Recombinant Human Insulin-like Growth Factor Research Articles

Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

Sex-based differences in growth-related IGF1 signaling in response to PAPP-A2 deficiency: comparative effects of rhGH, rhIGF1 and rhPAPP-A2 treatments

BackgroundChildren with pregnancy-associated plasma protein-A2 (PAPP-A2) mutations resulting in low levels of bioactive insulin-like growth factor-1 (IGF1) and progressive postnatal growth retardation have improved growth velocity and height following recombinant human (rh)IGF1 treatment. The present study aimed to evaluate whether Pappa2 deficiency and pharmacological manipulation of GH/IGF1 system are associated with sex-specific differences in growth-related signaling pathways.MethodsPlasma, hypothalamus, pituitary gland and liver of Pappa2ko/ko mice of both sexes, showing reduced skeletal growth, and liver of these mice treated with rhGH, rhIGF1 and rhPAPP-A2 from postnatal day (PND) 5 to PND35 were analyzed.ResultsReduced body and femur length of Pappa2ko/ko mice was associated with increases in: (1) components of IGF1 ternary complexes (IGF1, IGFBP5/Igfbp5, Igfbp3, Igfals) in plasma, hypothalamus and/or liver; and (2) key signaling regulators (phosphorylated PI3K, AKT, mTOR, GSK3β, ERK1/2 and AMPKα) in hypothalamus, pituitary gland and/or liver, with Pappa2ko/ko females having a more prominent effect. Compared to rhGH and rhIGF1, rhPAPP-A2 specifically induced: (1) increased body and femur length, and reduced plasma total IGF1 and IGFBP5 concentrations in Pappa2ko/ko females; and (2) increased Igf1 and Igf1r levels and decreased Ghr, Igfbp3 and Igfals levels in the liver of Pappa2ko/ko females. These changes were accompanied by lower phospho-STAT5, phospho-AKT and phospho-ERK2 levels and higher phospho-AMPK levels in the liver of Pappa2ko/ko females.ConclusionsSex-specific differences in IGF1 system and signaling pathways are associated with Pappa2 deficiency, pointing to rhPAPP-A2 as a promising drug to alleviate postnatal growth retardation underlying low IGF1 bioavailability in a female-specific manner.

In vitro maturation of African catfish (Clarias gariepinus, Burchell, 1822) oocytes results in viable larvae

In this study, we describe a protocol for in vitro maturation and ovulation of African catfish (Clarias gariepinus) postvitellogenic ovarian follicles, resulting in fertilizable and developmentally competent eggs. Such culture systems enable detailed studies of fish oocyte development and present a useful tool in assisted reproduction of fish. The maturation-inducing effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), human chorionic gonadotropin (hCG) and recombinant human insulin-like growth factor 1 (rhIGF-1) were evaluated based on their ability to promote ooplasm translucency and germinal vesicle breakdown (GVBD), which indicates meiosis resumption. Among the tested groups, only follicles exposed to DHP underwent GVBD in a time-dependent manner, with the highest rates (>80%) observed within 10 to 12 h of incubation. Furthermore, adding rhIGF-1 or hCG prior to or during incubations with DHP did not influence the GVBD outcome. This suggested that the oocytes acquired maturational competence prior to isolation, as high concentrations of DHP (1 μg/ml) were alone sufficient to induce complete nuclear and cytoplasmic maturation; however, only 4 ± 3% of these oocytes ovulated in culture. To promote ovulation, we incorporated prostaglandins (PGs) as an additional incubation step following DHP-induced maturation. Exposure to both prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) led to increased rupture of the follicular layer, most notably in groups with 5 μg/ml PGF2α (71 ± 8%). In addition to identifying optimal hormonal stimulation, effects of culture media pH in combination with media supplementation—in the form of bovine serum albumin (BSA) and fetal bovine serum (FBS)—were considered. Compared to results in media with pH of 7.5, significantly less follicles matured and ovulated in a more alkaline environment (pH 8.5), regardless of supplement type and concentration. Although the presence of FBS (5–20%) slightly improved ovulation rates, this benefit was not reflected in the fertilization outcomes. In vitro matured and ovulated oocytes obtained in non-supplemented media (pH 7.5) maintained their developmental competence and were successfully fertilized. The hatching rate was 39%, after which the survival rate of larvae was 8% at 72 h post fertilization (hpf).

Gut development following insulin-like growth factor-1 supplementation to preterm pigs

BackgroundReduced insulin-like growth factor-1 (IGF-1) levels may contribute to impaired organ development in preterm infants. Using preterm pigs as a model, we hypothesized that IGF-1 supplementation improves health and gut development during the first three weeks of life.MethodsFirst, clinical and organ endpoints were compared between artificially-reared, cesarean-delivered preterm pigs and vaginally-delivered, sow-reared term pigs at 5, 9 and 19 days. Next, preterm pigs were treated with recombinant human IGF-1 for 19 days (2.25 mg/kg/day, systemically).ResultsRelative to term pigs, preterm pigs had lower body weight, fat, bone contents, relative weights of liver and spleen and a longer and thinner intestine at 19 days. Preterm birth reduced intestinal villi heights and peptidase activities, but only at 5 and 9 days. In preterm pigs, IGF-1 reduced mortality primarily occurring from gastrointestinal complications and with a tendency towards salvaging smaller pigs. IGF-1 supplementation also increased spleen and kidney weights, small intestine length and maltase to lactase activity, reflecting gut maturation.ConclusionPreterm birth affects body composition and gut maturation in the first 1–2 weeks, but differences are marginal thereafter. Supplemental IGF-1 may improve gut health in pigs and infants in the first few weeks after preterm birth.ImpactInsulin-like growth factor 1 (IGF-1) supplementation may improve gut health and development in prematurity, but whether the effects are sustained beyond the immediate postnatal period is unclear.In preterm pigs, the prematurity effects on IGF-1 and gut health deficiencies are most pronounced during the first week of life and diminishes thereafter.In preterm pigs, IGF-1 supplementation beyond the first week of life reduced mortality.The present study provides evidence of a sustained effect of IGF-1 supplementation on the gastrointestinal tract after the immediate postnatal period.

Evaluation of recombinant human IGF-1/IGFBP-3 on intraventricular hemorrhage prevention and survival in the preterm rabbit pup model

Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates processes of vascular maturation. The pathogenesis of intraventricular hemorrhage (IVH) relates to the fragility of the immature capillaries in the germinal matrix, and its inability to resist fluctuations in cerebral blood flow. In this work, using different experimental setups, we aimed to (i) establish an optimal time-point for glycerol-induction of IVH in relation to time-point of recombinant human (rh) IGF-1/rhIGFBP-3 administration, and (ii) to evaluate the effects of a physiologic replacement dose of rhIGF-1/rhIGFBP-3 on prevention of IVH and survival in the preterm rabbit pup. The presence of IVH was evaluated using high-frequency ultrasound and post-mortem examinations. In the first part of the study, the highest incidence of IVH (> 60%), occurred when glycerol was administered at the earliest timepoint, e.g., 6 h after birth. At later time-points (18 and 24 h) the incidence decreased substantially. In the second part of the study, the incidence of IVH and mortality rate following rhIGF-1/rhIGFBP-3 administration was not statistically different compared to vehicle treated animals. To evaluate the importance of maintaining intrauterine serum levels of IGF-1 following preterm birth, as reported in human interventional studies, additional studies are needed to further characterize and establish the potential of rhIGF-1/rhIGFBP-3 in reducing the prevalence of IVH and improving survival in the preterm rabbit pup.

Insulin-like growth factor-1 short-period therapy improves Non-Alcoholic Fatty Liver Disease (NAFLD) in obese mice

This study seeks to evaluate Insulin-like Growth Factor 1 (IGF-1) short-period therapy in Non-Alcoholic Fatty Liver Disease (NAFLD) as it relates to western diet-induced obesity. For this purpose, 21-day-old male Swiss mice were divided into a control group (CG, N=8), which was fed a standard diet, and an obese group (GO, N=16), which was fed a western diet, rich in saturated fat and simple carbohydrates, for 12 weeks. In the 11th week, part of the animals in the obese group (N=8) received a daily subcutaneous injection of recombinant human IGF-1 (100µg/kg/day) during seven consecutive days (GO+IGF-1). Biometric and metabolic parameters, intraperitoneal glucose tolerance test (IGTT), quantitative analysis of liver steatosis, quantitative analysis of collagen in liver and expression of immunoperoxidase of alpha-smooth muscle actin (a-SMA) were analyzed. Our data demonstrated that IGF-1 short-term treatment was able to improve obesity-related biometric and metabolic parameters. In addition, it promoted the recovery of liver parenchyma, thereby reducing steatosis and fibrosis, thus demonstrating an important hepatoprotective action.

Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3

Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates essential processes of vascular maturation and stabilization. Importantly, preterm birth is associated with reduced serum levels of IGF-1 as compared to in utero levels. Using a preterm rabbit pup model, we investigated the uptake of systemic recombinant human (rh) IGF-1 in complex with its main binding protein IGF-binding protein 3 (BP-3) to the brain parenchyma via the choroid plexus. Five hours after subcutaneous administration, labeled rhIGF-1/rhIGFBP-3 displayed a widespread presence in the choroid plexus of the lateral and third ventricle, however, to a less degree in the fourth, as well as in the perivascular and subarachnoid space. We found a time-dependent uptake of IGF-1 in cerebrospinal fluid, decreasing with postnatal age, and a translocation of IGF-1 through the choroid plexus. The impact of systemic rhIGF-1/rhIGFBP-3 on IGF-1 receptor activation in the choroid plexus decreased with postnatal age, correlating with IGF-1 uptake in cerebrospinal fluid. In addition, choroid plexus gene expression was observed to increase with postnatal age. Moreover, using choroid plexus in vitro cell cultures, gene expression and protein synthesis were further investigated upon rhIGF-1/rhIGFBP-3 stimulation as compared to rhIGF-1 alone, and found not to be differently altered. Here, we characterize the uptake of systemic rhIGF-1/rhIGFBP-3 to the preterm brain, and show that the interaction between systemic rhIGF-1/rhIGFBP-3 and choroid plexus varies over time.

Frequency and Predictive Factors of Hypoglycemia in Patients Treated With rhIGF-1: Data From the Eu-IGFD Registry.

The European Increlex® Growth Forum Database (Eu-IGFD) is an ongoing surveillance registry (NCT00903110) established to collect long-term safety and effectiveness data on the use of recombinant human insulin-like growth factor-1 (rhIGF-1, mecasermin, Increlex) for the treatment of children/adolescents with severe primary insulin-like growth factor-1 deficiency (SPIGFD). This analysis of Eu-IGFD data aimed to identify the frequency and predictive factors for hypoglycemia adverse events (AEs) in children treated with rhIGF-1. Data were collected from December 2008 to May 2021. Logistic regression was performed to identify predictive risk factors for treatment-induced hypoglycemia AEs. Odds ratios (ORs) are presented with 95% CIs for each factor. In total, 306 patients were enrolled in the registry; 84.6% were diagnosed with SPIGFD. Patients who experienced ≥ 1 hypoglycemia AE (n = 80) compared with those with no hypoglycemia AEs (n = 224) had a lower mean age at treatment start (8.7 years vs 9.8 years), a more frequent diagnosis of Laron syndrome (27.5% vs 10.3%), and a history of hypoglycemia (18.8% vs 4.5%). Prior history of hypoglycemia (OR 0.25; 95% CI: [0.11; 0.61]; P = .002) and Laron syndrome diagnosis (OR 0.36; 95% CI: [0.18; 0.72]; P = .004) predicted future hypoglycemia AEs. Total hypoglycemia AEs per patient per treatment year was 0.11 and total serious hypoglycemia AEs per patient per treatment year was 0.01. Hypoglycemia occurs more frequently in patients with prior history of hypoglycemia and/or Laron syndrome compared with patients without these risk factors, and these patients should be carefully monitored for this AE throughout treatment.

Effects of IGF1 and IGF2 on In Vitro Ovarian Follicle Maturation in Rainbow Trout, Oncorhynchus mykiss

Ovarian follicle maturation (OFM), including the resumption of meiosis in the oocyte, is under hormonal regulation. Insulin-like growth factors (IGF) have been shown to participate in the regulation of OFM with species-specific actions in teleost fishes. In the present study, in vitro treatment of rainbow trout ovarian follicles with recombinant human insulin-like growth factor-1 (rhIGF1) or rhIGF2 did not induce germinal vesicle breakdown (GVBD), a marker for the resumption of meiosis, in the oocytes. Co-incubation of follicle-enclosed oocytes with rhIGF1 and the maturation-inducing steroid (MIS) in rainbow trout, 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP), also did not induce GVBD in follicles from rainbow trout that were not able to respond to the MIS alone suggesting IGFs cannot induce oocyte maturational competence (OMC), which is the ability of the oocyte to respond to the MIS. Nevertheless, the addition of rhIGF1 with the MIS increased the proportion of oocytes completing GVBD compared with MIS alone, although this potentiation was small and varied greatly among clutches of follicles from fish with oocytes at different stages of germinal vesicle migration. Collectively, these observations suggest IGFs may have synergistic actions with the MIS but cannot induce resumption of meiosis directly at the oocyte and are not potent inducers of OMC in rainbow trout. Rainbow trout are the first teleost fish in which IGFs were found to induce neither OMC nor resumption of meiosis in vitro.

DSC2 suppresses the growth of gastric cancer through the inhibition of nuclear translocation of γ-catenin and PTEN/PI3K/AKT signaling pathway.

Globally, gastric cancer (GC) is still a major leading cause of cancer-associated deaths. Downregulated desmocollin2 (DSC2) is considered to be closely related to tumor progression. However, the underlying mechanisms of DSC2 in GC progression require further exploration. We initially constructed different GC cells based on DSC2 contents, established the mouse tumor xenografts, and subsequently performed clonal formation, MTT, Caspase-3 activity, and sperm DNA fragmentation assays to detect the functions of DSC2 in GC growth. Subsequently, we performed western blot, Co-IP, and immunofluorescence assays to investigate the underlying mechanisms through pretreatment with PI3K inhibitor, LY294002, and its activator, recombinant human insulin-like growth factor I (IGF1). DSC2 could significantly inhibit the viability of GC cells at both in vitro and in vivo levels. The underlying mechanism may be that DSC2 binds the γ-catenin to decrease its nuclear level, thereby downregulating the anti-apoptotic factor BCL-2 expression and upregulating the pro-apoptotic factor P53 expression, which adjusts the PTEN/PI3K/AKT signaling pathway to promote the cancer cell apoptosis. Our finding suggests that DSC2 might be a potential therapeutic target for the treatment of cancers, most especially GC.

Insulin-like growth factor 1 supplementation supports motor coordination and affects myelination in preterm pigs.

Preterm infants have increased risk of impaired neurodevelopment to which reduced systemic levels of insulin-like growth factor 1 (IGF-1) in the weeks after birth may play a role. Hence, we hypothesized that postnatal IGF-1 supplementation would improve brain development in preterm pigs, used as a model for preterm infants. Preterm pigs delivered by cesarean section received recombinant human IGF-1/IGF binding protein-3 complex (rhIGF-1/rhIGFBP-3, 2.25 mg/kg/day) or vehicle from birth to postnatal day 19. Motor function and cognition were assessed by monitoring of in-cage and open field activities, balance beam test, gait parameters, novel object recognition and operant conditioning tests. Collected brains were subject to magnetic resonance imaging (MRI), immunohistochemistry, gene expression analyses and protein synthesis measurements. The IGF-1 treatment increased cerebellar protein synthesis rates (both in vivo and ex vivo). Performance in the balance beam test was improved by IGF-1 but not in other neurofunctional tests. The treatment decreased total and relative caudate nucleus weights, without any effects to total brain weight or grey/white matter volumes. Supplementation with IGF-1 reduced myelination in caudate nucleus, cerebellum, and white matter regions and decreased hilar synapse formation, without effects to oligodendrocyte maturation or neuron differentiation. Gene expression analyses indicated enhanced maturation of the GABAergic system in the caudate nucleus (decreased NKCC1:KCC2 ratio) with limited effects in cerebellum or hippocampus. Supplemental IGF-1 during the first three weeks after preterm birth may support motor function by enhancing GABAergic maturation in the caudate nucleus, despite reduced myelination. Supplemental IGF-1 may support postnatal brain development in preterm infants, but more studies are required to identify optimal treatment regimens for subgroups of very or extremely preterm infants.

Insulin-like growth factor-1 supplementation promotes brain maturation in preterm pigs.

Very preterm infants show low levels of insulin-like growth factor-1 (IGF-1), which is associated with postnatal growth restriction and poor neurological outcomes. It remains unknown whether supplemental IGF-1 may stimulate neurodevelopment in preterm neonates. Using cesarean-delivered preterm pigs as a model of preterm infants, we investigated the effects of supplemental IGF-1 on motor function and regional and cellular brain development. Pigs were treated with 2.25 mg/kg/day of recombinant human IGF-1/IGF binding protein 3 complex from birth until Day 5 or 9 before collection of brain samples for quantitative immunohistochemistry (IHC), RNA-seq and qPCR analyses. Brain protein synthesis was measured using in vivo labeling with [2H5] phenylalanine. We showed that the IGF-1 receptor was widely distributed in the brain and largely coexisted with immature neurons. Region-specific quantification of IHC labeling showed that IGF-1 treatment promoted neuronal differentiation, increased subcortical myelination, and attenuated synaptogenesis in a region- and time-dependent manner. The expression levels of genes involved in neuronal and oligodendrocyte maturation, angiogenic and transport functions were altered, reflecting enhanced brain maturation in response to IGF-1 treatment. Cerebellar protein synthesis was increased by 19% at Day 5 and 14% at Day 9 after IGF-1 treatment. Treatment had no effect on Iba1-positive microglia or regional brain weights and did not affect motor development or the expression of genes related to IGF-1 signaling. In conclusion, the data show that supplemental IGF-1 promotes brain maturation in newborn preterm pigs. The results provide further support for IGF-1 supplementation therapy in the early postnatal period in preterm infants.SIGNIFICANCE STATEMENTDeficiency of systemic insulin-like growth factor-1 (IGF-1) is associated with delayed neurological development in preterm infants. Here, we show that the IGF-1 receptor is primarily expressed in immature neurons in the developing brain of the translational preterm pig model. Supplementation with IGF-1 accelerates neuron differentiation in the hippocampus and promotes myelination in subcortical white matter regions in a time-dependent way. Furthermore, systemic IGF-1 supplementation stimulates cerebral protein synthesis. Our study suggests that IGF-1 therapy in the early postnatal period might be supportive for neurodevelopment in preterm infants.

Hormonal imbalance in patients with chronic renal failure in the pre-dialysis and dialysis periods (part1)

The review describes the problem of studying progressive changes of hormones concentrations (parathyroid hormone, insulin, somatotropin, prolactin) in patients with chronic renal failure on the pre-dialysis and dialysis stages. The pathogenetic relationships between kidney function deterioration and hormone concentrations as well as changes of their biological effects were evaluated. Parathyroid hormone is considered as an uraemic toxin, since its concentration in the blood begins to increase when the glomerular filtration rate decreases below 50 ml/min. All stages of chronic kidney disease are accompanied by disorders of calcium-phosphorus metabolism. Prolonged excess of parathyroid hormone leads to bone loss and to the progression to secondary hyperparathyroidism that is a frequent complication in patients with the later stages of chronic renal failure and, especially, in those on dialysis treatment. The elevation of insulin level in chronic renal failure is the consequence of progressive decrease in glomerular filtration rate and insulin excretion by proximal tubules. So, it results in insulin half-life prolongation. Long-term dialysis therapy eliminates factors that reduce the degradation of insulin by extrarenal tissues, which results in an improvement of their insulin sensitivity. Experimental and clinical studies have shown that an excess of somatotropin can adversely affect the kidneys that leads to glomerular hyperfiltration and the progression to glomerulosclerosis. The risk of possible side effects on kidneys should be taken into account when prescribing recombinant human insulin-like growth factor. The prolactin concentration is usually increased in chronic kidney disease due to reduced clearance and increased secretion. Hyperprolactinemia manifests as galactorrhea and hypogonadism. Dialysis therapy can’t normalize the increased concentration of prolactin. Modern options for pathogenetic treatment of endocrine disorders in patients with chronic renal failure are outlined in this article. It was found that kidneys play an important role in regulating hormones concentrations in the blood. Endocrine disorders are one of the most important components of the uraemic syndrome, which requires further clinical studies, aimed on the searching of better treatment strategies and prevention of hormonal imbalance on the pre-dialysis and dialysis stages of chronic kidney disease.

Insulin-like Growth Factor-1 Prevents Hypoxia/Reoxygenation-Induced White Matter Injury in Sickle Cell Mice

Occlusion of cerebral blood vessels causes acute cerebral hypoxia-an important trigger of ischemic white matter injury and stroke in sickle cell disease (SCD). While chronic hypoxia triggers compensatory neuroprotection via insulin-like growth factor-1 (IGF-1) and hypoxia inducible factor-1α (HIF-1α), severe bouts of acute hypoxia and subsequent restoration of blood flow (hypoxia/reoxygenation, H/R) overwhelm compensatory mechanisms and cause neuroaxonal damage-identified as white matter lesions-in the brain. The neuroprotective role of IGF-1 in the pathogenesis of white matter injury in SCD has not been investigated; however, it is known that systemic IGF-1 is reduced in individuals with SCD. We hypothesized that IGF-1 supplementation may prevent H/R-induced white matter injury in SCD. Transgenic sickle mice homozygous for human hemoglobin S and exposed to H/R developed white matter injury identified by elevated expression of non-phosphorylated neurofilament H (SMI32) with a concomitant decrease in myelin basic protein (MBP) resulting in an increased SMI32/MBP ratio. H/R-challenge also lowered plasma and brain IGF-1 expression. Human recombinant IGF-1 prophylaxis significantly induced HIF-1α and averted H/R-induced white matter injury in the sickle mice compared to vehicle-treated mice. The expression of the IGF-1 binding proteins IGFBP-1 and IGFBP-3 was elevated in the IGF-1-treated brain tissue indicating their potential role in mediating neuroprotective HIF-1α signaling. This study provides proof-of-concept for IGF-1-mediated neuroprotection in SCD.

Insulin-like growth factor 1 reduces coronary atherosclerosis in pigs with familial hypercholesterolemia.

Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque's fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis, consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress, and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis showed that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1's effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.

Elevated Insulin-Like Growth Factor-1-Induced Female Rats Perpetuate the Polycystic Ovary Syndrome Phenotype: Pathological Mechanism of Insulin-Like Growth Factor-1 in Polycystic Ovary Syndrome

Objectives: This study was designated to establish a polycystic ovary syndrome (PCOS) rat model with recombinant human insulin-like growth factor-1 (RH-IGF-1). We made assessment on the characteristics of hyperinsulinemia and hyperandrogenism in the rat model. Design: This study performed the characteristics of PCOS upon RH-IGF-1 injection and evaluated the disease process of PCOS syndrome caused by the insulin-resistant pathological condition of IGF-1 based on the comparative study of in vivo test. Setting: The experiment was conducted in the experimental research center of Yinzhou NO.2 hospital, Ningbo, Zhejiang Province, China. Materials and Methods: Thirty-four female Sprague Dawley immature rats aged 21 days were randomly divided into two groups. Those treated with RH-IGF-1 2 mg/100 g daily were in RH-IGF-1 group (n = 20), and those with 0.9% sodium chloride 0.2 mL/100 g daily were in the saline group (n = 14). The experiment was carried out in two stages. In stage I, rats were anesthetized upon the first estrous cycle in the saline group with tissue and blood samples collected (n = 7), and rats in the RH-IGF-1-treated group were anesthetized on the 5th day after vaginal opening (VO) (n = 10). In stage II, rats in the saline group were anesthetized after three complete cycles (n = 7), meanwhile, while on the 15th day after VO (n = 10) for those in the RH-IGF-1 group. Results: We have found that compared with the control group, rats injected with RH-IGF-1 expressed an early VO, disordered estrous cycle, polycystic ovaries, and significantly increased ovarian weight/body weight ratio. And from the perspective of hormone secretion, their androgen increased significantly and the insulin resistance index also elevated distinctly, possessing main characteristics similar to PCOS. Limitations: In this study, we were limited by the inability to examine IGF-1 in hypothalamus. IGF-1 in hypothalamus and in vitro experiments would be taken into consideration for further study in the future. Conclusions: These findings suggest that IGF-1 may be a key factor in the pathogenesis of PCOS, and the increase of androgen may be the pathological result, not the cause of PCOS.

Five-Year Therapy with Recombinant Human Insulin-Like Growth Factor-1 in a Patient with PAPP-A2 Deficiency

Introduction: The metalloproteinase pregnancy-associated plasma protein A2 (PAPP-A2) cleaves insulin-like growth factor (IGF)-binding proteins 3 and 5 to release bioactive IGF-I from its ternary complex. Patients with mutations in PAPP-A2 have growth failure and low free IGF-I despite elevated total IGF-I. We describe 5-year treatment response to recombinant human IGF-1 (rhIGF-1) in a patient with PAPP-A2 deficiency, and the phenotype of PAPP-A2 deficiency in three siblings. Methods: Two siblings (P2, P3) with PAPP-A2 deficiency were recruited for rhIGF-1 therapy at 120 μg/kg subcutaneous twice daily, along with a third sibling (P1) for phenotyping. We evaluated efficacy and safety of rhIGF-1 therapy, including effect on metabolic measures and bone mineral density (BMD). Results: Treatment with rhIGF-1 was started in 10.4-year- (P3) and 14.5-year (P2)-old brothers. P2 discontinued therapy due to pseudotumor cerebri. P3 continued rhIGF-1 for 5 years; height velocity increased (3.0 cm/year at baseline; 5.0–7.6 cm/year thereafter) as did height SDS (+0.6). P3’s pubertal onset was at 12.4 year. BMD height-adjusted Z-score modestly improved for lumbar spine (+0.4), and decreased in forearm (−0.2) and hip (−0.3). All siblings had hyperinsulinemia. Impaired glucose tolerance (IGT) resolved in P1. P2 showed worsening glucose tolerance (2-h glucose: 225 mg/dL). Impaired fasting glucose and hyperinsulinemia initially resolved for P3, but IGT (2-h glucose: 152 mg/dL) developed during puberty. Conclusion: Therapy with rhIGF-1 modestly improved linear growth in one patient with PAPP-A2 deficiency, but without true catch-up. Therapy was associated with pseudotumor cerebri in a sibling. Initial improvement in BMD and glycemic pattern on rhIGF-1 was not sustained during puberty.

Insulin-Like Growth Factor1 Preserves Gastric Pacemaker Cells and Motor Function in Aging via ERK1/2 Activation

Impaired gastric motor function in the elderly causes reduced food intake leading to frailty and sarcopenia. We previously found that aging-related impaired gastric compliance was mainly owing to depletion of interstitial cells of Cajal (ICC), pacemaker cells, and neuromodulator cells. These changes were associated with reduced food intake. Transformation-related protein 53-induced suppression of extracellular signal-regulated protein kinase (ERK)1/2 in ICC stem cell (ICC-SC) cell-cycle arrest is a key process for ICC depletion and gastric dysfunction during aging. Here, we investigated whether insulin-like growth factor 1 (IGF1), which can activate ERK in gastric smooth muscles and invariably is reduced with age, could mitigate ICC-SC/ICC loss and gastric dysfunction in klotho mice, a model of accelerated aging. Klotho mice were treated with the stable IGF1 analog LONG R3 recombinant human (rh) IGF1 (150 μg/kg intraperitoneally twice daily for 3 weeks). Gastric ICC/ICC-SC and signaling pathways were studied by flow cytometry, Western blot, and immunohistochemistry. Gastric compliance was assessed in exvivo systems. Transformation-related protein 53 was induced with nutlin 3a and ERK1/2 signaling was activated by rhIGF-1 in the ICC-SC line. LONG R3 rhIGF1 treatment prevented reduced ERK1/2 phosphorylation and gastric ICC/ICC-SC decrease. LONG R3 rhIGF1 also mitigated the reduced food intake and impaired body weight gain. Improved gastric function by LONG R3 rhIGF1 was verified by invivo systems. In ICC-SC cultures, rhIGF1 mitigated nutlin 3a-induced reduced ERK1/2 phosphorylation and cell growth arrest. IGF1 can mitigate age-related ICC/ICC-SC loss by activating ERK1/2 signaling, leading to improved gastric compliance and increased food intake in klotho mice.

Lab-scale Preparation of Recombinant Human Insulin-like Growth Factor-1 in Escherichia coli and its Potential Safety on Normal Human Lung Cell Line.

Insulin-like growth factor-1 (IGF-1) is structurally similar to insulin and acts as an endocrine hormone secreted by the liver. Production of recombinant human IGF-1 (rhIGF-1) in Escherichia coli (E.coli) and evaluation of its proliferation stimulatory activity. hIGF-1 gene cloned into pBSK (+) simple vector was transformed into TOP 10 chemically competent cells of E. coli. Polymerase chain reaction (PCR) was achieved using specific hIGF-1 gene primers to confirm the successful transformation. To express the rhIGF-1 in E. coli (Rosetta (DE3) pLysS); the hIGF-1 gene was cloned into the pET-15b expression vector and then the recombinant pET-15b/IGF-1 vector was transformed into a chemically prepared competent expression bacterial cells; Rosetta (DE3) pLysS. The rhIGF-1 was expressed as insoluble aggregates called inclusion bodies (IBs) using a 2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer. IBs were solubilized in a denatured form using 6 M guanidinium hydrochloride (GdmCl), followed by in vitro protein refolding using the rapid dilution method. The refolded hIGF-1 was purified using the HiTrap- ANX anion exchange column. Western blot and ELISA using rabbit polyvalent anti-hIGF- 1 were performed to confirm the protein antigenic identity. Cell proliferation activity of rhIGF-1 was testified on normal human lung cell line (WI-38). rhIGF-1 was purified from the HiTrap-ANX column at a concentration of 300 μg/ml. Western blot showed a single 7.6 kDa band obtained in the induced Rosetta (DE3) pLYsS. ELISA confirmed the molecular identity of the rhIGF-1 epitope, the concentration of purified rhIGF-1 obtained from the ELISA standard curve using rhIGF-1 reference protein as a standard was 300 μg/ml, and activity on WI-38 cells was 2604.17I U/mg. Biologically active native rhIGF-1 protein was successfully expressed. Patents related to the preparation of IGF-1 were mentioned along the text.

Pilot dose-ranging of rhIGF-1/rhIGFBP-3 in a preterm lamb model of evolving bronchopulmonary dysplasia.

Low levels of insulin-like growth factor-1 (IGF-1) protein in preterm human infants are associated with bronchopulmonary dysplasia (BPD). We used our preterm lamb model of BPD to determine (1) dosage of recombinant human (rh) IGF-1 bound to binding protein-3 (IGFBP-3) to reach infant physiologic plasma levels; and (2) whether repletion of plasma IGF-1 improves pulmonary and cardiovascular outcomes. Group 1: normal, unventilated lambs from 128 days gestation through postnatal age 5 months defined normal plasma levels of IGF-1. Group 2: continuous infusion of rhIGF-1/rhIGFBP-3 (0.5, 1.5, or 4.5 mg/kg/day; n = 2) for 3 days in mechanically ventilated (MV) preterm lambs determined that 1.5 mg/kg/day dosage attained physiologic plasma IGF-1 concentration of ~125 ng/mL, which was infused in four more MV preterm lambs. Group 1: plasma IGF-1 protein increased from ~75 ng/mL at 128 days gestation to ~220 ng/L at 5 months. Group 2: pilot study of the optimal dosage (1.5 mg/kg/day rhIGF-1/rhIGFBP-3) in six MV preterm lambs significantly improved some pulmonary and cardiovascular outcomes (p < 0.1) compared to six MV preterm controls. RhIGF-1/rhIGFBP-3 was not toxic to the liver, kidneys, or lungs. Three days of continuous iv infusion of rhIGF-1/rhIGFBP-3 at 1.5 mg/kg/day improved some pulmonary and cardiovascular outcomes without toxicity. Preterm birth is associated with rapid decreases in serum or plasma IGF-1 protein level. This decline adversely impacts the growth and development of the lung and cardiovascular system. For this pilot study, continuous infusion of optimal dosage of rhIGF-1/rhIGFBP-3 (1.5 mg/kg/day) to maintain physiologic plasma IGF-1 level of ~125 ng/mL during mechanical ventilation for 3 days statistically improved some structural and biochemical outcomes related to the alveolar formation that would favor improved gas exchange compared to vehicle-control. We conclude that 3 days of continuous iv infusion of rhIGF-1/rhIGFBP-3 improved some physiological, morphological, and biochemical outcomes, without toxicity, in mechanically ventilated preterm lambs.