Canine MAS1: monoallelic expression is suggestive of an imprinted gene

Imprinted genes are epigenetically modified in a parent‐of‐origin dependent manner and as a consequence are differentially expressed, with one allele typically expressed while the other is repressed. In canine, the insulin like growth factor 2 receptor gene (IGF2R) is imprinted with predominant expression of the maternally inherited allele. Because imprinted genes usually occur in clusters, we examined the allelic expression pattern of the gene encoding the canine Mas receptor (MAS1), which is located upstream of IGF2R on canine chromosome 1 and is highly conserved in mammals. In this report we describe monoallelic expression of canine MAS1 in the neonatal umbilical cord of several individuals and we identify the expressed allele as maternally inherited. These data suggest that canine MAS1 is an imprinted gene.     

pkc-1 regulates daf-2 insulin/IGF signalling-dependent control of dauer formation in Caenorhabditis elegans

In Caenorhabditis elegans, the insulin/IGF pathway participates in the decision to initiate dauer development. Dauer is a diapause stage that is triggered by environmental stresses, such as a lack of nutrients. Insulin/IGF receptor mutants arrest constitutively in dauer, an effect that can be suppressed by mutations in other elements of the insulin/IGF pathway or by a reduction in the activity of the nuclear hormone receptor daf‐12. We have isolated a pkc‐1 mutant that acts as a novel suppressor of the dauer phenotypes caused by insulin/IGF receptor mutations. Interactions between insulin/IGF mutants and the pkc‐1 suppressor mutant are similar to those described for daf‐12 or the DAF‐12 coregulator din‐1. Moreover, we show that the expression of the DAF‐12 target daf‐9, which is normally elevated upon a reduction in insulin/IGF receptor activity, is suppressed in a pkc‐1 mutant background, suggesting that pkc‐1 could link the daf‐12 and insulin/IGF pathways. pkc‐1 has been implicated in the regulation of peptide neurosecretion in C. elegans. Although we demonstrate that pkc‐1 expression in the nervous system regulates dauer formation, our results suggest that the requirement for pkc‐1 in neurosecretion is independent of its role in modulating insulin/IGF signalling. pkc‐1 belongs to the novel protein kinase C (nPKC) family, members of which have been implicated in insulin resistance and diabetes in mammals, suggesting a conserved role for pkc‐1 in the regulation of the insulin/IGF pathway.     

Single nucleotide polymorphisms in the imprinted bovine insulin-like growth factor 2 receptor gene (IGF2R) are associated with body size traits in Irish Holstein-Friesian cattle

The regulation of the bioavailability of insulin‐like growth factors (IGFs) is critical for normal mammalian growth and development. The imprinted insulin‐like growth factor 2 receptor gene (IGF2R) encodes a transmembrane protein receptor that acts to sequester and degrade excess circulating insulin‐like growth factor 2 (IGF‐II) – a potent foetal mitogen – and is considered an important inhibitor of growth. Consequently, IGF2R may serve as a candidate gene underlying important growth‐ and body‐related quantitative traits in domestic mammalian livestock. In this study, we have quantified genotype–phenotype associations between three previously validated intronic bovine IGF2R single nucleotide polymorphisms (SNPs) (IGF2R:g.64614T>C, IGF2R:g.65037T>C and IGF2R:g.86262C>T) and a range of performance traits in 848 progeny‐tested Irish Holstein‐Friesian artificial insemination sires. Notably, all three polymorphisms analysed were associated (P ≤ 0.05) with at least one of a number of performance traits related to animal body size: angularity, body depth, chest width, rump width, and animal stature. In addition, the C‐to‐T transition at the IGF2R:g.65037T>C polymorphism was positively associated with cow carcass weight and angularity. Correction for multiple testing resulted in the retention of two genotype–phenotype associations (animal stature and rump width). None of the SNPs analysed were associated with any of the milk traits examined. Analysis of pairwise r² measures of linkage disequilibrium between all three assayed SNPs ranged between 0.41 and 0.79, suggesting that some of the observed SNP associations with performance may be independent. To our knowledge, this is one of the first studies demonstrating associations between IGF2R polymorphisms and growth‐ and body‐related traits in cattle. These results also support the increasing body of evidence that imprinted genes harbour polymorphisms that contribute to heritable variation in phenotypic traits in domestic livestock species.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA‐122‐5p (miR‐122) regulates the hepatic IR in vitro. We first found that the miR‐122 level was upregulated in the liver of rats fed with a high‐fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin‐like growth factor 1 receptor (IGF‐1R), a potential target of miR‐122, was downregulated in the diabetic liver. In vitro, glucosamine‐induced IR was introduced in HepG2 hepatic cells, and the levels of miR‐122 and IGF‐1R were further assessed. An increase of miR‐122 level and a decrease of IGF‐IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF‐1R as a direct target of miR‐122. Moreover, in IR HepG2 cells, antagonizing miR‐122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6‐phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR‐122 inhibitor in IR hepatic cells were impaired when IGF‐1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR‐122 inhibitor transfection. In conclusion, our study demonstrates that miR‐122 is able to regulate IR in hepatic cells by targeting IGF‐1R.     

Insulin and Insulin-like Growth Factor-1 Receptors Act as Ligand-specific Amplitude Modulators of a Common Pathway Regulating Gene Transcription

Insulin and insulin-like growth factor-1 (IGF-1) act on highly homologous receptors, yet in vivo elicit distinct effects on metabolism and growth. To investigate how the insulin and IGF-1 receptors exert specificity in their biological responses, we assessed their role in the regulation of gene expression using three experimental paradigms: 1) preadipocytes before and after differentiation into adipocytes that express both receptors, but at different ratios; 2) insulin receptor (IR) or IGF1R knock-out preadipocytes that only express the complimentary receptor; and 3) IR/IGF1R double knock-out (DKO) cells reconstituted with the IR, IGF1R, or both. In wild-type preadipocytes, which express predominantly IGF1R, microarray analysis revealed ∼500 IGF-1 regulated genes (p < 0.05). The largest of these were confirmed by quantitative PCR, which also revealed that insulin produced a similar effect, but with a smaller magnitude of response. After differentiation, when IR levels increase and IGF1R decrease, insulin became the dominant regulator of each of these genes. Measurement of the 50 most highly regulated genes by quantitative PCR did not reveal a single gene regulated uniquely via the IR or IGF1R using cells expressing exclusively IGF-1 or insulin receptors. Insulin and IGF-1 dose responses from 1 to 100 nm in WT, IRKO, IGFRKO, and DKO cells re-expressing IR, IGF1R, or both showed that insulin and IGF-1 produced effects in proportion to the concentration of ligand and the specific receptor on which they act. Thus, IR and IGF1R act as identical portals to the regulation of gene expression, with differences between insulin and IGF-1 effects due to a modulation of the amplitude of the signal created by the specific ligand-receptor interaction.     

Tumor Necrosis Factor‐α Stimulates Cell Proliferation in Adipose Tissue‐Derived Stromal‐Vascular Cell Culture: Promotion of Adipose Tissue Expansion by Paracrine Growth Factors

Objective: Elevated levels of tumor necrosis factor‐α (TNF‐α) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF‐α has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF‐α, alone or together with insulin‐like growth factor‐I (IGF‐I), support preadipocyte development. Research Methods and Procedures: Cultured stromal‐vascular cells from rat inguinal fat depots were exposed to serum‐free media containing insulin and 0.2 nM TNF‐α, 2.0 nM TNF‐α, or 0.2 nM TNF‐α + 1.0 nM IGF‐I at different times during 7 days of culture. Results: TNF‐α inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte‐specific antigen (AD3; early differentiation marker) and glycerol‐3‐phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF‐α did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF‐α from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF‐α + 1 nM IGF‐I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. Discussion: These data suggest that paracrine levels (0.2 nM) of TNF‐α alone or in combination with IGF‐I may support adipose tissue development by increasing the total number of stromal‐vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.     

Study of the differential transcription in liver of growth hormone receptor (GHR), insulin-like growth factors (IGF1, IGF2) and insulin-like growth factor receptor (IGF1R) genes at different postnatal developmental ages in pig breeds

The objective of this study was to determine hepatic expression levels of GHR, IGF1R, IGF1 and IGF2 genes in young growing gilts at different developmental ages (60–210 days) in five pig breeds: Polish Large White (PLW), Polish Landrace (PL), Pulawska (Pul), Duroc (Dur) and Pietrain (Pie). We studied the differences among pig breeds as well as within each breed for pigs in different developmental ages. Obtained results revealed major differences among breeds in hepatic gene expression of porcine GHR, IGF1R, IGF1 and IGF2 genes in different developmental ages. The differences among breeds of GHR expression were significantly higher in PLW, PL at the age of 60, 90, 120 days as compared to Pul, Dur and Pie. In turn, the highest level of IGF1R expression was observed in PL at age of 150, 180 and 210 days, whereas in case of IGF1 the highest level was recorded in Pie gilts at the age of 60 and 90 days. Moreover trait associated study revealed highly significant correlations between hepatic expressions of IGF1R and IGF2 genes and carcass composition traits (P < 0.01) The results of study suggest that porcine GHR, IGF1R, IGF1 and IGF2 genes may be potential candidate genes for postnatal growth and carcass composition traits. Therefore, the implementation of the hepatic expression of GH/IGF genes into the pig breeding and gene assisted selection program in different pig breeds should be considered. However, further population wide study is needed to clarify the hepatic expression association with economic traits, such as body growth, meat quality and carcass composition traits.     

Temporal pattern of neuronal insulin release during Caenorhabditis elegans aging: Role of redox homeostasis

The insulin‐IGF‐1/DAF‐2 pathway has a central role in the determination of aging and longevity in Caenorhabditis elegans and other organisms. In this paper, we measured neuronal insulin secretion (using INS‐22::Venus) during C. elegans lifespan and monitored how this secretion is modified by redox homeostasis. We showed that INS‐22::Venus secretion fluctuates during the organism lifetime reaching maximum levels in the active reproductive stage. We also demonstrate that long‐lived daf‐2 insulin receptor mutants show remarkable low levels of INS‐22::Venus secretion. In contrast, we found that short‐lived mutant worms that lack the oxidation repair enzyme MSRA‐1 show increased levels of INS‐22::Venus secretion, specifically during the reproductive stage. MSRA‐1 is a target of the insulin‐IGF‐1/DAF‐2 pathway, and the expression of this antioxidant enzyme exclusively in the nervous system rescues the mutant insulin release phenotype and longevity. The msra‐1 mutant phenotype can also be reverted by antioxidant treatment during the active reproductive stage. We showed for the first time that there is a pattern of neuronal insulin release with a noticeable increment during the peak of reproduction. Our results suggest that redox homeostasis can modulate longevity through the regulation of insulin secretion, and that the insulin‐IGF‐1/DAF‐2 pathway could be regulated, at least in part, by a feedback loop. These findings highlight the importance of timing for therapeutic interventions aimed at improving health span.     

The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen‐thawed, unsorted, and sex‐sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non‐sexed (NS), sexed for X‐sperm (SX), and sexed for Y‐sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P = 0.09) or IGF2R genes (P = 0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex‐sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed. Mol. Reprod. Dev. 79:77–84, 2012. © 2011 Wiley Periodicals, Inc.     

Reduced Expression of FOXC2 and Brown Adipogenic Genes in Human Subjects with Insulin Resistance

Objective: We investigated subcutaneous adipose tissue expression of FOXC2 and selected genes involved in brown adipogenesis in adult human subjects in whom we have previously identified a reduced potential of precursor cell commitment to adipose‐lineage differentiation in relation to insulin resistance. Research Methods and Procedure: Gene expression was studied using quantitative real time polymerase chain reaction. The relation between the expression of brown adipogenic genes and the genes involved in progenitor cell commitment, adipose cell size, and insulin sensitivity in vivo was analyzed. Results: The expression of FOXC2, MASK, MAP3K5, retinoblastoma protein (pRb), peroxisome proliferator‐activated protein gamma (PPARγ), and retinoid X receptor gamma (RXRγ) was decreased in the insulin‐resistant compared with insulin‐sensitive subjects, whereas PPARγ‐2 and CCAAT/enhancer binding protein alpha (C/EBPα) showed no differential expression. The FOXC2 expression correlated with that of Notch and Wnt signaling genes, as well as of the genes studied participating in brown adipogenesis, including MASK, MAP3K5, PPARγ, pRb, RXRγ, and PGC‐1. A second‐level correlation between PPARγ and UCP‐1 was also significant. In addition, the expression of MASK, MAP3K5, pRb, RXRγ, and PGC‐1 inversely correlated with adipose cell mass and also correlated with the glucose disposal rate in vivo. Discussion: Our results suggest that a reduced brown adipose phenotype is associated with insulin resistance and that a basal brown adipose phenotype may be important for maintaining normal insulin sensitivity.     

SUMO‐modified insulin‐like growth factor 1 receptor (IGF‐1R) increases cell cycle progression and cell proliferation

Increasing number of studies have shown nuclear localization of the insulin‐like growth factor 1 receptor (nIGF‐1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF‐1R have, however, still not been disclosed. Previously, we reported that IGF‐1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple‐SUMO‐site‐mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R‐). Cell clones (R‐WT and R‐TSM) expressing equal amounts of IGF‐1R were selected for experiments. Phosphorylation of IGF‐1R, Akt, and Erk upon IGF‐1 stimulation was equal in R‐WT and R‐TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R‐WT proliferated substantially faster than R‐TSM, which did not differ significantly from the empty vector control. Upon IGF‐1 stimulation G1‐S‐phase progression of R‐WT increased from 12 to 38%, compared to 13 to 20% of R‐TSM. The G1‐S progression of R‐WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO‐IGF‐1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO‐IGF‐1R dependent mechanisms seem important.     

Genes in the insulin and insulin-like growth factor pathway and odds of metachronous colorectal neoplasia

Insulin and insulin-like growth factor (IGF) genes are implicated in colorectal carcinogenesis. Gene-by-gene interactions that influence the insulin/IGF pathways were hypothesized as modifiers of colorectal neoplasia risk. We built a classification tree to detect interactions in 18 IGF and insulin pathway-related genes and metachronous colorectal neoplasia among 1,439 subjects pooled from two chemoprevention trials. The probability of colorectal neoplasia was greatest (71.8%) among carriers of any A allele for rs7166348 (IGF1R) and AA genotype for rs1823023 (PIK3R1). In contrast, carriers of any A at rs7166348 (IGF1R), any G for the PIK3R1 variant, and AA for rs10426094 (INSR) had the lowest probability (14.3%). Logistic regression modeling showed that any A at rs7166348 (IGF1R) with the AA genotype at rs1823023 (PIK3R1) conferred the highest odds of colorectal neoplasia (OR 3.7; 95% CI 2.2–6.5), compared with carriage of GG at rs7166348 (IGF1R). Conversely, any A at rs7166348 (IGFR1), any G allele at rs1823023 (PIK3R1), and the AA genotype at rs10426094 (INSR) conferred the lowest odds (OR 0.22; 95% CI 0.07–0.66). Stratifying the analysis by parent study and intervention arm showed highly consistent trends in direction and magnitude of associations, with preliminary evidence of genotype effects on measured IGF-1 levels in a subgroup of subjects. These results were compared to those from multifactor dimensionality reduction, which identified different single nucleotide polymorphisms in the same genes (INSR and IGF1R) as effect modifiers for colorectal neoplasia. These results support a role for genetic interactions in the insulin/IGF pathway genes in colorectal neoplasia risk.