InsR Expression Research Articles

PiRNA array analysis provide insight into the mechanism of DEHP-induced testicular toxicology in pubertal male rats

Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, could cause male reproductive toxicity by disrupting spermatogenesis. Piwi-interacting RNAs (piRNAs) are a small non-coding RNAs specifically highly expressed in the germline and interact with PIWI proteins to regulate spermatogenesis. Accumulating studies have confirmed that environmental poisons could induce male reproductive injury via altering piRNA expression. However, it remains unclear whether DEHP causes male reproductive dysfunction by perturbing piRNA levels. In this study, we conducted piRNA microarray expression analyses on testes of DEHP-exposed and control male rats and performed some in vitro and in vivo studies to explore the role of piRNA on DEHP-induced male reprouctive toxicity. Our results showed that DEHP exposure leaded to changed expression profile of piRNAs in pubertal male rat testes. And bioinformatics analyses revealed that down-regulated piR-rno-26751 probably targeted Insr mRNA expression regulation. Results from gene and protein expression tests demonstrated that DEHP caused decreased expression of INSR mainly in spermatogonia. Moreover, MEHP, the main metabolite of DEHP resulted in cell apoptosis and down-regulation of INSR and its downstream p-IRS1, p-PI3K, p-AKT and p-FOXO1 in GC-1spg cells. Conversely, overexpression of INSR restored cell apoptosis the down-regulation of the above proteins in GC-1spg cells. In conclusion, these findings suggest that DEHP-induced down-regulation of piR-rno-26751 targets the suppression of INSR, leading to apoptosis of spermatogonia in pubertal male rats.

KLF3 impacts insulin sensitivity and glucose uptake in skeletal muscle.

Skeletal muscle is one of the predominant sites involved in glucose disposal, accounting for approximately 80% of postprandial glucose uptake, and plays a critical role in maintaining glycemic homeostasis. Dysregulation of energy metabolism in skeletal muscle is involved in developing insulin resistance and type 2 diabetes (T2D). Transcriptomic responses of skeletal muscle to exercise found that the expression of Klf3 was increased in T2D Goto-Kakizaki (GK) rats and decreased after exercise with improved hyperglycemia and insulin resistance, implying that Klf3 might be associated with insulin sensitivity and glucose metabolism. We also found that knockdown of Klf3 promoted basal and insulin-stimulated glucose uptake in L6 myotubes, while overexpression of Klf3 resulted in the opposite. Through pairwise comparisons of L6 myotubes transcriptome, we identified 2256 and 1988 differentially expressed genes in Klf3 knockdown and overexpression groups, respectively. In insulin signaling, the expression of Slc2a4, Akt2, Insr and Sorbs1 was significantly increased by Klf3 knockdown and decreased with klf3 overexpression; Ptprf and Fasn were markedly downregulated in klf3 reduced group and upregulated in klf3 overexpressed group. Moreover, downregulation of Klf3 promoted the expression of GLUT4 and AKT proteins, as well as the translocation of GLUT4 to the cell membrane in the basal situation, and enhanced insulin sensitivity, characterized by increased insulin-stimulated GLUT4 translocation and AKT, TBC1D1 and TBC1D4 phosphorylation, while overexpression of Klf3 showed contrary results. These results suggest that Klf3 affects glucose uptake and insulin sensitivity via insulin signal transduction and intracellular metabolism, offering a novel potential treatment strategy for T2D.

Huangqin-Huanglian Decoction Protects Liver against Non-alcoholic Fatty Liver Disease in High Fat-diet Mice.

Traditional Chinese medicine (TCM) has the advantage of low toxicity of natural ingredients, multiple targets and effects, and low medication costs. It has unique advantages for metabolic and chronic diseases. Huangqin-Huanglian decoction (HQHLD) is composed of Scutellariae Radix, Coptidis Rhizoma, Rehmanniae Radix, and Gentianae Radix Et Rhozima; it has great potential for the treatment of NAFLD with the modern pharmacological research and TCM theory, but there is still a relative lack of research on the potential targets and pharmacological effects of HQHLD. In this work, we have used network pharmacology to predict the targets and signaling pathways of HQHLD, and validated NAFLD-related targets using the HFD model in order to explore more therapeutic drugs and methods for NAFLD. We collected the HQHLD ingredients and NAFLD targets through TCMSP, ETCM, DisGeNET, HGMD, MalaCards, OMIM, and TTD, built ingredients-target networks by Cytoscape, and screened key ingredients in HQHLD. DAVID and Metascape databases were used for GO functional enrichment analysis and KEGG pathway enrichment analysis, respectively. Molecular docking of the key ingredients and key targets was performed by AutoDock. We verified the effect of HQHLD on high-fat diet (HFD) mice by measuring the weight, liver weight index, and the level of TG, TC, LDL-C, and HDLC. HE staining and oil-red staining were performed to detect the damage and fat accumulation in the liver. The changes in INSR, PPAR-α, PPAR-γ, TNF-α, and caspase3 were experimented with WB. With the network pharmacology analysis, we found quercetin, baicalein, sitosterol, wogonin, oroxylin-A, glycyrrhizin, hydroberberine, berberine, sesamin, and carotene to be the main ingredients in HQHLD. According to KEGG pathway analysis, INSR, AKT, JNK1, PPAR-α, PPAR-γ, and the other 16 targets are the main targets of HQHLD in the treatment of NAFLD. We took HFD mice as the in vivo model of NAFLD. Our results showed that HQHLD could reduce liver weight, and TG and LDL-C levels, and increase HDL-C level in serum. By HE and oil red staining, we found that HQHLD could protect the morphology of hepatocytes and reduce fat in the liver. We also found HQHLD to protect the liver by increasing the expression of INSR and PPAR-α, and reducing the expression of PPAR-γ, TNF-α, and caspase3 in the liver. In conclusion, our study has firstly studied the main ingredients and key targets of HQHDL in treating NAFLD by network pharmacology analysis, and preliminarily confirmed that HQHLD could alleviate NAFLD in a multi-target way by lowering fatty acids, and decreasing insulin resistance, inflammation, and apoptosis in the liver.

Abstract 7191: Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma

Abstract Background: Resistance to proteasome inhibitors (PI) bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of multiple myeloma (MM); thus, identification of novel therapeutic options is an unmet medical need. ALK inhibitors have been shown to exhibit anti-MM activity in monotherapy or in combination with CFZ. Our data showed that MM cells were negative for ALK; thus, we aimed to identify the mechanism of action of the ALK inhibitor ceritinib in MM cells and the mechanism of synergy between ceritinib and CFZ. Methods: A set of PI-naïve and PI-resistant MM cells was used in this study. Genome-wide CRISPR/Cas9-based screening using the Brunello library was performed in the AMO-1 cell line. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30 database. RNA sequencing was used to determine expression changes after treatment, and RNA-seq data from patients included in the CoMMpass study were analyzed. Western blotting was performed to assess the levels of total and phosphorylated protein. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and CFZ on the intracellular metabolites. An in vivo mouse model based on the orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Among approved ALK inhibitors, ceritinib has been identified as the most synergistic cytotoxic combination with CFZ in PI-naïve and PI-resistant MM cells. An in silico search of the ChEMBL database identified InsR and IGF1R as receptor tyrosine kinase that are inhibited by ceritinib. PI-naïve, PI-resistant cell lines, as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and the transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, FOXO1 inhibition using a selective chemical inhibitor protected the cells from ceritinib-induced cytotoxicity. Next, ceritinib inhibited mTORC signaling and induced the expression of genes related to cell cycle arrest, which are downstream of FOXO1. Likewise, ceritinib, by targeting the InsR/IGF1R, impaired purine-pyrimidine and amino acid homeostasis, suggesting metabolic and proliferation shut-down and amino acid starvation due to downstream mTORC and Akt inhibition and FOXO1 induction. The combination of ceritinib and CFZ was superior to CFZ in PI-resistant MM in vivo and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by inhibiting InsR/IGF1R and downstream Akt signaling. Therefore, ceritinib is a promising treatment option for PI-resistant MM. Supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Andrej Besse, Tiberiu Totu, Marianne Kraus, Anthonius P. Janssen, Jana Veprkova, Max Mendez Lopez, Ondrej Slaby, Marija Buljan, Mario van der Stelt, Christoph Driessen, Lenka Besse. Targeting IGF1R/INSR pathway with approved ALK inhibitors inhibits AKT signaling and overcomes proteasome inhibitor resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7191.

A high fat diet potentiates neonatal iron overload-induced memory impairments in rats.

The present study aimed at evaluating possible synergistic effects between two risk factors for cognitive decline and neurodegenerative disorders, i.e. iron overload and exposure to a hypercaloric/hyperlipidic diet, on cognition, insulin resistance, and hippocampal GLUT1, GLUT3, Insr mRNA expression, and AKT phosporylation. Male Wistar rats were treated with iron (30mg/kg carbonyl iron) or vehicle (5% sorbitol in water) from 12 to 14th post-natal days. Iron-treated rats received a standard laboratory diet or a high fat diet from weaning to adulthood (9months of age). Recognition and emotional memory, peripheral blood glucose and insulin levels were evaluated. Glucose transporters (GLUT 1 and GLUT3) and insulin signaling were analyzed in the hippocampus of rats. Both iron overload and exposure to a high fat diet induced memory deficits. Remarkably, the association of iron with the high fat diet induced more severe cognitive deficits. Iron overload in the neonatal period induced higher insulin levels associated with significantly higher HOMA-IR, an index of insulin resistance. Long-term exposure to a high fat diet resulted in higher fasting glucose levels. Iron treatment induced changes in Insr and GLUT1 expression in the hippocampus. At the level of intracellular signaling, both iron treatment and the high fat diet decreased AKT phosphorylation. The combination of iron overload with exposure to a high fat diet only led to synergistic deleterious effect on emotional memory, while the effects induced by iron and by the high fat diet on AKT phosphorylation were comparable. These findings indicate that there is, at least to some extent, an additive effect of iron combined with the diet. Further studies investigating the mechanisms associated to deleterious effects on cognition and susceptibility for the development of age-associated neurodegenerative disorders are warranted.

Abstract B055: Hyperinsulinemia acts via acinar cell insulin receptors to drive obesity-associated pancreatic cancer initiation

Abstract The rising incidence of pancreatic cancer is largely driven by increased prevalence of obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of obesity and T2D, and is associated with increased cancer incidence and mortality. Whether insulin alone plays a causal role in increasing cancer risk by directly affecting the tumor cell of origin remains unclear. Our previous studies demonstrated that genetically reducing production of insulin suppressed formation of pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice with mutant Kras. However, consistent with a role for insulin in modulating the immune system, we saw changes in fibrosis, as well as changes in the transcriptomes of immune cells in the pancreas in mice with reduced insulin. Thus, it remained unclear whether hyperinsulinemia exerted its effects directly on the cells that give rise to PanINs or indirectly on the tumor microenvironment. Here, we tested whether insulin receptor (Insr) in KrasG12D-expressing pancreatic acinar cells was necessary for the effects of diet-induced hyperinsulinemia on pancreatic cancer development. Loss of Insr in KrasG12D-expressing acinar cells did not halt the development of hyperinsulinemia or weight gain associated with high fat diet consumption in mice. However, solely reducing Insr expression in KrasG12D-expressing acinar cells significantly reduced formation of PanIN and tumors. Mechanistically, proteomic analyses suggested that insulin and Insr coordinately regulate the production of digestive enzymes, the main function of acinar cells, by modulating the activity of the spliceosome, ribosome, and secretory machinery. Consistent with this adding insulin to TGF-alpha treated wild-type acini ex vivo increased their conversion into ductal structures and this was blocked by treatment with trypsin inhibitor. Additionally, loss of Insr also reduced the formation of duct-like structures in acinar explants after TGF-alpha stimulation and made them insensitive to insulin. Collectively, these data demonstrated that insulin action on the insulin receptor in acinar cells promotes conditions that cooperate with Kras signaling to increase the risk of developing pancreatic cancer especially in the context of hyperinsulinemia and obesity. Citation Format: Anni M.Y. Zhang, Yi Han Xia, Jeffrey S.H. Lin, Ken H. Chu, Wei Chuan K. Wang, Titine J.J. Ruiter, Jenny C.C. Yang, Nan Chen, Justin Chhuor, Shilpa Patil, Haoning H. Cen, Elizabeth J. Rideout, Vincent R. Richard, David F. Schaeffer, Rene P. Zahedi, Christoph H. Borchers, James D. Johnson, Janel L. Kopp. Hyperinsulinemia acts via acinar cell insulin receptors to drive obesity-associated pancreatic cancer initiation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B055.

Cortisol controls endoplasmic reticulum stress and hypoxia dependent regulation of insulin receptor and related genes expression in HEK293 cells.

Objective. Glucocorticoids are important stress-responsive regulators of insulin-dependent metabolic processes realized through specific changes in genome function. The aim of this study was to investigate the impact of cortisol on insulin receptor and related genes expression in HEK293 cells upon induction the endoplasmic reticulum (ER) stress by tunicamycin and hypoxia. Methods. The human embryonic kidney cell line HEK293 was used. Cells were exposed to cortisol (10 µM) as well as inducers of hypoxia (dimethyloxalylglycine, DMOG; 0.5 mM) and ER stress (tunicamycin; 0.2 µg/ml) for 4 h. The RNA from these cells was extracted and reverse transcribed. The expression level of INSR, IRS2, and INSIG2 and some ER stress responsive genes encoding XBP1n, non-spliced variant, XBP1s, alternatively spliced variant of XBP1, and DNAJB9 proteins, was measured by quantitative polymerase chain reaction and normalized to ACTB. Results. We showed that exposure of HEK293 cells to cortisol elicited up-regulation in the expression of INSR and DNAJB9 genes and down-regulation of XBP1s, XBP1n, IRS2, and INSIG2 mRNA levels. At the same time, induction of hypoxia by DMOG led to an up-regulation of the expression level of most studied mRNAs: XBP1s and XBP1n, IRS2 and INSIG2, but did not change significantly INSR and DNAJB9 gene expression. We also showed that combined impact of cortisol and hypoxia introduced the up-regulation of INSR and suppressed XBP1n mRNA expression levels. Furthermore, the exposure of HEK293 cells to tunicamycin affected the expression of IRS2 gene and increased the level of XBP1n mRNA. At the same time, the combined treatment of these cells with cortisol and inductor of ER stress had much stronger impact on the expression of all the tested genes: strongly increased the mRNA level of ER stress dependent factors XBP1s and DNAJB9 as well as INSR and INSIG2, but down-regulated IRS2 and XBP1n. Conclusion. Taken together, the present study indicates that cortisol may interact with ER stress and hypoxia in the regulation of ER stress dependent XBP1 and DNAJB9 mRNA expression as well as INSR and its signaling and that this corticosteroid hormone modified the impact of hypoxia and especially tunicamycin on the expression of most studied genes in HEK293 cells. These data demonstrate molecular mechanisms of glucocorticoids interaction with ER stress and insulin signaling at the cellular level.

In-silico Study of Epicathecin from Cinnamomum verum Against Insulin Receptor for Diabetes Mellitus

Obesity has become more common in recent years. Fatalism can raise the chance of fatal metabolic and cardiovascular diseases. Type 2 diabetes is one of the metabolic illnesses brought on by fat. Insulin receptor resistance, also known as non-insulin dependent type 2 diabetes, is a consequence of type 2 diabetes. Reduced insulin sensitivity and the inability of pancreatic beta cells to produce enough insulin to offset the development of resistance are the main causes of type 2 diabetes mellitus. Insulin receptor problems that decrease INSR expression on the cell surface and follow receptor abnormalities are the common mechanisms behind insulin receptor resistance. The single-nucleotide found in INSR. Increasing the expression of 4IBM is one strategy to combat its effects on insulin receptor function and increased risk of INSR-mediated illnesses. Based on earlier studies, cinnamon (Cinnamomum verum), a traditional plant, has been shown to have potential as a treatment for a number of illnesses, including diabetes mellitus. Using Autodock 4 and the Lamarckian genetic algorithm as a basis, this in-silico study intends to investigate the potential of active compounds found in cinnamon and their role as 4IBM protein inhibitors for therapy in type 2 diabetes mellitus. The binding energy ranged from -7.95 to -6.15 kcal/mol, according to docking data, with the compound epicatechin having the lowest binding energy and an inhibitory constant of 7.80. Further investigation into the active ingredients in cinnamon and their potential use in the treatment of diabetes mellitus can be based on the findings of this study.

Targeting IGF1R/Insr Pathway with Approved ALK-Inhibitors Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Background: The treatment of multiple myeloma (MM) has advanced rapidly with the discovery of proteasome inhibitors (PIs) bortezomib (BTZ) and carfilzomib (CFZ). PIs disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in MM cells, leading to apoptosis. However, despite this success, essentially all patients with MM eventually develop resistance to PIs. Therefore, identifying strategies to overcome PIs resistance is clinically important. ALK inhibitors exhibit anti-MM activity ex vivo. Previously, the ALK inhibitor ceritinib has been shown to inhibit IGF1R/InsR signaling. Signaling through the IGF1/IGF1R axis contributes to acquired resistance to BTZ, and PI activity is reduced in the presence of IGF1, suggesting that IGF1R inhibitors may enhance the cytotoxic effect of PIs. To date, IGF1R/InsR inhibitors have not been successful as single agents in clinical trials and no formally approved drugs are available. Importantly, the drug repurposing approach offers a promising strategy for drug development, particularly for MM. Therefore, we aimed to identify i) whether ALK inhibitors are cytotoxic in MM by targeting the IGF1R/InsR pathway, and ii) whether they can be combined with PIs to overcome PI resistance in vitro and in vivo. Methods: A set of PI-naïve and PI-resistant cells was used in this study. The cytotoxicity of drugs was determined by CCK8 assay in cell lines and by CellTiter-Glo assay in primary cells. Genome-wide CRISPR/Cas9-based loss-of-function screening using the Brunello library was performed in the AMO-1 cell line to identify the mechanism of action of ceritinib. Kinase inhibitor selectivity data were retrieved from ChEMBL, v30. Western blotting was performed to assess the levels of total and phosphorylated proteins. RNA sequencing was used to determine the number of transcripts in PI-sensitive and PI-resistant cells, and RNA-seq data from patients included in the CoMMpass study were analyzed. Unbiased LC-MS/MS was performed to determine the effects of ceritinib and carfilzomib on intracellular metabolites. An in vivo mouse model based on orthotopic injection of AMO-BTZ cells into the femur of NSG mice was used to determine the effect of the drug combination in vivo. Results: Initially, seven ALK inhibitors were tested in PI-naïve and PI-adapted MM cell lines. Based on IC 50 values, the most effective ALK inhibitors to induce cytotoxicity in MM were ceritinib > brigatinib > entrectinib. The combination of ceritinib, brigatinib, and entrectinib showed synergistic cytotoxicity with PI BTZ and CFZ and overcame PI-resistance in four different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in the negative regulation of mTORC signaling (DDIT4, NPRL2/3, TSC1/2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling and impaired purine-pyrimidine and amino acid production pathways, suggesting a metabolic and proliferation shut-down and amino acid starvation due to targeting of the upstream receptor tyrosine kinases (RTK), which are essential for cell viability and thus not identified by CRISPR screening. Further search in the ChemBL database identified InsR and IGF1R, as RTK that was potently inhibited by ceritinib. PI-naïve and PI-resistant cell lines as well as MM patients were negative for ALK, but positive for InsR and IGF1R expression. Accordingly, co-treatment of PI-resistant cells with a combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Importantly, the combination of ceritinib and CFZ was superior to CFZ in an orthotopic mouse model bearing PI-resistant MM cells and showed strong synergistic cytotoxicity in primary cells from MM patients progressing under or after PI-containing therapy. Conclusion: Ceritinib, an FDA-approved drug, overcomes PI resistance in MM by targeting InsR/IGF1R signaling, which is essential for PI resistance in MM. Therefore, ceritinib represents a promising, potential option for the treatment of PI-resistant MM.

Association between diabetes and stress-induced hyperglycemia with skeletal muscle gene expression of INSR in critically ill patients: A prospective cohort study

This study aimed to investigate the association between diabetes and stress-induced hyperglycemia with skeletal muscle gene expression of INSR of critically ill patients. Skeletal muscle biopsies were prospectively taken from the vastus muscle, and the expression level of INSR was analyzed using RT-qPCR. Fifty patients were included from April 2018 to September 2018. No significant differences in skeletal muscle gene expression were found between patients with or without diabetes. Similarly, there were no differences in gene expression between groups according to the presence of hypoglycemia 〈 70 mg/dl or hyperglycemia 〉 140 mg/dl. Patients with glycemic variability ≥ 40 mg/dl exhibited a downregulation of INSR compared to those with glycemic variability < 40 mg/dl (1.3 [0.01–5] vs. 2.1 [0.7 - 3.4] fold-changes, P = 0.045). The same pattern was observed when glycemic gap threshold of 80 mg/dl was used (1.4 [0.25–5] vs 1 [0.01 - 2.3] fold-changes in patients with glycemic gap < 80 mg/dl and glycemic gap ≥ 80 mg/dl respectively, P = 0.015). In conclusion, INSR was downregulated in the skeletal muscle of critically ill patients with stress-induced hyperglycemia.

Changes of insulin receptors in high fat and high glucose diet mice with insulin resistance

ABSTRACT This study aimed to observe the expression of insulin-signaling molecules in different organs of mice with insulin resistance (IR). Firstly, mice were fed a high-fat and high-sugar diet (HF group) to establish an IR model, and the controls (NF group) were fed with a normal diet. Next, the weight, fasting blood glucose (FBG), serum insulin and insulin tolerance were detected. Pathological changes of liver tissues were observed by H&E staining. The expressions of INSR, IRS-1 and IRS-2 in the liver, skeletal muscle and ovary were measured by qRT-PCR and western blotting. As a result, compared with the NF group, the HF group mice had increased weight, FBG, insulin and IR index after 6-week of feeding as well as a worse performance in the insulin tolerance test and H&E staining showed fatty liver-like changes after 12-week of feeding, exhibited lower expression of INSR, IRS-1 and IRS-2 in the liver of mice at 6 and 12 weeks. The expression of INSR and IRS-1 in skeletal muscle tissues exhibited the same trend, while those in ovary organs showed the opposite trend. These results suggested that the insulin signaling alters in the liver, skeletal muscle and ovary organs with the progress of IR.

Molecular Characteristics of Everolimus-resistant Renal Cell Carcinoma Cells Generated by Continuous Exposure to Everolimus.

Mammalian target of rapamycin (mTOR) inhibitors represent the standard of care for metastatic renal cell carcinoma (RCC). However, treatment outcomes are relatively poor, suggesting a potential problem with tolerating mTOR inhibitors. The aim of this study was to establish everolimus-resistant sublines and to compare their molecular characteristics with those of their counterparts. Human-derived RCC, Caki-2, and 786-O cells were continuously exposed to everolimus at 1 μM, and the established resistant sublines were designated as Caki/EV and 786/EV, respectively. Cellular characteristics were compared between both cells. Caki/EV and 786/EV cells showed a decrease in sensitivity to everolimus as well as other mTOR inhibitors. Expression of mTOR and its effectors exhibited no alteration in resistant sublines and their counterparts. However, phosphorylation of S6K, an index of mTOR activity, decreased in resistant sublines. PCR array analysis of mTOR signaling pathway-related factors indicated that the expression of INSR, TP53, and IGFBP3 increased in Caki/EV cells, whereas that of TELO2, HRAS, and SGK1 was up-regulated in 786/EV cells. The levels of DDIT4, DEPTOR, HIF1A, and PLD1 mRNAs decreased in both cell lines. The novel everolimus-resistant Caki/EV and 786/EV cells exhibited cross-resistance to other mTOR inhibitors and decreased mTOR activity. Furthermore, down-regulation of DDIT4, DEPTOR, HIF1A, and PLD1 may contribute to everolimus resistance.

Aerobic exercise improves cognitive impairment in mice with type 2 diabetes by regulating the MALAT1/miR-382-3p/BDNF signaling pathway in serum-exosomes

BackgroundIt has been documented that aerobic exercise (AE) has a positive effect on improving cognitive function in type 2 diabetes (T2DM) patients. Here, we tried to explore how AE regulates the expression of long non-coding RNA in serum-exosomes (Exos), thereby affecting cognitive impairment in T2DM mice as well as its potential molecular mechanism.MethodsT2DM mouse models were constructed, and serum-Exos were isolated for whole transcriptome sequencing to screen differentially expressed lncRNA and mRNA, followed by prediction of downstream target genes. The binding ability of miR-382-3p with a long non-coding RNA MALAT1 and brain-derived neurotrophic factor (BDNF) was explored. Then, primary mouse hippocampal neurons were collected for in vitro mechanism verification, as evidenced by the detection of hippocampal neurons' vitality, proliferation, and apoptosis capabilities, and insulin resistance. Finally, in vivo mechanism verification was performed to assess the effect of AE on insulin resistance and cognitive disorder.ResultsTranscriptome sequencing analysis showed that MALAT1 was lowly expressed and miR-382-3p was highly expressed in serum-Exos samples of T2DM mice. There were targeted binding sites between MALAT1 and miR-382-3p and between miR-382-3p and BDNF. In vitro experiments showed that MALAT1 upregulated BDNF expression by inhibiting miR-382-3p. Silencing MALAT1 or overexpressing miR-382-3p could reduce the expression of INSR, IRS-1, IRS-2, PI3K/AKT, and Ras/MAPK, inhibit neuronal proliferation, and promote apoptosis. In vivo experiments further confirmed that AE could increase the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thereby improving cognitive impairment in T2DM mice.ConclusionAE may upregulate the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thus improving cognitive impairment in T2DM mice.

Mucin1 induced trophoblast dysfunction in gestational diabetes mellitus via Wnt/β-catenin pathway

BackgroundTo elucidate the role of Mucin1 (MUC1) in the trophoblast function (glucose uptake and apoptosis) of gestational diabetes mellitus (GDM) women through the Wnt/β-catenin pathway.MethodsGlucose uptake was analyzed by plasma GLUT1 and GLUT4 levels with ELISA and measured by the expression of GLUT4 and INSR with immunofluorescence and Western blotting. Apoptosis was measured by the expression of Bcl-2 and Caspase3 by Western blotting and flow cytometry. Wnt/β-catenin signaling measured by Western blotting. In vitro studies were performed using HTR-8/SVneo cells that were cultured and treated with high glucose (HG), sh-MUC1 and FH535 (inhibitor of Wnt/β-catenin signaling).ResultsMUC1 was highly expressed in the placental trophoblasts of GDM, and the Wnt/β-catenin pathway was activated, along with dysfunction of glucose uptake and apoptosis. MUC1 knockdown resulted in increased invasiveness and decreased apoptosis in trophoblast cells. The initial linkage between MUC1, the Wnt/β-catenin pathway, and glucose uptake was confirmed by using an HG-exposed HTR-8/SVneo cell model with MUC1 knockdown. MUC1 knockdown inhibited the Wnt/β-catenin signaling pathway and reversed glucose uptake dysfunction and apoptosis in HG-induced HTR-8/SVneo cells. Meanwhile, inhibition of Wnt/β-catenin signaling could also reverse the dysfunction of glucose uptake and apoptosis.ConclusionsIn summary, the increased level of MUC1 in GDM could abnormally activate the Wnt/β-catenin signaling pathway, leading to trophoblast dysfunction, which may impair glucose uptake and induce apoptosis in placental tissues of GDM women.

Antidiabetic activity of eupafolin through peroxisome proliferator-activated receptor-gamma and PI3K/Akt signaling in Type 2 diabetic rats.

Eupafolin is a phyto compound of flavone that exerts anti-inflammatory, antioxidant, and antiproliferative properties. The main purpose of this study is to examine the antidiabetic effect of eupafolin on nicotinamide-streptozotocin (STZ)-induced Type 2 diabetes (T2D) rats. After nicotinamide (120 mg/kg) treatment, STZ (60 mg/kg) was administrated intravenously to induce T2D. Rats with fasting blood glucose (FBG) > 200 mg/dL are chosen for the study 7 days after T2D induction. The eupafolin treatment was continued for another 15 days. FBG and an oral glucose tolerance test (OGTT) were measured on the 21st day after T2D induction. The blood lipid, serum insulin, and homeostatic model assessment (HOMA-IR) were determined. In liver homogenate, oxidative stress indicators were measured. In addition, the effect of eupafolin on the expression of the proteins InsR, insulin receptor substrate (IRS)-2, GLUT4, PPARγ, and phosphatidylinositol 3-kinase (PI3K)/Akt was investigated using a western blot. As measured by OGTT and HOMA-IR, eupafolin treatment reduced FBG and insulin resistance (IR). Furthermore, when compared to diabetic rats, liver antioxidant enzymes were dramatically normalized. The level of glycogen in the liver of diabetic rats was increased by eupafolin treatment. In T2D rats, eupafolin dramatically increased the InsR, IRS-2, GLUT4, and PPARγ. Further, the eupafolin treatment activated the PI3K/Akt signaling in T2D rats. These findings imply that the antidiabetic mechanism of eupafolin may be related to the activation of the PPARγ and the PI3K/Akt signaling pathway in T2D rats. As a result, the flavonoid eupafolin could be an antidiabetic medication for T2D after a comprehensive clinical investigation.

Dysregulation of Inflammation, Oxidative Stress, and Glucose Metabolism-Related Genes and miRNAs in Visceral Adipose Tissue of Women with Type 2 Diabetes Mellitus.

BACKGROUND Human visceral adipose tissue (VAT), now identified as an endocrine organ, plays a significant role in impaired fasting glucose and diabetes through the deregulated metabolism and adipogenesis of visceral adipocytes in obesity. Our study focuses on exploring the link between inflammation, oxidative stress, and glucose metabolism-associated genes with corresponding miRNAs in human visceral adipocytes and VAT from individuals with glucose metabolism disorders. MATERIAL AND METHODS We examined the expression of ATM, NFKB1, SOD2, INSR, and TIGAR, along with their related miRNAs using PCR, in two contexts:1 - During the three-stage visceral adipogenesis under normal glucose levels (5.5 millimoles), intermittent, and chronic hyperglycemia (30 millimoles).2 - In visceral adipose tissue from subjects (34 F, 18 M) with normal glucose metabolism, impaired fasting glucose, and type 2 diabetes mellitus. RESULTS Both chronic and intermittent hyperglycemia similarly influenced ATM, NFKB1, TIGAR, SOD2, INSR gene expression in visceral adipocytes, with corresponding changes in a few tested miRNAs (eg, let-7g-5p, miR-145-5p, miR-21-5p). Anthropometric and biochemical parameters led us to focus on female subjects. Our results showed transactivation of NFKB1, TIGAR, miR-10b-5p, miR-132-3p, miR-20a-5p, miR-21-5p, and miR-26a-5p exclusively in type 2 diabetes mellitus. Upregulated molecules (excluding miR-10b-5p and miR-20a-5p) positively correlated with glucose metabolism markers. CONCLUSIONS The genes studied may undergo miRNA interferences and hyperglycemic memory in visceral adipocytes under hyperglycemic conditions. VAT from women with type 2 diabetes mellitus, but not with impaired fasting glucose, showed transactivated miRNAs and a molecular dysregulation of TIGAR and NFKB1, possibly enhancing inflammation, oxidative stress, and disrupted glucose metabolism. These findings highlight the epigenetic and molecular disturbances in VAT related to glucose metabolism abnormalities. However, additional research is necessary to further understand their biological significance.

Therapeutic exploration of polyherbal formulation against letrozole induced PCOS rats: A mechanistic approach

ObjectiveThis study aimed to develop an effective alternative medicine with multi potential herbs against polycystic ovarian syndrome (PCOS) in rats induced by letrozole treatment. Materials and methodPolyherbal syrup was prepared with a combination of S. asoca bark, G. sylvestre leaves, P. daemia aerial parts, C. zeylanium stem bark, C. bonduc seeds, and W. somnifera roots ethanolic extract. In vitro cell viability study, adenosine monophosphate-activated protein kinase (AMPK), and glucose transporter 4 (GLUT4) gene expression assay were carried out on the Chinese Hamster Ovarian (CHO) cell line. For the PCOS induction letrozole (1 mg/kg p. o.) was given for 21 consecutive days. The PCOS induction was confirmed by measuring estrus irregularity, insulin resistance by oral glucose tolerance test (OGTT), and hyperandrogenism by measuring serum total testosterone level 21 days after completion of letrozole treatment. After induction of PCOS, metformin (155 mg/kg p. o.), and polyherbal syrup (100 mg/kg, 200 mg/kg, and 400 mg/kg p. o.) were administered for further 28 days. The treatment efficacy was measured by measuring serum lipid profile, fasting insulin level, sex hormones level, ovarian steroidogenic enzymes, ovarian tissue insulin receptor, AMPK, and GLUT4 protein expression levels, and histomorphological studies. The post-treatment effect was confirmed by reproductive performance studies. ResultsLetrozole-induced PCOS rats showed significant estrus irregularity, abnormal sex hormones levels, and hyperandrogenism indicated by showing increased free androgenic index and decreased sex hormones binding globulin (SHBG) level. The insulin resistance in PCOS rats was indicated by increased fasting glucose levels with impaired glucose clearance in the OGT test. Homeostasis Model Assessment Index of Insulin Resistance (HOMA-IR) increased level, also decreases INSR, GLUT4, and AMPK mRNA expression in ovarian cells confirming the insulin resistance in PCOS rats. Ovarian histology in PCOS rats also showed many follicular cysts, atretic follicles, and the absence of corpus luteum. The administration of polyherbal syrup, in a dose-dependent manner, effectively restored these alterations. The treatment of polyherbal formulation 400 mg/kg possesses highly significant efficacy over the treatment of metformin in PCOS rats. It mainly acts by reducing peripheral and ovarian hyperandrogenism and improves insulin sensitivity via activating the insulin receptor and AMP-activated kinase-mediated transcription and translation of GLUT4 from the cytoplasm to the ovarian membrane improves glucose uptake and promotes the follicular development and ovulation. The higher fertility rate, delivery index, and survival of delivered pups confirm the broader and superior efficacy of PCOS. These beneficial actions are mainly attributable to the formulation's inclusion of the key secondary metabolites flavonoids and phytosterols. In conclusion, the prepared polyherbal syrup was found to be the safest and most effective alternative medicine for both endocrinal and metabolic complications of PCOS women.

Abstract 2851: ALK-inhibitors as a novel potential therapy for proteasome inhibitor-resistant multiple myeloma

Abstract Background: Proteasome inhibitors (PI) disrupt the equilibrium between production and disposal of excess and/or misfolded proteins in multiple myeloma (MM) cells, leading to apoptosis. Resistance to PI bortezomib (BTZ) and carfilzomib (CFZ) is a major obstacle to the successful treatment of MM. The drug repurposing approach offers a promising strategy to classical drug development in particular in MM. Previously, ALK-inhibitors were shown to have anti-MM activity ex vivo. Therefore, we aimed to identify the mechanism of action of ALK-inhibitors against MM cells and to identify promising drug therapy combinations of ALK-inhibitors for refractory MM. Methods: A set of PI-naïve and PI-resistant cells was used in the study. Viability of the cells was determined by CCK8 viability assay. Genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library was used in MM AMO-1 cell line to identify mechanism of action of ceritinib. Western blot was used to assess levels of total and phosphorylated proteins. RNA-seq was used to determine the amount of transcripts in PI-sensitive and PI-resistant cells. Results: Initially, 7 different ALK-inhibitors were tested across PI-naïve and PI-adapted MM cell lines. Based on the IC50 values, the most effective ALK-inhibitors to induce cytotoxicity in MM were ceritinib &amp;gt; brigatinib &amp;gt; crizotinib &amp;gt; entrectinib. The combination of ceritinib, brigatinib and entrectinib showed synergistic cytotoxicity with the PI BTZ and CFZ and overcame PI-resistance in 4 different sets of PI-adapted cells. The strongest synergistic cytotoxicity was observed between ceritinib and CFZ in CFZ-adapted cells. CRISPR/Cas9-screening identified genes involved in negative regulation of mTORC signalling (NPRL2, NPRL3, TSC1, TSC2) and transcription factor FOXO1 as the major resistance candidates to ceritinib. Subsequently, ceritinib treatment significantly inhibited mTORC signaling, suggesting a shut-down of cellular proliferation due to targeting of the upstream receptor-tyrosine kinases (RTK), essential for cell viability and thus not identified by the screening. Further search identified InsR and IGF1R as the overlapping RTK inhibited by ceritinib and brigatinib. Co-treatment of PI-resistant cells with the combination of InsR/IGF1R inhibitors and CFZ induced strong synergistic cytotoxicity, resembling the synergistic cytotoxicity observed for ceritinib and CFZ, whereas InsR/IGF1R inhibitors did not show any synergistic cytotoxicity with ceritinib, suggesting targeting the same pathway. Our data further show that PI-naïve and resistant cells are negative for ALK, but positive for InsR and IGF1R expression. Conclusion: Crizotinib inhibits InsR/IGF1R signaling in MM, which is essential for MM survival and together with CFZ it overcomes PI-resistance. Therefore, it represents a promising and already available therapy for PI-resistant MM patients. Citation Format: Lenka Besse, Andrej Besse, Marianne Kraus, Matej Jasik, Ondrej Slaby, Christoph Driessen. ALK-inhibitors as a novel potential therapy for proteasome inhibitor-resistant multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2851.

Characterization of Infiltrating Immune Cells and Secretory or Membrane-Associated Proteins in KRAS Lung Adenocarcinoma.

This study identified the expression and prognosis significance of secretory or membrane-associated proteins in KRAS lung adenocarcinoma (LUAD) and depicted the characteristics between the immune cell infiltration and the expression of these genes. Gene expression data of LUAD samples (n = 563) were accessed from The Cancer Genome Atlas (TCGA). The expression of secretory or membrane-associated proteins was compared among the KRAS-mutant, wild-type, and normal groups, as well as the subgroup of the KRAS-mutant group. We identified the survival-related differentially expressed secretory or membrane-associated proteins and conducted the functional enrichment analysis. Then, the characterization and association between their expression and the 24 immune cell subsets were investigated. We also constructed a scoring model to predict KRAS mutation by LASSO and logistic regression analysis. Secretory or membrane-associated genes with differential expression (n = 74) across three groups (137 KRAS LUAD, 368 wild-type LUAD, and 58 normal groups) were identified, and the results of GO and KEGG indicated that they were strongly associated with immune cell infiltrations. Among them, ten genes were significantly related to the survival of patients with KRAS LUAD. The expression of IL37, KIF2, INSR, and AQP3 had the most significant correlations with immune cell infiltration. In addition, eight DEGs from the KRAS subgroups were highly correlated with immune infiltrations, especially TNFSF13B. Using LASSO-logistic regression, a KRAS mutation prediction model based on the 74 differentially expressed secretory or membrane-associated genes was built, and the accuracy was 0.79. The research investigated the relationship between the expression of KRAS-related secretory or membrane-associated proteins in LUAD patients with prognostic prediction and immune infiltration characterization. Our study demonstrated that secretory or membrane-associated genes were closely associated with the survival of KRAS LUAD patients and were strongly correlated to immune cell infiltration.

Interactions of alcohol and combination antiretroviral (cART) drug in diabetic male Sprague Dawley rats: Hippocampal perturbations and toxicosis.

Hippocampal pathology in diabetes is constantly investigated but the resultant health impact of the concomitant presence of alcohol and combined antiretroviral therapy (cART) in diabetes requires further studies to delineate toxicities inimical to hippocampal normal function. Forty-eight male Sprague Dawley rats were divided into eight groups (n=6): negative control (NC), alcohol (AL), cART (AV), alcohol-cART (AA), diabetic control (DB), diabetes-alcohol (DAL), diabetes-cART (DAV), and diabetes-alcohol-cART (DAA) exposure groups. Following diabetes induction and sub-chronic (90 days) treatment exposure, hippocampal homogenates were profiled for pro-inflammatory cytokines and oxidative stress (MDA and GPx) using immunoassay, while apoptotic genes (BAX, Bcl2, and Caspase-3), insulin receptor genes (INSR and IRS-1), and blood-brain barrier (BBB) junctional proteins (claudin-5, and occludin) gene expression were assessed using qPCR. Histomorphology of hippocampal neuronal number, nuclei area, and volume of dentate gyrus and neurogenesis were accessed using Giemsa stain, Ki67, and DCX histochemistry respectively. A central hippocampal effect that underpins all treatments is the reduction of DG neuronal number and antioxidant (GPx), highlighting the venerability of the hippocampal dentate gyrus neurons to diabetes, alcohol, cART, and their combinatorial interactions. Additionally, elevated BAX, Bcl2, and IRS1 mRNA levels in the DAL group, and their downregulation in AA, suggests IRS-1-regulated apoptosis due to differential modulating effects of alcohol treatment in diabetes (DAL) in contrast to alcohol with cART (AA). Although the interaction in AA therapy ameliorated the independent alcohol and cART effects on MDA levels, pro-inflammatory cytokines, and DCX, the interaction in AA exacerbated a deficiency in the expression of INSR, IRS-1 (insulin sensitivity), and BBB mRNA which are implicated in the pathogenies of diabetes. Furthermore, the diabetic comorbidity groups (DAV, DAL, and DAA) all share a central effect of elevated hippocampal oxidative stress, BAX, and Caspase-3 mRNA expression with the reduced number of hippocampal neurons, dentate gyrus volume, and neurogenesis, highlighting neurodegenerative and cognitive deficiency implication of these comorbidity treatments. Considering these findings, assessment of hippocampal well-being in patients with these comorbidities/treatment combinations is invaluable and caution is advised particularly in alcohol use with cART prophylaxis in diabetes.