Upregulation Of Insulin Receptor Research Articles

Telmisartan versus metformin in downregulating myostatin gene expression and enhancing insulin sensitivity in the skeletal muscles of type 2 diabetic rat model.

Objective: Telmisartan is an angiotensin receptor blocker (ARB) that specifically blocks angiotensin II type-1 receptors (AT1R). Telmisartan has been proven to have antidiabetic effects via a variety of mechanisms, and it can be utilized in some diabetic patients due to its dual benefit for hypertensive patients with type 2 DM (T2DM) and when the other oral antidiabetic medications are intolerable or contraindicated. However, its precise underlying hypoglycemic mechanism is still obscure. Aim of work: We sought to establish a link between telmisartan administration and myostatin expression in skeletal muscles of T2DM rat model as a potential hypoglycemic mechanism of telmisartan. Materials and Methods: 32 male albino rats were included in the study; 8 rats served as controls (group I). T2DM was inducted in the other 24 rats, which were then randomly subdivided into 3 groups (8 in each): (group II) the Diabetic group and (groups III and IV) which were treated with either telmisartan (8mg/kg/day) or metformin (250mg/kg/day) respectively via oral gavage for a 4-week period. Results: Telmisartan administration resulted in a significant improvement in OGTT, HOMA-IR, glucose uptake, and muscle mass/body ratios in Telmisartan group as compared to Diabetic group (p < 0.05). Additionally, telmisartan induced a significant boost in adiponectin and IL-10 serum levels with a substantial drop in TNF-α and IL-6 levels in Telmisartan group compared to diabetic rats (p < 0.05). Moreover, telmisartan significantly boosted SOD and GSH, and decreased MDA levels in the skeletal muscles of telmisartan group. Furthermore, a significant downregulation of myostatin and upregulation of insulin receptor, IRS-1, and IRS-3 genes in the skeletal muscles of Telmisartan group were also detected. Histologically, telmisartan attenuated the morphological damage in the skeletal muscle fibers compared to diabetic rats, as evidenced by a considerable decrease in the collagen deposition area percentage and a reduction in NF-kB expression in the muscle tissues of group III. Conclusion: Telmisartan administration dramatically reduced myostatin and NF-kB expressions in skeletal muscles, which improved insulin resistance and glucose uptake in these muscles, highlighting a novel antidiabetic mechanism of telmisartan in treating T2DM.

Acquired Resistance to PRMT5 Inhibition in Mantle Cell Lymphoma Is Associated with Compensatory Activation of Multiple Signaling Pathways

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin's lymphoma. MCL patients who relapse on targeted therapies have a particularly poor prognosis. Protein arginine methyltransferase 5 (PRMT5), an enzyme that drives symmetric dimethylation of arginine (SDMA) residues, is overexpressed in MCL and promotes MCL growth and survival. Thus, PRMT5 has emerged as an attractive therapeutic target in MCL. We developed a small molecule PRMT5 inhibitor (PRT-382) that exhibits significant anti-MCL activity in cell lines (50-100 nM) and in vivo preclinical MCL murine models (10 mg/kg, daily, four days on; 3 days off). Despite the anti-tumor activity of PRMT5 inhibition, we have observed some animals develop drug resistance leading to rapid progression of MCL despite continued treatment with PRT-382. While treatment with PRT-382 significantly prolonged the survival of our MCL patient derived xenograft (PDX) model (83D vs. 48D, p-value: 0.0045), one mouse developed a rapid and much greater expansion of MCL cells both in the peripheral blood and in multiple organs when it reached early removal criteria at day 90. Despite the significantly increased tumor burden in this mouse while on PRMT5 inhibitory therapy, SDMA reduction in MCL tumor cells was still achieved in comparison to the vehicle control treated mice. MCL tumor cells from this PRMT5 inhibitor refractory mouse and PRMT5 inhibitor naïve mice were re-engrafted and treated with PRT-382. Compared to mice engrafted with treatment naïve cells, the mice engrafted with PRMT5 inhibitor refractory cells had decreased survival (p-value: 0.004). Thus, in vivo resistance was retained allowing generation of additional samples for mechanistic studies. In addition to in vivo models of resistance, some MCL cell lines (Maver, Mino, UPN1, and Jeko) show primary resistance to PRMT5 inhibition based on high IC50s of PRT-382 (mean: 800; range: 300-2000 nM) compared to the sensitivity to this drug in other MCL cell lines (mean: 80; range: 50-100 nM). Prolonged culture of four PRT-382 sensitive MCL lines (CCMCL, Rec-1, SP53, and Z-138) with drug escalation produced cell lines with acquired PRT-382 resistance with IC50s 3 to 5 times (200-500 nM) that of parental lines. We found this PRMT5 inhibitor resistance persisted after prolonged culture (30d) in the absence of drug. As observed in the MCL PDX PRMT5 inhibitor refractory mice, SDMA reduction was still achieved at the original PRT-382 doses. Utilizing single cell RNA sequencing (scRNA-seq) and bulk RNA-sequencing, these multiple in vivo and in vitro PRMT5 inhibitor resistant samples were evaluated in comparison with their parental/untreated counterparts to identify compensatory pro-survival pathways amplified with PRMT5 inhibitor resistance. ScRNA-seq analysis on MCL PDX samples demonstrated a strong shift in global gene expression depending on treatment duration and condition. Upregulation of Insulin Receptor, PI3K, MAPK, and MTOR signaling were identified using Ingenuity Pathway Analysis (p-value threshold of 0.001 and z-score of 1.1) based on differentially expressed genes with PRMT5 inhibitor treatment in the resistant cell lines and PDX in comparison to their sensitive or parental counterparts. In vitro drug combination analysis has demonstrated synergy inhibiting PRMT5 (PRT-382) in combination with PI3K/MTORC1 and 2 (Omipalisib), MTORC1 (Temsirolimus), and EIF1A (Silvestrol) in MCL cell lines with primary (Mino) and acquired (Rec-1, SP53, and Z-138) resistance to PRMT5 inhibition. We are currently mechanistically validating these pathways in our in vitro models and invivo preclinical MCL models.

Insulin dysregulation as a prominent driver of cell cycle re-entry in Alzheimer's disease.

Cell Cycle Re-entry (CCR) is observed in several neurological disorders and contributes to cognitive impairment in the early stages. The CCR markers were monitored and correlated functionally with upregulation of Insulin Receptor (INSR) and Insulin like Growth factor 1 Receptor (IGF1R) in Alzheimer's Disease (AD) cell model and mouse brain tissue. The AD cell model was developed as per Majumder et al. (2019) and checked for Cyclin A, D and E Levels after 6 and 11 hours of model development in differentiated SHSY5Y cell line (with 10 µM RA and BDNF for cholinergic neuron like cells) by qPCR. We then checked for expression and activity of INSR and IGF1R in these cells and mouse brain (extracted from FFPE slides) through western blot and qPCR. To assess the correlation of the two events, we exploited overexpression based in-vitro studies for real-time assessment. Levels of INSR and IGF1R both increase on mRNA and protein levels in both cell and animal model. The cyclins A2, D and E however decline when the receptors are overexpressed in a serum free media for 6 hours and 11 hours. The time points were chosen to mimic the average mammalian cell cycle and showed that the upregulation of the receptors negatively impact and reduce cyclin D levels up to 4 fold whereas Cyclin A levels and Cyclin E levels are marginally reduced by 0.48 and 0.5 fold respectively. However, when stimulated with conventional ligands, Insulin and IGF1 or amyloid beta (1-42) the receptors positively correlate with the Cyclin levels. Western blots show that amyloid beta binding phosphorylates the pY1135/1136 of IGF1R, pY1150/1151 of INSR rather than conventional pY1158, 1162 and 1163. The reduction in conventional ligands, Insulin and IGF1 with ageing and build-up of amyloid beta which preferentially phosphorylates a different site could have implications that determines cellular fate after the re-entry. Reference: Majumder, P., Roy, K., Bagh, S. and Mukhopadhyay, D., 2019. Receptor tyrosine kinases (RTKs) consociate in regulatory clusters in Alzheimer's disease and type 2 diabetes. Molecular and cellular biochemistry, 459(1), pp.171-182.

Dietary energy level affects adipose depot mass but does not impair in vitro subcutaneous adipose tissue response to short-term insulin and tumor necrosis factor-α challenge in nonlactating, nonpregnant Holstein cows

We assessed effects of overfeeding energy to nonlactating and nonpregnant Holstein cows during a length of time similar to a typical dry period on body lipid storage and the abundance of genes related to insulin signaling, inflammation, and ubiquitination in subcutaneous adipose tissue (SAT) in vitro challenged with insulin and recombinant bovine tumor necrosis factor-α. Fourteen cows were randomly assigned to either a high-energy (OVE; net energy for lactation = 1.60 Mcal/kg of dry matter; n = 7) or control (CON; net energy for lactation = 1.30 Mcal/kg of dry matter; n = 7) diet for 6 wk. Immediately after slaughter, liver, kidneys, and mammary gland were separated and weighed. The adipose tissue mass in the omental, mesenteric, and perirenal depots was dissected and weighed. Subcutaneous adipose tissue was collected from the tail-head region and was used as follows: control, bovine insulin (INS) at 1 µmol/L, tumor necrosis factor-α at 5 ng/mL (TNF), and their combination. Despite a lack of difference in final body condition score, OVE cows had greater energy intake and were heavier than CON cows. Furthermore, overfeeding led to greater mass of mesenteric and perirenal adipose, liver, and mammary gland. Overall, SAT incubated with INS had an upregulation of insulin receptor (INSR), interleukin-10 (IL10), small ubiquitin-like modifier 3 (SUMO3), and ubiquitin conjugating enzyme E2I (UBC9), whereas TNF upregulated peroxisome proliferator-activated receptor gamma (PPARG), diacylglycerol O-acyltransferase 2 (DGAT2), interleukin-6 (IL6), nuclear factor kappa B subunit 1 (NFKB1), small ubiquitin-like modifier 2 (SUMO2), and UBC9. Regardless of in vitro treatment, feeding OVE upregulated PPARG, fatty acid synthase (FASN), and insulin induced gene 1 (INSIG1). Abundance of PPARG was greater in SAT of OVE cows cultured individually with INS and TNF. The interaction between diet and in vitro treatment revealed that sterol regulatory element binding transcription factor 1 (SREBF1) had greater abundance in SAT from the CON group in response to culture with INS, whereas SAT from OVE cows had greater SREBF1 abundance in response to culture with TNF. The mRNA abundance of IL6 and NFKB1 was greater in response to TNF treatment and overall in CON cows. Furthermore, SAT from these cows had greater IL10 abundance when cultured with INS and TNF. Overall, data highlighted that overfeeding energy increases adipose tissue mass in part by stimulating transcription of key genes associated with insulin signaling, adipogenesis, and lipogenesis. Because SAT thickness or mass was not measured, the lack of effect of overfeeding on body condition score limits its use to predict overall body lipid storage. An overt inflammatory response in SAT after a 6-wk period of over-consumption of energy could not be discerned.

Abstract 4248: Functional imaging markers for blockade of breast cancer metastasis by IGF1R and insulin receptor targeted drugs using novel MRI and targeted iron oxide nanoparticles

Abstract IGF1R and insulin receptor (IR) regulate biology of estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. The results of initial clinical trials with anti-IGF1R drugs have been disappointing due to feedback upregulation of insulin receptor (IR) signaling and absence of biomarkers for these drugs. We evaluated the effects of BMS-754807, a dual tyrosine kinase inhibitor of IGF1R/IR on ER+ and TNBC. In ER+ MCF-7 cells, it inhibited xenograft growth of MCF-7 tumors (n = 10 per treatment) compared to vehicle. Interestingly, while tumor growth was suppressed over a period of five weeks, eventually the tumors displayed resistance to BMS-754807. In TNBC, it inhibited motility in vitro. In contrast to ER+ cells, BMS-754807 did not inhibit primary tumor growth of TNBC cells injected into the mammary fat pad of mice. But at a dose of 50 mg/kg daily inhibited metastasis of TNBC cells, MDA-231-LM2 and MDA-MB435A/LCC6 cells, in the orthotopic and tail vein models of metastasis compared to vehicle (n = 10/group). Our data indicate that regulation of metastasis and tumor growth by IGF1R can be discrete events and functional imaging to identify biological properties of metastatic breast cancer regulated by IGF1R/IR are needed to better define treatments. While MRI is a powerful tool for detecting and imaging cancer, its utility in imaging metastasis to the lung is limited due to the challenges of lung MRI with conventional 3D gradient echo (GRE). MRI does not visualize lung well, mainly due to the abundance of air-tissue interfaces, which cause the MR signal to decay too rapidly for conventional MRI pulse sequences to capture. Clinically metastases are monitored by CT or PET but exposure of patients to ionizing radiation is a concern and problematic in longitudinal studies monitoring response to a targeted drug. Therefore, we recently reported the utility of a novel MR sequence called sweep imaging with Fourier transformation (SWIFT), where the data is acquired quasi-simultaneously with the radiofrequency pulse, to image lung metastasis of breast cancer. Here, we monitored response to IGF1R and IGF1R/IR targeted drugs in preclinical models of lung metastasis of breast cancer. We used MDA-231-LM2 cells with the tail vein injection model of metastasis. Mice with breast cancer metastases in the lungs were treated with either huEM164, an antibody against IGF1R, or BMS-754807. Metastasis was monitored by BLI and SWIFT MRI weekly. SWIFT was more sensitive in detecting inhibition of metastasis by these drugs. Thus, dual inhibition of IGF1R and IR is effective in blocking growth of ER+ and metastasis of TNBC. However, combination of this therapeutic strategy with other agents may be necessary to prevent or delay onset of resistance. Further, noninvasive biomarkers of response to IGF1R/IR targeted drugs can be developed with SWIFT imaging. Citation Format: Deepali Sachdev, Huy Donguyen, Naoharu Kobayashi, Sidath C. Kumarapperuma, Joeseph C. Weber. Functional imaging markers for blockade of breast cancer metastasis by IGF1R and insulin receptor targeted drugs using novel MRI and targeted iron oxide nanoparticles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4248.

Corn gluten hydrolysate and capsaicin have complimentary actions on body weight reduction and lipid-related genes in diet-induced obese rats

The aim of this study was to test the hypothesis that a combination of corn gluten hydrolysate (CGH) and capsaicin may have an additive or synergistic effect on body weight reduction. For 13 weeks, male Sprague-Dawley rats were provided a diet to induce obesity. Afterward, the rats were randomly divided into 5 dietary groups: the normal control (n = 5), the high-fat control (n = 8), the high-fat diet (HFD) containing 35% CGH (n = 7), the HFD containing 0.02% capsaicin (HF-P) (n = 8), and the HFD containing both CGH and capsaicin (HF-CP) (n = 7) for an additional 4 weeks. Administration of CGH plus capsaicin, along with a HFD, led to significant decreases in body weight, fat mass, lipids in the liver, and plasma leptin as well as increases in plasma adiponectin. The pattern of gene expression was different in each target organ. In the liver, up-regulation of peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1α, and acyl-coenzyme A oxidase was found in the HF-CP group. In contrast, down-regulation of peroxisome proliferator-activated receptor γ was found in both the HFD containing 35% CGH and HF-CP groups. In skeletal muscle, up-regulation of insulin receptor and uncoupling protein 3 was found in the HF-P group only, whereas up-regulation of the glucose transporter 4 gene was observed in both the HF-CP and HF-P groups. In adipose tissue, up-regulation of peroxisome proliferator-activated receptor γ and hormone-sensitive lipase was only found in the HF-CP group. In summary, this study suggests that CGH and capsaicin perform complementary actions on food intake, lipid metabolism, and insulin sensitivity by a coordinated control of energy metabolism in the liver, adipose tissue, and skeletal muscle, thus exerting an additive effect on body weight reduction.

Abstract B9: Activated MAPK Pathway Mediates Resistance to Romidepsin via Bim Degradation in Romidepsin-Selected HuT 78 Cells

Abstract Acquired and intrinsic resistance to histone deacetylase inhibitors (HDIs), a new targeted group of anti-tumor agents, limits their clinical efficacy. A detailed understanding of the mechanisms of resistance to HDIs may lead to strategies designed to increase clinical efficacy. To investigate molecular mechanisms of resistance to the HDI romidepsin (Dp), we studied a cutaneous T-cell lymphoma (CTCL) cell line, HuT 78, independently selected in verapamil (Vp) or valspodar (PSC833) to prevent the emergence of P-glycoprotein (Pgp), a known resistance mechanism. The HuT 78 sublines DpVp 50 and DpP 75 display 100-200-fold resistance to romidepsin, not due to Pgp expression. A custom-made Taqman low density gene expression array detected increased expression of insulin receptor (IR) in the resistant cells. Real-time PCR analysis confirmed the results of gene array and detected more than 50-fold upregulation of IR in the romidepsin-selected cells compared to the parental cells. Increased phosphorylation (5- to 8- fold) of mitogen activated protein kinase kinase (MEK), a downstream effector of the IR pathway, was also observed in the resistant cells compared to the parental cells. While HuT 78 cells were insensitive to MEK inhibition, resistant cells were found to be exquisitely sensitive to MEK inhibition (IC50 &amp;lt; 10 nM) but not to phosphatidylinositol 3-kinase (PI3K) inhibition. Combined treatment of romidepsin with low concentrations (1- 3 nM) of MEK inhibitor also resulted in increased cell death in romidepsin-resistant HuT 78 cells. The exquisite sensitivity to MEK inhibition in the resistant sublines was found to correlate with restoration of the expression of Bim (BCL2L11), a Bcl-2-homology domain-3 only (BH-3) proapoptotic protein. In the resistant cells, the MAPK pathway appeared to regulate Bim expression posttranslationally as we did not detect any gene induction of BCL2L11 or FOXO3, a transcription factor known to activate BCL2L11 expression, following MEK inhibition. These findings implicate increased activation of the MAPK pathway as a mechanism of resistance to romidepsin, and suggest that combining romidepsin with MEK inhibitors may be an effective strategy to overcome resistance.

Abstract 4825: Inhibition of AKT abrogates resistance to endocrine therapy in estrogen receptor positive breast cancer

Abstract Estrogen receptor α-positive (ER+) breast cancers eventually adapt to hormone deprivation and acquire resistance to aromatase inhibitors. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Herein we investigated the effects of AKT inhibition on the hormone-independent growth of four ER+ breast cancer cell lines (MCF-7, ZR75-1, MDA-MB-361 and HCC-1428) with acquired resistance to estrogen deprivation (termed long-term estrogen-deprived cells, LTED). Treatment with the ATP-competitive AKT inhibitor AZD5363 inhibited phosphorylation of the AKT/mTOR substrates PRAS40, GSK3β/α, and S6K while inducing hyperphosphorylation of AKT at S473 and T308. AZD5363 suppressed the hormone-independent growth of 3/4 LTED lines. AZD5363 prevented the emergence of hormone-independent MCF-7, ZR75-1, and MDA-361 cells. Further, treatment of ovariectomized athymic mice with AZD5363 suppressed the hormone-independent growth of ER+/PI3K mutant MCF-7 xenografts. Combined treatment with AZD5363 and the ER downregulator fulvestrant more effectively suppressed xenograft growth than either drug alone. AZD5363 also caused dose-dependent inhibition of an ER+ breast cancer explant model unresponsive to fulvestrant or tamoxifen. We next examined whether AKT inhibition results in feedback upregulation of mitogenic signaling pathways. Treatment with AZD5363 resulted in upregulation and phosphorylation of the HER3 receptor tyrosine kinase (RTK), as well as upregulation of insulin receptor (InsR) protein levels and phosphorylation of Src at Y416. A Src family kinase inhibitor suppressed AZD5363-induced upregulation of P-HER3 in MCF-7/LTED cells. Src inhibition or siRNA-mediated knockdown of InsR or IGF-IR significantly enhanced the growth inhibitory effects of AZD5363. Following treatment with AZD5363, phospho-RTK array analysis revealed increased phosphorylation of multiple RTKs in MCF-7/LTED and/or ZR75-1/LTED cells, including InsR, IGF-IR, EGFR, HER2, HER3, HER4, Dtk, and FGFR3. Treatment with AZD5363 also upregulated RTK mRNA levels. Further, treatment of MCF-7/LTED cells with AZD5363 resulted in marked translocation of AKT PH-GFP to the membrane, reflective of increased PIP3 production and, thus, AKT phosphorylation at T308. Pre-treatment with the PI3K inhibitor BKM120 or the IGF-IR/InsR TKI AEW541 prevented AZD5363-induced membrane localization of PH-GFP. These results suggest that 1) AKT signaling is critical for hormone-independent growth of ER+ breast cancer cells in vitro and in vivo; and 2) AKT inhibition in these cells induces feedback upregulation of RTK expression and activity. Thus, inhibitors of AKT merit evaluation as a potential treatment for endocrine-resistant breast cancer. The combination of these agents with inhibitors of upstream RTKs are under evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4825. doi:1538-7445.AM2012-4825

Excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents

Although many animal studies indicate insulin has cardioprotective effects, clinical studies suggest a link between insulin resistance (hyperinsulinemia) and heart failure (HF). Here we have demonstrated that excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents. Chronic pressure overload induced hepatic insulin resistance and plasma insulin level elevation. In contrast, cardiac insulin signaling was upregulated by chronic pressure overload because of mechanical stretch-induced activation of cardiomyocyte insulin receptors and upregulation of insulin receptor and Irs1 expression. Chronic pressure overload increased the mismatch between cardiomyocyte size and vascularity, thereby inducing myocardial hypoxia and cardiomyocyte death. Inhibition of hyperinsulinemia substantially improved pressure overload-induced cardiac dysfunction, improving myocardial hypoxia and decreasing cardiomyocyte death. Likewise, the cardiomyocyte-specific reduction of insulin receptor expression prevented cardiac ischemia and hypertrophy and attenuated systolic dysfunction due to pressure overload. Conversely, treatment of type 1 diabetic mice with insulin improved hyperglycemia during pressure overload, but increased myocardial ischemia and cardiomyocyte death, thereby inducing HF. Promoting angiogenesis restored the cardiac dysfunction induced by insulin treatment. We therefore suggest that the use of insulin to control hyperglycemia could be harmful in the setting of pressure overload and that modulation of insulin signaling is crucial for the treatment of HF.

Iron Depletion by Deferoxamine Up-Regulates Glucose Uptake and Insulin Signaling in Hepatoma Cells and in Rat Liver

Iron depletion improves insulin resistance in patients with nonalcoholic fatty liver disease and diabetes and also stabilizes the hypoxia-inducible factor (HIF)-1, resulting in increased glucose uptake in vitro. This study investigated the effect of iron depletion by deferoxamine on insulin signaling and glucose uptake in HepG2 hepatocytes and in rat liver. In HepG2 cells, deferoxamine stabilized HIF-1alpha and induced the constitutive glucose transporter Glut1 and the insulin receptor. Up-regulation of insulin receptor by deferoxamine was mimicked by the intracellular iron chelator deferasirox and the hypoxia inducer CoCl2 and required the HIF-1 obligate partner ARNT/HIF-1beta. Iron depletion increased insulin receptor activity, whereas iron supplementation had the opposite effect. Deferoxamine consistently increased the phosphorylation status of Akt/PKB and its targets FoxO1 and Gsk3beta, which mediate the effect of insulin on gluconeogenesis and glycogen synthesis, and up-regulated genes involved in glucose uptake and utilization. Iron depletion of Sprague-Dawley rats increased HIF-1alpha expression, improved glucose clearance, and was associated with up-regulation of insulin receptor and Akt/PKB levels and of glucose transport in hepatic tissue. Conversely, gluconeogenic genes were not affected. In rats with fatty liver because of a high-calorie and high-fat diet, glucose clearance was increased by iron depletion and decreased by iron supplementation. Thus, iron depletion by deferoxamine up-regulates glucose uptake, and increases insulin receptor activity and signaling in hepatocytes in vitro and in vivo.

Rescue and regeneration of injured peripheral nerve axons by intrathecal insulin

Insulin peptide, acting through tyrosine kinase receptor pathways, contributes to nerve development or repair. In this work, we examined the direction, impact and repertoire of insulin signaling in vivo during peripheral nerve regeneration in rats. First, we demonstrated that insulin receptor is expressed on lumbar dorsal root ganglia neuronal perikarya using immunohistochemistry. Immunoblots and polymerase chain reactions confirmed the presence of both α and β insulin receptor subunits in dorsal root ganglia. In vivo and in vitro assessment of dorsal root ganglion neurons showed preferential localization of insulin receptor to perikaryal sites. In vivo, intrathecal delivery of fluorescein isothiocyanate-labeled insulin identified localization around dorsal root ganglia neurons. The direction and impact of potential insulin signaling was evaluated by concurrently delivering insulin or carrier over a 2 week period using mini-osmotic pumps, either intrathecally, near nerve, or with both deliveries, following a selective sural nerve crush injury. Only intrathecal insulin increased the number and maturity of regenerating sensory sural nerve axons distal to the crush site. As well, only intrathecal insulin rescued retrograde loss of sural axons after crush. In a separate experiment, insulin also rescued retrograde loss and atrophy of deep peroneal, largely motor, axons post-injury. Intrathecal insulin increased the expression of calcitonin-gene-related peptide in regenerating sprouts, increased the number of visualized regenerating fiber clusters, and reduced downregulation of calcitonin-gene-related peptide in dorsal root ganglia neurons. Insulin delivered intrathecally does not appear to influence expression of insulin-like growth factor-1 at dorsal root ganglion neurons or near peripheral nerve injury, but was associated with upregulation of insulin receptor α subunit in dorsal root ganglia. Intrathecal insulin delivery was associated with greater recovery of thermal sensation and longer distances to stimulus response with the pinch test following sural nerve crush. Insulin signaling at neuron perikarya can drive distal sensory axon regrowth, rescue retrograde alterations of axons and alter axon peptide expression. Moreover, such actions are associated with upregulation of its own receptor.

C-peptide levels and the duration of lactational amenorrhea

Changes in insulin sensitivity play an important role in directing metabolic energy toward milk production in lactating mammals. In rats, prolactin causes upregulation of insulin receptors in mammary tissue together with an increase in peripheral insulin resistance, leading to a preferential allocation of energy to support milk production (1, 2). In humans, the shift in energy allocation during lactation away from peripheral tissues and toward milk production is correlated with lower insulin levels (2–4).

A disturbance in the neuronal insulin receptor signal transduction in sporadic Alzheimer's disease.

Disturbances of glucose and energy metabolism are hypothesized as pathogenetic factors in sporadic dementia of Alzheimer type (SDAT). Insulin and is receptors play an important role in the regulation of brain glucose metabolism and neuronal growth. In postmortem brain cortex in SDAT, the densities of brain insulin receptors were decreased compared to adult controls, but were increased in relation to aged controls. Tyrosine kinase activity, a signal transduction mechanism common to insulin and IGF-1 receptors, was reduced in SDAT in comparison to middle-aged and age-matched control groups. The data are consistent with a neurotrophic role of insulin in the human brain and an upregulation of insulin receptors is SDAT brain as a compensatory mechanism, possibly due to impaired signal transduction mechanism.

P–35 - A role of PKC-α isoform in up-regulation of insulin receptor caused by phorbol esters in adrenal chromaffin cells

P–35 - A role of PKC-α isoform in up-regulation of insulin receptor caused by phorbol esters in adrenal chromaffin cells

Up-regulation of insulin receptors with dexamethasone in cultured human urinary bladder carcinoma cells.

Cultured human urinary bladder carcinoma cells ( JTC -32) were used to investigate the regulation of insulin receptors by dexamethasone. When the cells were preincubated with dexamethasone at 37 degrees C, insulin binding sites increased up to 24 h. A large increase in insulin binding sites took place for 14 h of preincubation with dexamethasone. At lower concentrations of dexamethasone (less than 1 nM), no significant increase in insulin binding sites was observed, but the maximal increase was observed at more than 10 nM. Scatchard plots showed that dexamethasone increased the number of high affinity insulin binding sites (2.8 fold) without any change in the apparent equilibrium constant in JTC -32 cells. In addition, this steroid hormone also increased the number of low affinity insulin binding sites (1.6 fold) with a small change in the apparent equilibrium constant. Although insulin and dexamethasone did not affect the number of cells or the amount of cellular proteins per dish, dexamethasone plus insulin slightly increased them.

Up-regulation of insulin receptors in rat liver plasma membranes.

Kinetic experiments (uptake versus time) were utilized to examine the effects of occupancy on insulin receptor availability in rat liver plasma membranes in vitro. The following observations were made: 1) at 4 degrees C, a 3-h exposure of membranes to 100 nM native insulin, followed by removal of unbound hormone, resulted in a subsequent decrease of 125I-insulin binding at 4 degrees C. In a similar experiment at 23 degrees C, no decrease of 125I-insulin binding was observed. 2) At 23 degrees C, 131I-insulin (5 nM) was bound to membranes in a slowly reversible manner after a 3-h association. After removal of free hormone, the 131I-insulin-treated membranes displayed similar binding of 125I-insulin (1 nM) relative to controls despite persistent high level occupancy of receptors by 131I-insulin. 3) At 23 degrees C, phospholipase pretreatment of membranes enhanced 125I-insulin uptake (approximately 40%). Phospholipase-digested membranes exposed to 100 nM native insulin for 3 h bound more 125I-insulin (approximately 40%) than did nondigested membranes preincubated without native insulin. The results allowed speculation that rat liver membranes up-regulated insulin receptors after treatment with insulin and that this was mediated by exposure of cryptic binding sites.