 0.03 U/mL, respectively, and interestingly the enzyme activities increased in the pigs from 178 days old to 789 days old. From the feed the amount of gross energy, crude protein and crude fat absorbed by the transgenic pigs was significantly higher than the non-transgenic pigs. Meanwhile the moisture content of the feces was significantly reduced in transgenic pigs compared with the non-transgenic pigs. Furthermore, in all positive G1 pigs, ß-glucanase activity was detectable and the highest enzyme activity reached 3.5 U/mL in saliva. Also, crude protein digestion was significantly higher in G1 transgenic pigs than in control pigs. Taken together, our data showed that the transgenic ß-glucanase exerted its biological catalytic function in vivo in the saliva, and the improved performance of the transgenic pigs could be accurately passed on to the offspring, indicating a promising alternative approach to improving nutrient availability was established to improve utilization of livestock feed through transgenic animals.

Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis.

BACKGROUND: Milk is a complex liquid that provides nutrition to newborns. Recent reports demonstrated that milk is enriched in maternal-derived exosomes that are involved in fetal physiological and pathological conditions by transmission of exosomal mRNAs, miRNAs and proteins. Until now, there is no such research relevant to exosomal mRNAs and proteins in porcine milk, therefore, we have attempted to investigate porcine milk exosomal mRNAs and proteins using RNA-sequencing and proteomic analysis. RESULTS: A total of 16,304 (13,895 known and 2,409 novel mRNAs) mRNAs and 639 (571 known, 66 candidate and 2 putative proteins) proteins were identified. GO and KEGG annotation indicated that most proteins were located in the cytoplasm and participated in many immunity and disease-related pathways, and some mRNAs were closely related to metabolisms, degradation and signaling pathways. Interestingly, 19 categories of proteins were tissue-specific and detected in placenta, liver, milk, plasma and mammary. COG analysis divided the identified mRNAs and proteins into 6 and 23 categories, respectively, 18 mRNAs and 10 proteins appeared to be involved in cell cycle control, cell division and chromosome partitioning. Additionally, 14 selected mRNAs were identified by qPCR, meanwhile, 10 proteins related to immunity and cell proliferation were detected by Western blot. CONCLUSIONS: These results provide the first insight into porcine milk exosomal mRNA and proteins, and will facilitate further research into the physiological significance of milk exosomes for infants.

miR-146a-5p inhibits TNF-α-induced adipogenesis via targeting insulin receptor in primary porcine adipocytes.

TNF-α is a multifunctional cytokine participating in immune disorders, inflammation, and tumor development with regulatory effects on energy metabolism. Our work focused on the function of TNF-α in adipogenesis of primary porcine adipocytes. TNF-α could suppress the insulin receptor (IR) at the mRNA and protein levels. Microarray analysis of TNF-α-treated porcine adipocytes was used to screen out 29 differentially expressed microRNAs (miRNAs), 13 of which were remarkably upregulated and 16 were intensely downregulated. These 29 differentially expressed miRNAs were predicted to mainly participate in the insulin signaling pathway, adipocytokine signaling pathway, and type 2 diabetes mellitus pathway by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. miR-146a-5p, reportedly involved in immunity and cancer relevant processes, was one of the most highly differentially expressed miRNAs after TNF-α treatment. Red Oil O staining and TG assay revealed that miR-146a-5p suppressed adipogenesis. A dual-luciferase reporter and siRNA assay verified that miR-146a-5p targeted IR and could inhibit its protein expression. miR-146a-5p was also validated to be involved in the insulin signaling pathway by reducing tyrosine phosphorylation of insulin receptor substrate-1. Our study provides the first evidence of miR-146a-5p targeting IR, which facilitates future studies related to obesity and diabetes using pig models.

The aflatoxin-detoxifizyme specific expression in mouse parotid gland.

The aflatoxin-detoxifizyme (ADTZ) gene derived from Armillariella tabescens was cloned into parotid gland-specific expression vector (pPSPBGPneo) to construct the parotid gland-specific vector expressing ADTZ (pPSPBGPneo-ADTZ). Transgenic mice were generated by microinjection and identified by using PCR and Southern blotting analysis. PCR and Southern blotting analysis showed that total six transgenic mice carried the ADTZ gene were generated. RT-PCR analysis indicated that the expression of ADTZ mRNA could be detected only in parotid glands of the transgenic mice. The ADTZ activity in the saliva was found to be 3.72 ± 1.64 U/mL. After feeding a diet containing aflatoxin B1 (AFB1) for 14 days, the effect of ADTZ on serum biochemical indexes and AFB1 residues in serum and liver of mice were evaluated. The results showed that total protein and globulin contents in the test treatment (transgenic mice) produced ADTZ were significantly higher than that of the positive control, while alanine aminotransferase and aspartate aminotransferase activity in serum of the test treatment (transgenic mice) were remarkably lower compared to that of the positive control (P < 0.05). Moreover, AFB1 residues in serum and liver of the test treatment (transgenic mice) were significantly lower compared with that of the positive control (P < 0.05). These results in the study confirmed that ADTZ produced in transgenic mice could reduce, even eliminate the negative effects of AFB1 on mice.

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

Exploration of microRNAs in porcine milk exosomes.

BACKGROUND: Breast milk contains complex nutrients and facilitates the maturation of various biological systems in infants. Exosomes, membranous vesicles of endocytic origin found in different body fluids such as milk, can mediate intercellular communication. We hypothesized that microRNAs (miRNAs), a class of non-coding small RNAs of 18-25 nt which are known to be packaged in exosomes of human, bovine and porcine milk, may play important roles in the development of piglets. RESULTS: In this study, exosomes of approximately 100 nm in diameter were isolated from porcine milk through serial centrifugation and ultracentrifugation procedures. Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component. Solexa sequencing showed a total of 491 miRNAs, including 176 known miRNAs and 315 novel mature miRNAs (representing 366 pre-miRNAs), which were distributed among 30 clusters and 35 families, and two predicted novel miRNAs were verified targeting 3'UTR of IGF-1R by luciferase assay. Interestingly, we observed that three miRNAs (ssc-let-7e, ssc-miR-27a, and ssc-miR-30a) could be generated from miRNA-offset RNAs (moRNAs). The top 10 miRNAs accounted for 74.5% (67,154 counts) of total counts, which were predicted to target 2,333 genes by RNAhybrid software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using DAVID bioinformatics resources indicated that the identified miRNAs targeted genes enriched in transcription, immunity and metabolism processes, and 14 of the top 20 miRNAs possibly participate in regulation of the IgA immune network. CONCLUSIONS: Our findings suggest that porcine milk exosomes contain a large number of miRNAs, which potentially play an important role in information transfer from sow milk to piglets. The predicted miRNAs of porcine milk exosomes in this study provide a basis for future biochemical and biophysical function studies.

Pituitary-derived small extracellular vesicles promote liver repair by its cargo miR-143-3p.

The small Extracellular vesicles (sEV) has been recognized to be significant for intercellular communication due to their ability to transfer important cellular cargoes like miRNAs through circulation. The pituitary gland has not been clearly known about the role of its secreted sEV under normal physiological conditions. And Liver disease is a global public health burden. The present study is the first to investigate the effect of pituitary sEV on the liver. Sequencing and qRT-PCR revealed miR-143-3p is one of the richest in the pituitary sEV. MiR-143 Knockout (KO) mice resulted in a remarkable decrease in insulin-like growth factor 1 (IGF-1) levels and a significant increase in insulin-like growth factor binding protein 5 (IGFBP5) levels along with a reduction in liver primary cell growth. More importantly, compared with miR-143-KO-sEV, WT-sEV possesses a more robust capacity to improve miR-143 KO mice liver repair through the Wnt/ß-catenin pathway after an acute injury caused by carbon tetrachloride (CCl4). Our results indicate that pituitary-derived sEV promotes hepatocyte proliferation and liver repair by its cargo miR-143-3p and provides new insight into the regulation mechanism of the pituitary-liver axis, and open a new window for endocrine regulation by using sEV.

ß-Glucanase specific expression in the parotid gland of transgenic mice.

The feasibility of using the pig parotid secretory protein promoter to drive the ß-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing ß-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the ß-glucanase gene and the ß-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The ß-glucanase activity in the saliva was found to be 0.18 U/mL. After feeding a diet containing 2 % ß-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign ß-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of ß-glucans in feed.

miRNAs derived from milk small extracellular vesicles inhibit porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration, and high mortality in neonatal piglets. It has caused huge economic losses to animal husbandry worldwide. Current commercial PEDV vaccines do not provide enough protection against variant and evolved virus strains. No specific drugs are available to treat PEDV infection. The development of more effective therapeutic anti-PEDV agents is urgently needed. Our previous study suggested that porcine milk small extracellular vesicles (sEV) facilitate intestinal tract development and prevent lipopolysaccharide-induced intestinal injury. However, the effects of milk sEV during viral infection remain unclear. Our study found that porcine milk sEV, which was isolated and purified by differential ultracentrifugation, could inhibit PEDV replication in IPEC-J2 and Vero cells. Simultaneously, we constructed a PEDV infection model for piglet intestinal organoids and found that milk sEV also inhibited PEDV infection. Subsequently, in vivo experiments showed that milk sEV pre-feeding exerted robust protection of piglets from PEDV-induced diarrhea and mortality. Strikingly, we found that the miRNAs extracted from milk sEV inhibited PEDV infection. miRNA-seq, bioinformatics analysis, and experimental verification demonstrated that miR-let-7e and miR-27b, which were identified in milk sEV targeted PEDV N and host HMGB1, suppressed viral replication. Taken together, we revealed the biological function of milk sEV in resisting PEDV infection and proved its cargo miRNAs, miR-let-7e and miR-27b, possess antiviral functions. This study is the first description of the novel function of porcine milk sEV in regulating PEDV infection. It provides a better understanding of milk sEV resistance to coronavirus infection, warranting further studies to develop sEV as an attractive antiviral.

Roles of α-linolenic acid on IGF-I secretion and GH/IGF system gene expression in porcine primary hepatocytes.

The main purposes of this study were to investigate the effects of α-linolenic acid (ALA) on the insulin-like growth factor (IGF) system of porcine primary hepatocytes with or without growth hormone (GH) or insulin and the potential role of peroxisome proliferator-activated receptor α and -γ (PPARα/γ) pathway. We found that 1 µM ALA increased IGF-I secretion from hepatocytes at 48 and 72 h. Expression of hepatocytes IGF-I, IGF-II, GH receptor (GHR), insulin receptor (IR), IGF-binding protein 3 (IGFBP3), and IGFBP4 mRNAs was up-regulated by ALA treatment. GH (15 nM) alone or co-treated with ALA increased hepatocytes IGF-I secretion and the expression of GHR and IGFBP1 mRNAs, but down-regulated IGFBP5 mRNA compared with appropriate control across ALA. GH also enhanced the ALA-induced increase in the transcript levels of IGF-II and GHR, but tended to attenuate that of IGFBP4. Insulin (1 µM) alone or co-treated with ALA improved IGF-I secretion and the expression of IGFBP3 mRNA, but decreased IGFBP1 mRNA versus appropriate control across ALA. Insulin also up-regulated the expression of GHR, IR, IGFBP3, and IGFBP4 mRNAs, and tended to prevent the transcript levels of IGF-I and IGFBP4 improved by ALA. Both PPARγ agonist rosiglitazone and its antagonist GW9662 could elevated the IGF-I secretion in dose-dependent manner but they had no interaction with ALA. However, GW7647, a PPARα agonist, increased IGF-I secretion dose-dependently, but the antagonist GW6471 was without effect. Moreover, GW6471 prevented the IGF-I promoting effect of ALA. This suggests that the IGF-I promoting effect of ALA may be mediated by the PPARα pathway.