IGF-1 Group Research Articles

Exploring the Mechanism of KLF15 on the Biological Activity and Autophagy of Gastric Cancer Cells based on PI3K/Akt/Mtor Signaling Pathway.

To explore the mechanism of KLF15 on the biological activity and autophagy of gastric cancer cells based on the PI3K/Akt/mTOR signaling pathway. The gastric cancer AGS cells were divided into the Con group, pcDNANC group, pcDNA-KLF15 group, LY294002 group and IGF-1 group. RT-PCR was used to detect the expression of KLF15 in human gastric mucosal cells and gastric cancer cells; MTT method to detect cell proliferation; Transwell method to detect cell invasion; flow cytometry to detect cell apoptosis; Western blotting to detect PI3K, Akt, mTOR in cells, LC3, Beclin1, p62 protein expression.P<0.05 was used to indicate statistical significance. Compared with the human gastric mucosal cell line GES-1 cells, the expression of KLF15 in human gastric cancer cell lines MKN-28, MFC, SCG-7901 and AGS cells was significantly decreased, And the expression of KLF15 in AGS cells, was the lowest (P=0.006). Compared with the Con group, The expression of KLF15 in the cells of the PCDNA-KLF15 group was significantly increased (P=0.018); There was no significant difference in the expression of KLF15 between the Con group and the PCDNA-NC group (P=0.225). Compared with the Con group, the proliferation and invasion abilities of the cells in the pcDNA-KLF15 group were significantly reduced, And the apoptosis ability was significantly increased (P=0.019). The ratio of LC3II/LC31 and the expression of Beclin1 Protein in the control group were significantly higher than those in the Con group (P=0.017). Overexpression of KLF15 can inhibit the proliferation and invasion of Gastric cancer cells and promote cell apoptosis and autophagy, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

Abstract 11537: IGF-1 Reduces Atherosclerosis in Rapacz Pigs

Introduction: Cardiovascular disease, including atherosclerosis is the leading cause of mortality in the Western world. We have reported that insulin-like growth factor I (IGF-1) reduced atherosclerosis in the murine model, however it is unknown whether IGF-1 exerts protective effect on human-like atherosclerosis. Hypothesis: Rapacz pigs with familial hypercholesterolemia (FH pigs) fed with high-cholesterol diet develop human-like advanced plaques in coronary arteries. We hypothesize that IGF-1 suppresses atherosclerosis in FH pigs. Methods: Human recombinant IGF-1 ( 50 ug/kg) or saline (control) was injected daily into high-fat diet fed FH swine (n=9/group) for 6 months. Atherosclerotic burden was quantified by intravascular ultrasound (IVUS) at basal level (T0), after 3 months on diet (T3) and at sacrificing (T6). Results: IGF-1-injected pigs had 2.6-fold increase in total plasma IGF-1 (ELISA, p&lt;0.001 vs. control pigs). Pigs in IGF-1 and control groups had a gradual increase in vessel volume in the right coronary artery (RCA), and left anterior descending artery (LAD) with time as quantified by IVUS. However, IGF-1-injected pigs had a larger increase in vessel volume after 3 and 6 months compared to controls suggesting vascular hypertrophy. The atherosclerotic burden was increased in RCA, and LAD in both groups after 3 and 6 months of injections compared to the pre-injection time point. We found a significant reduction in atherosclerotic burden in RCA and LAD in IGF-1 group compared to control at 6 months’ time-point (RCA: IGF-1, 203±13%, vs. control, 244±8; LAD: IGF-1, 200±6%, vs. control, 228±11%). The lumen volume of coronaries was decreased in both groups, however, coronaries in IGF-1-injected pigs had on average 24±3% larger lumen volume compared to control pigs at 6 months’ time-point. Conclusions: We found that IGF-1 increased total volume of pig coronaries suggesting hypertrophy and these data are consistent with growth-stimulating effects of IGF-1. We found reduction in atherosclerotic burden and increase in lumen volume in coronaries in the IGF-1 group after 6 months of injections consistent with an anti-atherosclerotic effect of IGF-1. Our data support the feasibility of using IGF-1 for as an anti-atherogenic therapy.

PMON61 Evaluation of elevated IGF-1 in patients without clinical evidence of acromegaly

Abstract Introduction Insulin-like growth factor-1 (IGF-1) acts downstream of growth hormone mediating many of its effects. It is the recommended screening test for acromegaly, in evaluating pituitary incidentalomas and possible hypopituitarism. Case-based observations suggest that elevated IGF-1 may occur in patients without acromegaly. The aims were to 1) identify the frequency of elevated IGF-1 without evidence of GH excess, and 2) to examine potential differences in relevant medications and comorbidities between people with an elevated IGF-1 compared to a control group matched for age, sex, gonadal and pituitary status. Methods All people whose IGF-1 was measured at a single reference laboratory between Dec 1st 2018 – Dec 1st 2020 were identified. The electronic records of those with at least one IGF1 &amp;gt;1.1x the upper limit of the age-matched reference range were appraised to determine; 1) documentation of acromegalic features, 2) presence of relevant comorbidities and medication use, and 3) further investigation to exclude pathological GH excess. Results There were 2759 IGF-1 levels measured in 1963 people over the specified period. Out of these, 204 were in patients over 18 years of age and were &amp;gt;1.1 times the upper limit of the age-matched reference range. We excluded 48 with diagnosed acromegaly, 4 who had further workup for acromegaly planned, 9 on GH substitution for proven GH deficiency, 3 with Cushing's syndrome and 5 who were pregnant. A further 33 were excluded based on insufficient data available. A total of 102 cases (61M, 41F) were included in the analysis. Of these 102 people, 67 had pituitary MRI scans, 24 had a 75g oral glucose tolerance test, 28 had a subsequent repeat IGF-1 that had normalised and 13 had random GH &amp;lt;1ug/L. The characteristics of these 102 cases were compared to those of the matched control group (n=102) with normal IGF-1 assayed in the same period. There was no significant difference between the elevated and normal IGF-1 groups in age (mean 46.7 vs 47.0 years, P=0.90), sex (P&amp;gt;0.99), gonadal status (P=0.87) or pituitary status (P=0.15). Variables that were significantly different between cases and controls included dopamine agonist therapy (19/102 cases vs 6/102 controls, OR=3.66, 95% CI: 1.45-9.29, P=0.009) and chronic kidney disease (14/102 cases vs 4/102 controls, OR = 3.90, 95% CI: 1.28-11.14, P=0.024). Conclusion Out of 1963 patients having IGF-1 measured at a reference laboratory, a significantly elevated IGF-1 occurred in 204. In 102 (50%) of these patients, there was no known acromegaly, GH replacement or glucocorticoid excess, and no documented definitive acromegalic features. There were significant associations with dopamine agonist use and CKD. While assay-related issues and accuracy of the age-matched reference ranges probably represent the main influences on the prevalence of elevated IGF-1, we have identified two additional factors which should be considered. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

IGF-1 Gene Transfer Modifies Inflammatory Environment and Gene Expression in the Caudate-Putamen of Aged Female Rat Brain.

Brain aging is characterized by chronic neuroinflammation caused by activation of glial cells, mainly microglia, leading to alterations in homeostasis of the central nervous system. Microglial cells are constantly surveying their environment to detect and respond to diverse signals. During aging, microglia undergoes a process of senescence, characterized by loss of ramifications, spheroid formation, and fragmented processes, among other abnormalities. Therefore, the study of changes in microglia during is of great relevance to understand age-related declines in cognitive and motor function. We have targeted the deleterious effects of aging by implementing IGF-1 gene transfer, employing recombinant adenoviral vectors (RAds) as a delivery system. In this study, we performed intracerebroventricular (ICV) RAd-IGF-1 or control injection on aged female rats and evaluated its effect on caudate-putamen unit (CPu) gene expression and inflammatory state. Our results demonstrate that IGF-1 overexpression modified aged microglia of the CPu towards an anti-inflammatory condition increasing the proportion of double immuno-positive Iba1+Arg1+ cells. We also observed that phosphorylation of Akt was increased in animals treated with RAd-IGF-1. Moreover, IGF-1 gene transfer was able to regulate CPu pro-inflammatory environment in female aged rats by down-regulating the expression of genes typically overexpressed during aging. RNA-Seq data analysis identified 97 down-modulated DEG in the IGF-1 group as compared to the DsRed one. Interestingly, 12 of these DEG are commonly overexpressed during aging, and 9 out of 12 are expressed in microglia/macrophages and are involved in different processes that lead to neuroinflammation and/or neuronal loss. Finally, we observed that IGF-1 overexpression led to an improvement in motor functions. Although further studies are necessary, with the present results, we conclude that IGF-1 gene transfer is modifying both the pro-inflammatory environment and activation of microglia/macrophages in CPu. In this regard, IGF-1 gene transfer could counteract the neuroinflammatory effects associated with aging and improve motor functions in senile animals.

The effect of Shendan Sanjie capsule on angiogenesis in mice with colitis associated cancer and mechanism

Objective: To investigate the effects and the mechanism of Shendansanjie capsules on angiogenesis of colitis associated cancer(CAC) mice. Methods: Azoxymethane and dextran sulfact sodium were used to construct a mice model with CAC. Ten mice were divided into the normal group, model group, Shendan Sanjie capsule group, MK-2206 group, and Shendan Sanjie capsule + IGF-1 group, respectively. Immunohistochemistry was used to detect the microvessel density (MVD) in the colon tissue of each group of mice. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the mRNA levels of basic fibroblast growth factor (bFGF) and angiopoietin 2 (Ang2) in colon tissue. Western blot was used to detect the expressions of Akt, p-Akt, vascular endothelial growth factor A (VEGFA), hypoxia-inducible factor-1α (HIF-1α). Results: The number of MVD in the colon tissue of mice in the model group, Shendan Sanjie capsule group, MK-2206 group, Shendan Sanjie capsule + IGF-1 group were 63.3±3.3, 36.6±2.3, 36.6±2.2, 50.3±2.5, significantly higher than 2.0±0.1 in the normal group (P<0.05). The number of MVD in Shendan Sanjie capsule group, MK-2206 group and Shendan Sanjie capsule+ IGF-1 group are lower than that in model group (P<0.05), while Shendan Sanjie capsule+ IGF-1 group is higher than Shendan Sanjie Capsule group (P<0.05). The relative expressions of bFGF mRNA in the colon cancer tissue of mice in the model group, Shendan Sanjie capsule group, MK-2206 group and Shendan Sanjie capsule+ IGF-1 group were 4.55±0.31, 2.46±0.37, 2.49±0.33, 3.34±0.21, respectively, and the relative mRNA expressions of Ang2 were 5.78±0.19, 2.21±0.14, 2.26±0.17 and 3.67±0.32, respectively, which were significantly higher than 1.01±0.05 and 0.99±0.07 in the normal group (P<0.05). The mRNA levels of bFGF and Ang2 in Shendan Sanjie capsule group, MK-2206 group and Shendan Sanjie capsule+ IGF-1 group were lower than those in the model group (P<0.05), while Shendan Sanjie capsule+ IGF-1 group is higher than Shendan Sanjie capsule group (P<0.05). The relative expression levels of p-Akt/Akt, VEGFA and HIF-1α in colon cancer tissues of the model group were 4.75±0.18, 4.64±0.22 and 4.84±0.12, respectively, which were significantly higher than 1.01±0.07, 0.95± 0.08 and 0.98±0.05 in the normal group (P<0.05). The relative expressions of p-Akt/Akt, VEGFA and HIF-1α in colon cancer tissues in the Shendan Sanjie capsule group were 2.24±0.22, 3.15±0.26 and 2.07±0.18, respectively, which were significantly lower than those in the model group (P<0.05). However, compared with the MK-2206 group, the difference was not statistically significant (P>0.05). The relative expression levels p-Akt/Akt, VEGFA and HIF-1α in colon cancer tissue of the Shendan Sanjie capsule+ IGF-1 group were 3.37±0.15, 4.02±0.11, 3.52±0.24, respectively, which were significantly higher than those in the Shendan Sanjie capsule group (P<0.05). Conclusion: Shendan Sanjie capsules may inhibit Akt/HIF-1α/VEGFA signaling pathway, and then reduce the expression of microvascular growth factors bFGF and Ang2, thereby inhibit the tumor angiogenesis of CAC.

Jolkinolide B attenuates laryngeal cancer cell growth and induces apoptosis via PTEN/PI3K/Akt signaling pathway.

Jolkinolide B (JB) is a bioactive diterpenoid, isolated from the root of Euphorbia fischeriana Steud, and has been reported to have anti-tumor and anti-inflammation function by regulation of cell migration, invasion, apoptosis, and cell cycle. We aimed to evaluate the effect of JB on laryngeal cancer cells. Human normal larynx epithelial (HBE) cells and cancer cell lines TU212, TU177, and Hep-2 were cultured; MTT assay was used to assess cell proliferation. LY294002 (a PI3K/Akt inhibitor) and IGF-1 (a PI3K/Akt activator) were employed to investigate the expression of PI3K/Akt pathway. Cell migration and invasion activities were detected by scratch wound healing and transwell assay, respectively. Flow cytometry assay was used to assess cell apoptosis. The expression levels of proteins were assessed by immunofluorescence and Western blotting assay. JB inhibited TU212, TU177, and Hep-2 cell viability with an IC50 value of 54.57 ± 0.53μg/mL, 44.82 ± 0.32μg/mL, and 49.63 ± 0.47μg/mL, respectively. Compared with control group, the proliferation, migration, and invasion of cells significantly decreased after JB and LY294002 treatment, while cell apoptosis increased. In IGF-1 group, the results were opposite compared to the JB and LY294002 groups. Western blotting results showed that JB and LY294002 treatment significantly inhibited the levels of Bcl-2, p-PI3K, and p-Akt while the levels of Bax, cleaved caspase-3, and PTEN protein significantly increased. Our study suggested that JB exhibits an inhibition effect on laryngeal cancer cell growth in vitro.

Associations of genetically predicted circulating insulin-like growth factor-1 and insulin-like growth factor binding protein-3 with bladder cancer risk.

Insulin-like growth factors (IGF) play important roles in carcinogenesis. The associations of circulating IGF-1 and insulin-like growth factor-binding protein-3 (IGFBP-3) with the risks of bladder cancer remain unclear. In this large case control study of 2011 bladder cancer cases and 2369 heathy controls, we assessed the associations of circulating IGF-1 and IGFBP-3 with bladder cancer risks using a Mendelian randomization approach, which uses genetic variants as instruments to study causal relationship between risk factors and diseases. We first constructed a weighted genetic risk score (GRS) predictive of circulating IGF-1 and IGFBP-3 using 413 genome-wide association study-identified single nucleotide polymorphisms (SNPs) associated with IGF-1 and four SNPs with IGFBP-3, respectively. We found that higher GRS for IGF-1 was associated with a significantly reduced bladder cancer risk (odds ratio [OR] = 0.66 per SD increase, 95% confidence interval [CI], 0.54-0.82, p < 0.001). We then used a summary statistics-based MR method, inverse-variance weighting (IVW), and found a similar risk estimate (OR = 0.67 per SD increase, 95% CI = 0.54-0.83, p < 0.001). When we categorized individuals into high and low IGF-1 groups using the median GRS value in the controls, the high GRS group had a 21% reduced bladder cancer risk (OR = 0.79, 95% CI = 0.70-0.89) compared to the low GRS group. Genetically predicted circulating IGFBP-3 was not associated with bladder cancer risk. In conclusion, our data demonstrated for the first time a strong inverse relationship between circulating IGF-1 level and bladder cancer risk.

Insulin‐like growth factor I reduces atherosclerosis in Rapacz pigs

Cardiovascular disease is the leading cause of mortality in the Western world and its manifestations result in large part from atherosclerotic lesion development and destabilization. We have reported previously that administration of insulin-like growth factor I (IGF-1) reduced atherosclerosis in the murine model. To test the effect of IGF-1 on human-like atherosclerosis we injected 50 ug/kg recombinant human IGF-1 or saline (control) (n=9/group) into high-fat diet fed Rapacz familial hypercholesterolemic (FH) swine for 6 months. We quantified vessel volume, atherosclerotic burden (% of volume of atherosclerotic plaque + media per vessel volume), lumen volume and assessed plaque phenotype in coronary arteries using intravascular ultrasound (IVUS) at time 0 and after 3 and 6 months on the diet. IGF-1-injected pigs had on average 2.6-fold increase in total plasma IGF-1 (ELISA, p<0.001 vs. control pigs). Pigs in the IGF-1 and control group gradually gained weight over the time of experiment, however pigs in the IGF-1 group had a larger increase in BW compared to controls at 6 months (IGF-1, 60.6±7.4% increase vs. saline, 44.3±4.0% increase, p<0.05) consistent with growth-stimulating effects of IGF-1. Pigs in both groups had a gradual increase in vessel volume in the right coronary artery (RCA), left anterior descending artery (LAD) and left circumflex artery (LCX) with time as quantified by serial IVUS. However, IGF-1-injected pigs had a larger increase in vessel volume after 3 and 6 months compared to controls suggesting vascular hypertrophy. The atherosclerotic burden was increased in RCA, LAD and LCX in both groups after 3 and 6 months of injections compared to the pre-injection time point. We found a significant reduction in atherosclerotic burden in RCA and LAD in IGF-1 group compared to control at 6 months’ time-point (RCA: IGF-1, 203±13%, vs. control, 244±8; LAD: IGF-1, 200±6%, vs. control, 228±11%). The lumen volume of coronaries was decreased in both groups, however, coronaries in IGF-1-injected pigs had on average 24±3% larger lumen volume compared to control pigs at 6 months’ time-point. We used virtual histology (VH) post-IVUS image processing to assess plaque composition and to classify plaque sub-types. Pigs in both groups had a progressive increase in plaque calcification, necrotic core area, fibrous plaque and fibro-fatty plaque area. IGF-1 did not significantly change plaque composition. In summary, we report that IGF-1 increased pig BW, and total volume of coronaries suggesting hypertrophy and these data are consistent with growth-stimulating effects of IGF-1. We found a significant reduction in atherosclerotic burden and increase in lumen volume in coronaries in the IGF-1 group after 6 months of injections consistent with an anti-atherosclerotic effect of IGF-1. Our data support the feasibility of using IGF-1 as an anti-atherogenic therapy.

Targeting IRS-1/mPGES-1/NOX2 to inhibit the inflammatory response caused by insulin-like growth factor-I-induced activation of NF-κB and NLRP3 in cancer cells.

The levels of insulin-like growth factor-l (IGF-1) and reactive oxygen species (ROS) are abnormally elevated in various tumour tissues, and IGF-1 has been reported to be associated with the development and progression of inflammation in cancers. In this study, we found that IGF-1 activated nuclear factor-κB (NF-κB) and NLRP3 inflammatory signalling via IRS-1/mPGES-1/NOX2-regulated ROS. Additionally, in the B16-F10 tumour-bearing mouse model, the number of tumours, tumour growth, invasion of tissues and expression of proinflammatory factors in peripheral blood were significantly decreased by treatment with an inhibitor combination compared with those of the IGF-1 group. Taken together, targeting IRS-1/mPGES-1/NOX2 to inhibit inflammation related to NF-κB and NLRP3 is a potential strategy for controlling the development and progression of cancer.

The effect of growth factors on induction of mesenchymal stem cell to the skeletal muscle cells.

The objective of this study was to evaluate the effect of IGF1 and FGF2 growth factors on the differentiation of human adipose tissue‑derived mesenchymal stem cells (MSCs) into skeletal muscle cells. MSCs were divided into four groups. Group I (control group) was incubated only in myogenic medium, group II was incubated by adding 100 ng/ml FGF2 to the myogenic medium, group III was incubated by adding 20 ng/ml IGF1 to the myogenic medium, group IV was incubated by adding 100 ng/ml FGF2 and 20 ng/ml IGF1 to the myogenic medium. Cells dyed with MyoD1 antibodies were analyzed by flow cytometry so as to determine the myogenic differentiation on day 28. It was confirmed that 11.5 % of the control group, 39.2 % of the FGF2 group, 23.1 % of the IGF1 group, and 39.3 % of the IGF1-FGF2 group showed differentiation. Our results show that IGF1 and FGF2 have a positive contribution to myogenic differentiation of MSCs. They contribute to the studies related to muscle diseases and their treatment by the fact that growth factors support the feature of regeneration capacity and differentiation (Tab. 2, Fig. 7, Ref. 23).

Insulin and IGF-1 does not alter the morphokinetics of mouse embryo development

Objective To assess if there is a benefit to adding either IGF-1 or insulin to culture medium in terms of mouse embryo development using time-lapse morphokinetics. Design Prospective study. Materials and methods A total of 305 commercially obtained frozen mouse embryos (B6D2F1 & B6C3F1 hybrid) were thawed and cultured in (1) One-Step medium only, (2) One-Step medium with 100ng/mL insulin, and (3) One-Step medium with 100ng/mL IGF-1, using an EmbryoScope time lapse incubator at 37°C, 5.5% CO2, and 6.0% O2. The EmbryoScope was set to record images of each embryo every 10 minutes for 6 days of culture. The following time points were annotated: 2cell (t2), 3cell (t3), 4cell (t4), 5cell (t5), 6cell (t6), 7cell (t7), 8cell (t8), start of compaction (tSC), morula (tsM), start of blastulation (tSB), blastocyst (tB), expanded blastocyst (tEB) and hatching blastocyst (tHB). The 2-cell stage was considered as time zero since the exact time of insemination was unknown. All time points were statistically compared between each of the three groups. Blastocyst development rates for each group were also compared. Results A total of 304 blastocysts developed from the 316 embryos cultured, yielding an overall blastocyst development rate of 96.2%.When comparing the blastocyst development rate between the 3 groups, there were no significant differences between the One-Step and IGF-1 media groups (99.1% vs 96.2%). However, the insulin group showed significantly lower blastocyst rates when compared to the controls (93.3% vs 99.1%; p=0.02). There were no statistically significant differences in any of the time points measured between the One-Step, insulin and IGF-1 groups. Conclusions The data indicates that there is no beneficial effect of adding insulin or IGF-1 to culture media for mouse embryo development. Neither insulin not IGF-1 had any effect on the morphokinetics of these embryos. However, culture of mouse embryos in insulin supplemented media resulted in a lower blastocyst development rate compared to One-Step media. Ongoing studies are underway using a more sensitive mouse strain to see whether any changes in morphokinetics may be detected. Disclosures Nothing to disclose Funding None

Role of Programmed Cell Death 4 (PDCD4)-Mediated Akt Signaling Pathway in Vascular Endothelial Cell Injury Caused by Lower-Extremity Ischemia-Reperfusion in Rats.

BackgroundWe aimed to investigate the role of PDCD4-mediated Akt signaling pathway in vascular endothelial cell injury caused by ischemia-reperfusion in the lower extremities.Material/MethodsTen rats were used as control, while 50 rats were used for creating disease models and were assigned to 5 groups: model group (no injection), NC group (injected with vectors containing PDCD negative control sequence), sh-PDCD4 group (injected with vectors containing sh-PDCD4 sequence), IGF-1 group (injected with IGF-1), and sh-PDCD4+IGF-1 group (injected with IGF-1 and vectors containing sh-PDCD4 sequence).ResultsCompared with the control group, the expression levels of PDCD4 mRNA and protein, as well as levels of circulating endothelial cells, von Willebrand factor, thrombomodulin, and malondialdehyde, increased in the other 5 groups, while the mRNA and protein expression levels of Akt and eNOS, the protein expression levels of p-Akt and p-eNOS, and superoxide dismutase content decreased in these groups (all P<0.05). Compared with the model group, the sh-PDCD4 and sh-PDCD4+1GF-1 groups had lower mRNA and protein expressions of PDCD4 (all P<0.05), whereas the IGF-1 group had similar levels (all P>0.05). These 3 groups had lower levels of circulating endothelial cells, von Willebrand factor, thrombomodulin, and malondialdehyde, and higher mRNA and protein expressions of Akt and eNOS, protein expressions of p-Akt and p-eNOS, and superoxide dismutase content (all P<0.05). The NC group did not differ from the model group (all P>0.05).ConclusionsPDCD4 gene silencing can activate the Akt signaling pathway and attenuate vascular endothelial cell injury caused by ischemia-reperfusion in the lower extremities in rats.

Effect and mechanism of adipocyte co-culture on aquaporin-9 expression in HepG2 cells

Objective: To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action. Methods: Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples. Results: The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (t= 5.225,P= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (t= 13.708, 11.225,P= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (t= 27.136,P< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (t= 2.431,P= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (t= 5.082,P= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (t= 10.311,P< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (t= 33.573, 5.598,P< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (t=14.953, 28.931,P= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (t= 5.478, 5.369,P= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (t= 16.311, 8.769,P< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (t= 33.573, 9.146,P< 0.001 and 0.001). Conclusion: Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.

IGF-1 defends against chronic-stress induced depression in rat models of chronic unpredictable mild stress through the PI3K/Akt/FoxO3a pathway

This study aims to investigate the role of IGF-1 in chronic-stress induced depression through the PI3K/Akt/FoxO3a pathway. A rat model of chronic unpredictable mild stress (CUMS) was established. In total, 48 rats were randomized into control (normal rats), CUMS (CUMS modeled rats) and CUMS + IGF-1 (injection of IGF-1 before CUMS modeling) groups. Body weight, horizontal (number of horizontal crossing) and vertical activity (rearing times), and sucrose consumption were identified one day before and after the open-field test. The mRNA and protein expression of PI3K, Akt, FoxO3a and Bim in the hippocampus was measured by RT-qPCR and Western blotting, respectively. Compared with the control group, a lower body weight, a decreased number of horizontal crossings, reduced rearing times and lower sucrose consumption were observed in the CUMS and CUMS + IGF-1 groups after the test. However, a higher body weight, number of horizontal crossings, rearing times and sucrose consumption were found in the CUMS + IGF-1 group than those in the CUMS group. Compared with the control group, mRNA and protein expression of PI3K, Akt and FoxO3a was decreased, and Bim mRNA and protein expression was increased in the CUMS + IGF-1 and CUMS groups. Meanwhile, in comparison to the CUMS group, mRNA and protein expression of PI3K, Akt and FoxO3a was elevated, and Bim mRNA and protein expression was reduced in the CUMS + IGF-1 group. The results suggested that IGF-1 exerted an antidepressant-like effect on chronic-stress induced depression through the PI3K/Akt/FoxO3a pathway.

Preliminary Study of Mechanisms of Fluoride-Induced Suppression of Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells.

This study was aimed to determine if excessive exposure to fluoride could suppress the synthesis of nitric oxide (NO) and to detail the mechanisms involved. With the exception of the control group, human umbilical vein endothelial cells (HUVECs) were treated with sodium fluoride (NaF) (1.2μg/mL) for 24h, with or without a 2-h pretreatment with 100nM insulin-like growth factor 1 (IGF-1, a PI3K/AKT agonist), or 10μM histamine (HIS, a eNOS agonist). The levels of NO in culture fluids, as well as the expressions of eNOS, p-eNOS, PI3K, AKT, and p-AKT, were compared. The levels of NO significantly decreased in all experimental groups; however, the levels of NO were obviously higher in the NaF + HIS and NaF + IGF-1 groups, compared to the NaF group. The p-eNOS/eNOS ratios dropped clearly in NaF and NaF + HIS groups, while that in the NaF + HIS group was distinctly higher than that in the NaF group. The p-AKT/AKT ratios went down apparently in NaF and NaF + IGF-1 groups, while that in the NaF + IGF-1 group was overtly higher than that of the NaF group. Excessive exposure to fluoride inhibited the synthesis of NO. The PI3K/AKT/eNOS pathway played a crucial role in the reduced expression of NO caused by excessive fluoride exposure.

Role of PI3K/Akt signaling pathway in propofol-induced invasion of human liver cancer cell line HepG2

Objective To evaluate the role of phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt)signaling pathway in propofol-induced invasion of human liver cancer cell line HepG2. Methods HepG2 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a 5% CO2 incubator at 37 ℃.HepG2 cells at the logarithmic growth phase were divided into 6 groups(n=18 each)using a random number table: control group(group C), propofol group(group P), PI3K/Akt signaling pathway agonist IGF-1 group(group IGF), PI3K/Akt signaling pathway inhibitor LY294002 group(group LY), IGF-1 plus propofol group(group IGF+ P)and LY294002 plus propofol group(group LY + P). Propofol 120 μg/ml was added in group P. IGF-1 10 nmol/L was added in group IGF.LY294002 10 μmol was added in group LY.In group IGF+ P, 10 nmol/L IGF-1 was added, cells were incubated for 24 h, and then 120 μg/ml propofol was added.In group LY + P, 10 μmol LY294002 was added, cells were incubated for 24 h, and then 120 μg/ml propofol was added.The invasion of cells was measured by Transwell invasion assay at 24 h of incubation.The expression of PI3K and Akt mRNA in cells was determined by real-time polymerase chain reaction.The expression of Akt, PI3K and phosphorylated Akt(p-Akt)was detected by using Western blot. Results Compared with group C, the invasive cell count was significantly reduced, the expression of PI3K protein and mRNA was down-regulated, p-Akt/Akt ratio was decreased, and the expression of Akt mRNA was down-regulated in P, P+ IGF, LY and P+ LY groups, and the invasive cell count was significantly increased, the expression of PI3K protein and mRNA was up-regulated, p-Akt/Akt ratio was increased, and the expression of Akt mRNA was up-regulated in group IGF(P<0.05). Compared with group P, the invasive cell count was significantly increased, the expression of PI3K protein and mRNA was up-regulated, p-Akt/Akt ratio was increased, and the expression of Akt mRNA was up-regulated in group P+ IGF, and the invasive cell count was significantly reduced, the expression of PI3K protein and mRNA was down-regulated, p-Akt/Akt ratio was decreased, and the expression of Akt mRNA was down-regulated in group P+ LY(P<0.05). The invasive cell count was significantly reduced, the expression of PI3K protein and mRNA was down-regulated, p-Akt/Akt ratio was decreased, and the expression of Akt mRNA was down-regulated in group P+ IGF as compared with group IGF(P<0.05)and in group P+ LY as compared with group LY(P<0.05). Conclusion The mechanism by which propofol inhibits invasion of HepG2 cells is related to inhibiting activation of PI3K/Akt signaling pathways. Key words: Propofol; Cell line, tumor; Neoplasm invasiveness; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases

Possible alternative treatment for mandibular asymmetry by local unilateral IGF-1 injection into the mandibular condylar cavity: Experimental study in mice.

The purpose of this study was to investigate whether a local unilateral IGF-1 injection into the mandibular condylar cavity can induce unilateral endochondral mandibular growth without any systemic adverse effects. Seventy-five 3-week-old male Jcl:ICR mice were used in this study. The mice were divided into 2 groups: control group (n=22) and IGF-1 group (n=53). In the IGF-1 group, human IGF-1 was injected into the right mandibular condylar cavity, and phosphate-buffered saline solution was injected into the left cavity, 3 times per week for 10weeks. There was no significant difference in body weight, serum human IGF-1 concentration, and soft tissue thickness of the cheeks including the masseter muscles between the 2 groups. Unilateral IGF-1 injection induced a lateral shift of the mandible to the contralateral side, and microcomputed tomogtraphy analysis showed that unilateral IGF-1 injection induced endochondral growth in the condyle. Col2, Ihh, and Runx2 were extensively upregulated by the local unilateral IGF-1 injection in real-time reverse transcription polymerase chain reaction analysis. Proliferation marker KI67, IGF-1 signaling molecule AKT1, and chondrogenic differentiation marker Col2 were strongly expressed in the IGF-1 injected condyle by immunohistochemistry. Vital labeling showed that the distance between the labels was increased in the IGF-1 injection group compared with that of the control group. The results verified in this study indicated that local unilateral IGF-1 injection into the mandibular condylar cavity successfully induced unilateral endochondral mandibular growth in mice without any systemic adverse effects. Thus, local unilateral IGF-1 injection into the mandibular condylar cavity could be a useful alternative for mandibular asymmetry therapy during the growth period. However, additional experimental and clinical studies will be necessary to prove the real effect of this new therapy.

The single IGF-1 partial deficiency is responsible for mitochondrial dysfunction and is restored by IGF-1 replacement therapy

Background & aimsWe previously described in cirrhosis and aging, both conditions of IGF-1 deficiency, a clear hepatic mitochondrial dysfunction with increased oxidative damage. In both conditions, the hepatic mitochondrial function was improved with low doses of IGF-1. The aim of this work was to explore if the only mere IGF-1 partial deficiency, without any exogenous insult, is responsible for hepatic mitochondrial dysfunction. MethodsHeterozygous (igf1+/−) mice were divided into two groups: untreated and treated mice with low doses of IGF-1. WT group was used as controls. Parameters of hepatic mitochondrial function were determined by flow cytometry, antioxidant enzyme activities were determined by spectrophotometry, and electron chain transport enzyme levels were determined by immunohistochemistry and immunofluorescence analyses. Liver expression of genes coding for proteins involved in mitochondrial protection and apoptosis was studied by microarray analysis and RT-qPCR. ResultsHz mice showed a significant reduction in hepatic mitochondrial membrane potential (MMP) and ATPase activity, and an increase in intramitochondrial free radical production and proton leak rates, compared to controls. These parameters were normalized by IGF-1 replacement therapy. No significant differences were found between groups in oxygen consumption and antioxidant enzyme activities, except for catalase, whose activity was increased in both Hz groups. Relevant genes coding for proteins involved in mitochondrial protection and survival were altered in Hz group and were reverted to normal in Hz+IGF-1 group. ConclusionsThe mere IGF-1 partial deficiency is per se associated with hepatic mitochondrial dysfunction sensitive to IGF-1 replacement therapy. Results in this work prove that IGF-1 is involved in hepatic mitochondrial protection, because it is able to reduce free radical production, oxidative damage and apoptosis. All these IGF-1 actions are mediated by the modulation of the expression of genes encoding citoprotective and antiapoptotic proteins.

The PI3K-AKT-mTOR pathway activates recovery from general anesthesia.

We investigated roles of PI3K-AKT-mTOR pathway in recovery from general anesthesia. Sprague-Dawley rats divided into five groups: saline+artificial cerebrospinal fluid (ACSF; Group A), ketamine+ACSF (Group B), ketamine+IGF-1 (Group C), ketamine+PI3K inhibitor (Group D), and PI3K/Akt agonists (Group E). Proportion of δ waves on ECoGs was recorded. Rats were tested for duration of loss of righting reflex (LORR), ataxic period and behavior in Morris water maze. mRNA and protein expression of members of PI3K-AKT-mTOR pathway were measured by RT-qPCR and Western blots. Histopathologic changes in hippocampal tissues observed by HE staining. We found that the proportion of δ waves decreased in Group C, while increased in Group D compared with Group B; the durations of LORR and ataxic period were shorter in Group C, but longer in Group D. In Morris water maze, escape latency (EL) and duration and frequency of staying on platform was shorter in Group C and longer in Group D than in Group B. Group A exhibited low expression of proteins in PI3K-AKT-mTOR pathway, while p-AKT, p-mTOR and p-P70S6K expression increased in cerebral cortex, brain stem, and thalamus in Group C. By contrast, expression of those proteins was lower in Group D than Group B. Those proteins expressions were higher in Group E than in Group A. HE staining showed that anesthesia may induce cell apoptosis in rat hippocampal CA1 areas, and PI3K/Akt agonists could inhibit apoptosis. Our results suggest that activation of PI3K-AKT-mTOR pathway may promote recovery from general anesthesia and enhance spatial learning and memory.

Molecular Imaging for Comparison of Different Growth Factors on Bone Marrow-Derived Mesenchymal Stromal Cells' Survival and Proliferation In Vivo.

Introduction. Bone marrow-derived mesenchymal stromal cells (BMSCs) have emerged as promising cell candidates but with poor survival after transplantation. This study was designed to investigate the efficacy of VEGF, bFGF, and IGF-1 on BMSCs' viability and proliferation both in vivo and in vitro using bioluminescence imaging (BLI). Methods. BMSCs were isolated from β-actin-Fluc+ transgenic FVB mice, which constitutively express firefly luciferase. Apoptosis was induced by hypoxia preconditioning for up to 24 h followed by flow cytometry and TUNEL assay. 106 BMSCs with/without growth factors were injected subcutaneously into wild type FVB mice's backs. Survival of BMSCs was longitudinally monitored using bioluminescence imaging (BLI) for 5 weeks. Protein expression of Akt, p-Akt, PARP, and caspase-3 was detected by Western blot. Results. Hypoxia-induced apoptosis was significantly attenuated by bFGF and IGF-1 compared with VEGF and control group in vitro (P < 0.05). When combined with matrigel, IGF-1 showed the most beneficial effects in protecting BMSCs from apoptosis in vivo. The phosphorylation of Akt had a higher ratio in the cells from IGF-1 group. Conclusion. IGF-1 could protect BMSCs from hypoxia-induced apoptosis through activation of p-Akt/Akt pathway.