Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant.

BACKGROUND: Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy. METHODS: Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function. RESULTS: GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo. CONCLUSION: Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

A promising strategy for investigating the anti-aging effect of natural compounds: a case study of caffeoylquinic acids.

Caffeoylquinic acids, as plant-derived polyphenols, exhibit multiple biological activities such as antioxidant, anti-inflammatory, and neuroprotective activities. However, only limited information about their effect on longevity is available. In the current study, molecular docking was employed to explore the interactions between six representative caffeoylquinic acids and the insulin-like growth factor-1 receptor (IGFR), which is an important target protein for longevity. The results indicated that all six compounds were embedded well in the active pocket of IGFR, and that 3,5-diCQA exhibited the strongest affinity to IGFR. Moreover, ASP1153, GLU1080, ASP1086, and ARG1003 were the key amino acid residues during the interaction of these 6 compounds with IGFR. Furthermore, the lifespan extension effect of caffeoylquinic acids was evaluated in a Caenorhabditis elegans (C. elegans) model. The results revealed that all the caffeoylquinic acids significantly extended the lifespan of wild-type worms, of which 3,5-diCQA was the most potent compound. Meanwhile, 3,5-diCQA enhanced the healthspan by increasing the body bending and pharyngeal pumping rates and reducing the intestinal lipofuscin level. Further studies demonstrated that 3,5-diCQA induced longevity effects by downregulating the insulin/insulin-like growth factor signaling (IIS) pathway. This study suggested that the combination of molecular docking and genetic analysis of specific worm mutants could be a promising strategy to reveal the anti-aging mechanisms of small molecule natural compounds.

Insulin Receptor Isoforms Differently Regulate Cell Proliferation and Apoptosis in the Ligand-Occupied and Unoccupied State.

The insulin receptor (IR) presents two isoforms (IR-A and IR-B) that differ for the α-subunit C-terminal. Both isoforms are expressed in all human cells albeit in different proportions, yet their functional properties-when bound or unbound to insulin-are not well characterized. From a cell model deprived of the Insulin-like Growth Factor 1 Receptor (IGF1-R) we therefore generated cells exhibiting no IR (R-shIR cells), or only human IR-A (R-shIR-A), or exclusively human IR-B (R-shIR-B) and we studied the specific effect of the two isoforms on cell proliferation and cell apoptosis. In the absence of insulin both IR-A and IR-B similarly inhibited proliferation but IR-B was 2-3 fold more effective than IR-A in reducing resistance to etoposide-induced DNA damage. In the presence of insulin, IR-A and IR-B promoted proliferation with the former significantly more effective than the latter at increasing insulin concentrations. Moreover, only insulin-bound IR-A, but not IR-B, protected cells from etoposide-induced cytotoxicity. In conclusion, IR isoforms have different effects on cell proliferation and survival. When unoccupied, IR-A, which is predominantly expressed in undifferentiated and neoplastic cells, is less effective than IR-B in protecting cells from DNA damage. In the presence of insulin, particularly when present at high levels, IR-A provides a selective growth advantage.

Systemic Administration of Insulin Receptor Antagonist Results in Endothelial and Perivascular Adipose Tissue Dysfunction in Mice.

Hyperglycemia linked to diabetes results in endothelial dysfunction. In the present work, we comprehensively characterized effects of short-term hyperglycemia induced by administration of an insulin receptor antagonist, the S961 peptide, on endothelium and perivascular adipose tissue (PVAT) in mice. Endothelial function of the thoracic and abdominal aorta in 12-week-old male C57Bl/6Jrj mice treated for two weeks with S961 infusion via osmotic pumps was assessed in vivo using magnetic resonance imaging and ex vivo by detection of nitric oxide (NO) production using electron paramagnetic resonance spectroscopy. Additional methods were used to analyze PVAT, aortic segments and endothelial-specific plasma biomarkers. Systemic disruption of insulin signaling resulted in severe impairment of NO-dependent endothelial function and a loss of vasoprotective function of PVAT affecting the thoracic as well as abdominal parts of the aorta, however a fall in adiponectin expression and decreased uncoupling protein 1-positive area were more pronounced in the thoracic aorta. Results suggest that dysfunctional PVAT contributes to vascular pathology induced by altered insulin signaling in diabetes, in the absence of fat overload and obesity.

M2­like tumour­associated macrophage­secreted IGF promotes thyroid cancer stemness and metastasis by activating the PI3K/AKT/mTOR pathway.

M2­like tumour­associated macrophages (TAMs) have been demonstrated to promote the growth of anaplastic thyroid carcinoma (ATC). However, the underlying mechanism of M2­like TAMs in ATC remains unclear. Thus, in the present study, the role and mechanism of M2­like TAMs in ATC were investigated. M2­like TAMs were induced by treatment with PMA, plus IL­4 and IL­13, and identified by flow cytometry. Transwell and sphere formation assays were applied to assess the invasion and stemness of ATC cells. The expression levels of insulin­like growth factor (IGF)­1 and IGF­2 were examined by ELISA and reverse transcription­quantitative PCR. Proteins related to the epithelial­mesenchymal transition (EMT), stemness and the PI3K/AKT/mTOR pathway were examined via western blotting. Immunohistochemistry (IHC) was used to detect the expression of the M2­like TAM markers CD68 and CD206 in ATC tissues and thyroid adenoma tissues. It was found that treatment with PMA plus IL­4 and IL­13 successfully induced M2­like TAMs. Following co­culture with M2­like TAMs, the invasive ability and stemness of ATC cells were significantly increased. The expression levels of the EMT­related markers N­cadherin and Vimentin, the stemness­related markers Oct4, Sox2 and CD133, and the insulin receptor (IR)­A/IGF1 receptor (IGF1R) were markedly upregulated, whereas E­cadherin expression was significantly decreased. In addition, the production of IGF­1 and IGF­2 was significantly increased. Of note, exogenous IGF­1/IGF­2 promoted the invasion and stemness of C643 cells, whereas blocking IGF­1 and IGF­2 inhibited metastasis and stemness by repressing IR­A/IGF­1R­mediated PI3K/AKT/mTOR signalling in the co­culture system. IHC results showed that the expression of CD68 and CD206 was obviously increased in ATC tissues. To conclude, M2­like TAMs accelerated the metastasis and increased the stemness of ATC cells, and the underlying mechanism may be related to the section of IGF by M2­like TAMs, which activates the IR­A/IGF1R­mediated PI3K/AKT/mTOR signalling pathway.

FGF21 Normalizes Plasma Glucose in Mouse Models of Type 1 Diabetes and Insulin Receptor Dysfunction.

Fibroblast growth factor (FGF) 21 is a member of the FGF family of proteins. The biological activity of FGF21 was first shown to induce insulin-independent glucose uptake in adipocytes through the GLUT1 transporter. Subsequently, it was shown to have effects on the liver to increase fatty acid oxidation. FGF21 treatment provides beneficial metabolic effects in both animal models and patients with obesity, type 2 diabetes mellitus (T2D) and/or fatty liver disease. In this paper, we revisited the original finding and found that insulin-independent glucose uptake in adipocytes is preserved in the presence of an insulin receptor antagonist. Using a 40-kDa PEGylated (PEG) and half-life extended form of FGF21 (FGF21-PEG), we extended these in vitro results to 2 different mouse models of diabetes. FGF21-PEG normalized plasma glucose in streptozotocin-treated mice, a model of type 1 diabetes (T1D), without restoring pancreatic ß-cell function. FGF21-PEG also normalized plasma glucose levels and improved glucose tolerance in mice chronically treated with an insulin competitive insulin receptor antagonist, a model of autoimmune/type-B insulin resistance. These data extend the pharmacological potential of FGF21 beyond the settings of T2D, fatty liver, and obesity.

The cytoskeleton actin binding protein filamin A impairs both IGF2 mitogenic effects and the efficacy of IGF1R inhibitors in adrenocortical cancer cells.

Adrenocortical carcinomas (ACCs) overexpress insulin-like growth factor 2 (IGF2), that drives a proliferative autocrine loop by binding to IGF1R and IR, but IGF1R/IR-targeted therapies failed in ACC patients. The cytoskeleton actin-binding protein filamin A (FLNA) impairs IR signalling in melanoma cells. Aims of this study were to test FLNA involvement in regulating IGF1R and IR responsiveness to both IGF2 and inhibitors in ACC. In ACC cells H295R and SW13 and primary cultures (1ACC, 4 adenomas) we found that IGF1R and IR interacted with FLNA, and FLNA silencing increased IGF1R and reduced IR expression, with a downstream effect of increased cell proliferation and ERK phosphorylation. In addition, FLNA knockdown potentiated antiproliferative effects of IGF1R/IR inhibitor Linsitinib and IGF1R inhibitor NVP-ADW742 in H295R. Finally, Western blot showed lower FLNA expression in ACCs (n = 10) than in ACAs (n = 10) and an inverse correlation of FLNA/IGF1R ratio with ERK phosphorylation in ACCs only. In conclusion, we demonstrated that low FLNA levels enhance both IGF2 proliferative effects and IGF1R/IR inhibitors efficacy in ACC cells, suggesting FLNA as a new factor influencing tumor clinical behavior and the response to the therapy with IGF1R/IR-targeted drugs.

Identification of monoclonal antibodies suitable for blocking IGF-1 receptors in the horse.

Prolonged hyperinsulinemia is thought to be the cause of equine endocrinopathic laminitis, a common and crippling disease of the foot, for which there are no pharmacologic treatments other than pain relief. It has been suggested that insulin causes its effects on the lamellae by activating IGF-1 receptors (IGF-1R), as insulin receptors (InsR) are scarce in this tissue, whereas IGF-1R are abundant and become downregulated after prolonged insulin infusion. As a first step toward confirming this mechanism and beginning to develop a therapeutic anti-IGF-1R monoclonal antibody (mAb) for horses, it was necessary to identify available human IGF-1R mAbs that would recognize equine receptors. Four IGF-1R mAbs were tested using soluble equine IGF-1R, with ELISA and flow cytometry. Frozen equine lamellar and liver tissue was also used in radioligand binding assays. The results demonstrated that only one of the mAbs tested (mAb1) was able to compete effectively with IGF-1 for binding to its receptors in equine lamellar tissue, with an IC50 of 5 to 159 ng/mL. None of the 4 mAbs were able to bind to equine hepatic InsR. This study has generated valuable structure-activity information and has identified a prototype anti-IGF-1R mAb suitable for further development.

Linagliptin Ameliorates Hepatic Steatosis via Non-Canonical Mechanisms in Mice Treated with a Dual Inhibitor of Insulin Receptor and IGF-1 Receptor.

Abnormal hepatic insulin signaling is a cause or consequence of hepatic steatosis. DPP-4 inhibitors might be protective against fatty liver. We previously reported that the systemic inhibition of insulin receptor (IR) and IGF-1 receptor (IGF1R) by the administration of OSI-906 (linsitinib), a dual IR/IGF1R inhibitor, induced glucose intolerance, hepatic steatosis, and lipoatrophy in mice. In the present study, we investigated the effects of a DPP-4 inhibitor, linagliptin, on hepatic steatosis in OSI-906-treated mice. Unlike high-fat diet-induced hepatic steatosis, OSI-906-induced hepatic steatosis is not characterized by elevations in inflammatory responses or oxidative stress levels. Linagliptin improved OSI-906-induced hepatic steatosis via an insulin-signaling-independent pathway, without altering glucose levels, free fatty acid levels, gluconeogenic gene expressions in the liver, or visceral fat atrophy. Hepatic quantitative proteomic and phosphoproteomic analyses revealed that perilipin-2 (PLIN2), major urinary protein 20 (MUP20), cytochrome P450 2b10 (CYP2B10), and nicotinamide N-methyltransferase (NNMT) are possibly involved in the process of the amelioration of hepatic steatosis by linagliptin. Thus, linagliptin improved hepatic steatosis induced by IR and IGF1R inhibition via a previously unknown mechanism that did not involve gluconeogenesis, lipogenesis, or inflammation, suggesting the non-canonical actions of DPP-4 inhibitors in the treatment of hepatic steatosis under insulin-resistant conditions.

Coumarin-chalcone hybrids targeting insulin receptor: Design, synthesis, anti-diabetic activity, and molecular docking.

Four series of thirteen new coumarin-chalcone hybrids (DPCU 1-13, DPCT 1-13, DCCU 1-13 and DCCT 1-13) were designed and synthesized using Biginelli synthesis, Pechmann condensation, Acetylation, and Claisen-Schmidt reactions. Synthesized compounds were tested for insulin receptor in silico docking studies (PDB ID: 1IR3); DCCU 13 and DCCT 13 derivatives received the lowest docking score; Streptozocin (STZ) and Nicotinamide (NA) induced type II diabetes was tested for their anti-diabetic activity in rats. In vivo tests suggested that fasting blood glucose levels of animals treated with DCCU 13 (30 mg/kg body weight) and DCCT 13 (30 mg/kg body weight) were significantly and moderately suppressed, respectively, relative to fasting blood glucose levels of diabetic control animals. Similarly, therapy with DCCU 13 and DCCT 13 attenuated oxidative stress parameters such as lipid peroxidation (MDA), superoxide dismutase (SOD) and increased the glutathione (GSH) in the liver and pancreas in a dose-dependent manner. In comparison, therapy with DCCU 13 (30 mg/kg body weight) mitigated alterations in the histological architecture of the liver and pancreatic tissue. These results indicated that the hybrids DUUC 13 and DCCT 13 at 30 mg/kg had an anti-hyperglycemic and antioxidant impact on STZ + NA mediated type II diabetes in rats. Further detailed work could be required to determine the precise mode of action of the anti-diabetic behavior of hybrids.

Systems Analysis of Insulin and IGF1 Receptors Networks in Breast Cancer Cells Identifies Commonalities and Divergences in Expression Patterns.

Insulin and insulin-like growth factor-1 (IGF1), acting respectively via the insulin (INSR) and IGF1 (IGF1R) receptors, play key developmental and metabolic roles throughout life. In addition, both signaling pathways fulfill important roles in cancer initiation and progression. The present study was aimed at identifying mechanistic differences between INSR and IGF1R using a recently developed bioinformatics tool, the Biological Network Simulator (BioNSi). This application allows to import and merge multiple pathways and interaction information from the KEGG database into a single network representation. The BioNsi network simulation tool allowed us to exploit the availability of gene expression data derived from breast cancer cell lines with specific disruptions of the INSR or IGF1R genes in order to investigate potential differences in protein expression that might be linked to biological attributes of the specific receptor networks. Modeling-generated information was corroborated by experimental and biological assays. BioNSi analyses revealed that the expression of 75 and 71 genes changed during simulation of IGF1R-KD and INSR-KD, compared to control cells, respectively. Out of 16 proteins that BioNSi analysis was based on, validated by Western blotting, nine were shown to be involved in DNA repair, eight in cell cycle checkpoints, six in proliferation, eight in apoptosis, seven in oxidative stress, six in cell migration, two in energy homeostasis, and three in senescence. Taken together, analyses identified a number of commonalities and, most importantly, dissimilarities between the IGF1R and INSR pathways that might help explain the basis for the biological differences between these networks.

Development of a novel insulin receptor (IR) antagonist that exhibits anti-breast tumor activity.

Many reports have indicated that the insulin receptor (IR) causes tumorigenesis and the development of breast cancer. It has been considered a potential target for treating IR-related tumors. Traditionally, there are two categories of insulin receptor (IR) antagonists, they are small molecule antagonists and anti-IR antibodies. Here, we describe a new method (anti-idiotypic antibody strategy) for the development of IR antagonist. Hybridoma technology was employed to design and identify a series of anti-idiotypic antibodies against insulin. After repeated screening and identification, an anti-idiotypic antibody against IR (AK98) was obtained. Analysis through competitive ELISA and competitive receptor binding indicated that AK98 mimicked the receptor binding epitope of insulin. The interaction between AK98 and IR was determined using indirect immunofluorescence, immunoelectron microscopy, and Immunoprecipitation-Western (IP-WB). Further research using a tumor cell model revealed that AK98 inhibited insulin-IR binding and IR-mediated intracellular signaling pathways. Conclusively, the main purpose of this paper is that we proposed a new method (anti-idiotypic antibody strategy) to develop the insulin receptor (IR) antagonist (AK98), and a series of experiments showed that the anti-idiotypic antibody (AK98) exhibited good antagonistic activity against IR. This work suggests that the anti-idiotypic antibody may be a potential strategy to develop IR antagonists that can be used in treating breast cancer.

Taenia solium insulin receptors: promising candidates for cysticercosis treatment and prevention.

Insulin signaling pathway is an ancient and highly conserved pathway known to play critical roles in cell growth, control and metabolic regulation. In this study, we identified and characterized two insulin receptor genes (TsIR-1316 and TsIR-4810) from Taenia solium. TsIR-1316 was grouped with E. multilocularis insulin receptor (EmIR-1) and TsIR-4810 was closer to Taenia pisiformis insulin-like growth factor receptor (TpIR) on the same branch with a very high bootstrap value. TsIR-1316 was located on the integument of larvae and adult worms, as well as the ovary of adults and eggs. Alternatively, TsIR-4810 was located in the parenchyma and reproductive organs of the adult worms. By using in vitro cultivation systems with Cysticercus pisiformis as a model, we demonstrated that anti-TsIRs-LBD antibodies could effectively block the insulin signaling pathway, resulting in reduced phosphorylation of the insulin receptor as well as lower levels of glucose uptake and glycogen synthesis. The rabbits immunized with TsIR-1316-LBD, TsIR-4810-LBD and TsIR-1316-LBD + TsIR-4810-LBD produced protection against infection of T. pisiformis as demonstrated by a 94.6%, 96% and 80% reduction of establishment of larvae, respectively. These data suggested that TsIR-1316-LBD and TsIR-4810-LBD are promising vaccine candidates or novel drug targets against swine cysticercosis.

Intra-Pancreatic Insulin Nourishes Cancer Cells: Do Insulin-Receptor Antagonists such as PGG and EGCG Play a Role?

Harboring insulin-producing cells, the pancreas has more interstitial insulin than any other organ. In vitro, insulin activates both insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) to stimulate pancreatic cancer cells. Whether intra-pancreatic insulin nourishes pancreatic cancer cells in vivo remains uncertain. In the present studies, we transplanted human pancreatic cancer cells orthotopically in euglycemic athymic mice whose intra-pancreatic insulin was intact or was decreased following pretreatment with streptozotocin (STZ). In the next eight weeks, the tumor carriers were treated with one of the IR/IGF1R antagonists penta-O-galloyl-[Formula: see text]-D-glucose (PGG) and epigallocatechin gallate (EGCG) or treated with vehicle. When pancreatic tumors were examined, their fraction occupied with living cells was decreased following STZ pretreatment and/or IR/IGF1R antagonism. Using Western blot, we examined tumor grafts for IR/IGF1R expression and activity. We also determined proteins that were downstream to IR/IGF1R and responsible for signal transduction, glycolysis, angiogenesis, and apoptosis. We demonstrated that STZ-induced decrease in intra-pancreatic insulin reduced IR/IGF1R expression and activity, decreased the proteins that promoted cell survival, and increased the proteins that promoted apoptosis. These suggest that intra-pancreatic insulin supported local cancer cells. When tumor carriers were treated with PGG or EGCG, the results were similar to those seen following STZ pretreatment. Thus, the biggest changes in examined proteins were usually seen when STZ pretreatment and PGG/EGCG treatment concurred. This suggests that intra-pancreatic insulin normally combated pharmacologic effects of PGG and EGCG. In conclusion, intra-pancreatic insulin nourishes pancreatic cancer cells and helps the cells resist IR/IGF1R antagonism.

Optimized criteria for locomotion-based healthspan evaluation in C. elegans using the WorMotel system.

Getting a grip on how we may age healthily is a central interest of biogerontological research. To this end, a number of academic teams developed platforms for life- and healthspan assessment in Caenorhabditis elegans. These are very appealing for medium- to high throughput screens, but a broader implementation is lacking due to many systems relying on custom scripts for data analysis that others struggle to adopt. Hence, user-friendly recommendations would help to translate raw data into interpretable results. The aim of this communication is to streamline the analysis of data obtained by the WorMotel, an economically and practically appealing screening platform, in order to facilitate the use of this system by interested researchers. We here detail recommendations for the stepwise conversion of raw image data into activity values and explain criteria for assessment of health in C. elegans based on locomotion. Our analysis protocol can easily be adopted by researchers, and all needed scripts and a tutorial are available in S1 and S2 Files.

Luseogliflozin increases beta cell proliferation through humoral factors that activate an insulin receptor- and IGF-1 receptor-independent pathway.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors, which prevent the renal reabsorption of glucose, decrease blood glucose levels in an insulin-independent manner. We previously reported creating a mouse model of systemic inhibition of target receptors for both insulin and IGF-1 by treating animals with OSI-906, a dual insulin/IGF-1 receptor inhibitor, for 7 days. The OSI-906-treated mice exhibited an increased beta cell mass, hepatic steatosis and adipose tissue atrophy, accompanied by hyperglycaemia and hyperinsulinaemia. In the present study, we investigated the effects of an SGLT2 inhibitor, luseogliflozin, on these changes in OSI-906-treated mice. METHODS: We treated C57BL/6J male mice either with vehicle, luseogliflozin, OSI-906 or OSI-906 plus luseogliflozin for 7 days, and phenotyping was performed to determine beta cell mass and proliferation. Subsequently, we tested whether serum-derived factors have an effect on beta cell proliferation in genetically engineered beta cells, mouse islets or human islets. RESULTS: SGLT2 inhibition with luseogliflozin significantly ameliorated hyperglycaemia, but not hyperinsulinaemia, in the OSI-906-treated mice. Liver steatosis and adipose tissue atrophy induced by OSI-906 were not altered by treatment with luseogliflozin. Beta cell mass and proliferation were further increased by SGLT2 inhibition with luseogliflozin in the OSI-906-treated mice. Luseogliflozin upregulated gene expression related to the forkhead box M1 (FoxM1)/polo-like kinase 1 (PLK1)/centromere protein A (CENP-A) pathway in the islets of OSI-906-treated mice. The increase in beta cell proliferation was recapitulated in a co-culture of Irs2 knockout and Insr/IR knockout (ßIRKO) beta cells with serum from both luseogliflozin- and OSI-906-treated mice, but not after SGLT2 inhibition in beta cells. Circulating factors in both luseogliflozin- and OSI-906-treated mice promoted beta cell proliferation in both mouse islets and cadaveric human islets. CONCLUSIONS/INTERPRETATION: These results suggest that luseogliflozin can increase beta cell proliferation through the activation of the FoxM1/PLK1/CENP-A pathway via humoral factors that act in an insulin/IGF-1 receptor-independent manner.

UCP1-independent glucose-lowering effect of leptin in type 1 diabetes: only in conditions of hypoleptinemia.

The possibility to use leptin therapeutically for lowering glucose levels in patients with type 1 diabetes has attracted interest. However, earlier animal models of type 1 diabetes are severely catabolic with very low endogenous leptin levels, unlike most patients with diabetes. Here, we aim to test glucose-lowering effects of leptin in novel, more human-like murine models. We examined the glucose-lowering potential of leptin in diabetic models of two types: streptozotocin-treated mice and mice treated with the insulin receptor antagonist S961. To prevent hypoleptinemia, we used combinations of thermoneutral temperature and high-fat feeding. Leptin fully normalized hyperglycemia in standard chow-fed streptozotocin-treated diabetic mice. However, more humanized physiological conditions (high-fat diets or thermoneutral temperatures) that increased adiposity - and thus also leptin levels - in the diabetic mice abrogated the effects of leptin, i.e., the mice developed leptin resistance also in this respect. The glucose-lowering effect of leptin was not dependent on the presence of the uncoupling protein-1 and was not associated with alterations in plasma insulin, insulin-like growth factor 1, food intake or corticosterone but fully correlated with decreased plasma glucagon levels and gluconeogenesis. An important implication of these observations is that the therapeutic potential of leptin as an additional treatment in patients with type 1 diabetes is probably limited. This is because such patients are treated with insulin and do not display low leptin levels. Thus, the potential for a glucose-lowering effect of leptin would already have been attained with standard insulin therapy, and further effects on blood glucose level through additional leptin cannot be anticipated.

GPCRs and Insulin Receptor Signaling in Conversation: Novel Avenues for Drug Discovery.

Type 2 diabetes is a major health issue worldwide with complex metabolic and endocrine abnormalities. Hyperglycemia, defects in insulin secretion and insulin resistance are classic features of type 2 diabetes. Insulin signaling regulates metabolic homeostasis by regulating glucose and lipid turnover in the liver, skeletal muscle and adipose tissue. Major treatment modalities for diabetes include the drugs from the class of sulfonyl urea, Insulin, GLP-1 agonists, SGLT2 inhibitors, DPP-IV inhibitors and Thiazolidinediones. Emerging antidiabetic therapeutics also include classes of drugs targeting GPCRs in the liver, adipose tissue and skeletal muscle. Interestingly, recent research highlights several shared intermediates between insulin and GPCR signaling cascades opening potential novel avenues for diabetic drug discovery.

Exercise and metformin counteract altered mitochondrial function in the insulin-resistant brain.

Insulin resistance associates with increased risk for cognitive decline and dementia; however, the underpinning mechanisms for this increased risk remain to be fully defined. As insulin resistance impairs mitochondrial oxidative metabolism and increases ROS in skeletal muscle, we considered whether similar events occur in the brain, which - like muscle - is rich in insulin receptors and mitochondria. We show that high-fat diet-induced (HFD-induced) brain insulin resistance in mice decreased mitochondrial ATP production rate and oxidative enzyme activities in brain regions rich in insulin receptors. HFD increased ROS emission and reduced antioxidant enzyme activities, with the concurrent accumulation of oxidatively damaged mitochondrial proteins and increased mitochondrial fission. Improvement of insulin sensitivity by both aerobic exercise and metformin ameliorated HFD-induced abnormalities. Moreover, insulin-induced enhancement of ATP production in primary cortical neurons and astrocytes was counteracted by the insulin receptor antagonist S961, demonstrating a direct effect of insulin resistance on brain mitochondria. Further, intranasal S961 administration prevented exercise-induced improvements in ATP production and ROS emission during HFD, supporting that exercise enhances brain mitochondrial function by improving insulin action. These results support that insulin sensitizing by exercise and metformin restores brain mitochondrial function in insulin-resistant states.

Insulin potentiates the response to mechanical stimuli in small dorsal root ganglion neurons and thin fibre muscle afferents in vitro.

KEY POINTS: Insulin is known to activate the sympathetic nervous system centrally. A mechanical stimulus to tissues activates the sympathetic nervous system via thin fibre afferents. Evidence suggests that insulin modulates putative mechanosensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, we report the novel finding that insulin augments the mechanical responsiveness of thin fibre afferents not only at dorsal root ganglion, but also at muscle tissue levels. Our data suggest that sympathoexcitation is mediated via the insulin-induced mechanical sensitization peripherally. The present study proposes a novel physiological role of insulin in the regulation of mechanical sensitivity in somatosensory thin fibre afferents. ABSTRACT: Insulin activates the sympathetic nervous system, although the mechanism underlying insulin-induced sympathoexcitation remains to be determined. A mechanical stimulus to tissues such as skin and/or skeletal muscle, no matter whether the stimulation is noxious or not, activates the sympathetic nervous system via thin fibre afferents. Evidence suggests that insulin modulates putative mechanosensitive channels in the dorsal root ganglion (DRG) neurons of these afferents. Accordingly, we investigated whether insulin augments whole-cell current responses to mechanical stimuli in small DRG neurons of normal healthy mice. We performed whole-cell patch clamp recordings using cultured DRG neurons and observed mechanically-activated (MA) currents induced by mechanical stimuli applied to the cell surface. Local application of vehicle solution did not change MA currents or mechanical threshold in cultured DRG neurons. Insulin (500 mU mL-1 ) significantly augmented the amplitude of MA currents (P < 0.05) and decreased the mechanical threshold (P < 0.05). Importantly, pretreatment with the insulin receptor antagonist, GSK1838705, significantly suppressed the insulin-induced potentiation of the mechanical response. We further examined the impact of insulin on thin fibre muscle afferent activity in response to mechanical stimuli in normal healthy rats in vitro. Using a muscle-nerve preparation, we recorded single group IV fibre activity to a ramp-shaped mechanical stimulation. Insulin significantly decreased mechanical threshold (P < 0.05), although it did not significantly increase the response magnitude to the mechanical stimulus. In conclusion, these data suggest that insulin augments the mechanical responsiveness of small DRG neurons and potentially sensitizes group IV afferents to mechanical stimuli at the muscle tissue level, possibly contributing to insulin-induced sympathoexcitation.