Ann Clin Biochem. [23]. This dose was chosen on the basis of preliminary studies showing activation of the retinal insulin receptor signaling pathway without reducing blood glucose levels (Figure 1A). Quantitative PCR analysis was performed as described previously [26]. Yes We have shown that retinal insulin receptor signaling is disrupted in insulin-deficient diabetes but it was unclear whether it results from hyperglycemia and/or hypoinsulinemia. Fasting PG increased with age (r = 0.80, p < 0.01). A dose-response study of the blood glucose levels of non-diabetic control rats 30 minutes after subconjunctival administration of insulin was conducted and showed that doses over 0.0325 IU per 100 g of body weight significantly decreased blood glucose levels (A). Symptoms include frequent urination, lethargy, excessive thirst, and hunger. GnRH, gonadotropin-releasing hormone. Daily administration of phloridzin in diabetic animals restores glycemic levels close to normal by increasing renal glucose uptake and thus increased glucose excretion, leading to decreased blood glucose levels. 11 DiGiacomo JE, Hay WW Effect of hypoinsulinemia and hyperglycemia on fetal glucose utilization. Akt isoform-specific kinase assays were performed as previously described [25]. Cellular cholesterol and fatty acid homeostasis are transcriptionally regulated by three members of sterol regulatory element-binding protein (SREBP) family: SREBP-1a, SREBP-1c, and SREBP-2. min) was approximately 3 times higher than that previously observed by our group in the postabsorptive state in healthy subjects (15, 16). Thus, the retina responds to both tissue insulin availability and nutrient levels, similar to peripheral insulin-sensitive organs. Please enable it to take advantage of the complete set of features! Furthermore, insulin administered by subconjunctival injection was detected in the retinas as soon as 5 minutes after injection and was detectable for over an hour after injection (data not shown). Insulin-independent glycemic control was achieved by phloridzin therapy. Bethesda, MD 20894, Copyright Funding: This work was supported by National Institutes of Health -National Eye Institute grant EY20582, American Diabetes Association grant 7-08-RA-50, and the Jack and Nancy Turner Professorship at Penn State University. Diabetes pathology derives from the combination of hyperglycemia and hypoinsulinemia or insulin resistance leading to diabetic complications including diabetic neuropathy, nephropathy and retinopathy. Systemic insulin treatment allows for relatively good control of diabetes through mechanisms that can be attributed to normalization of the glycemic levels and activation of insulin signaling pathways in various tissues. However, the proportional contribution of each mechanism may vary, depending on underlying diabetes pathology or even the stage of disease. IRS, insulin receptor substrate. In most cases, the main cause is “insulin resistance.”. n≥4/group; *significantly different from control (P<0.05); #significantly different from diabetic (P<0.05). After performing the quality controls, background subtraction and intra-array normalization, GenomeStudio-exported files were imported into GeneSpring GX11.0 software (Agilent Technologies). Apoptosis was measured by two complementary approaches previously demonstrated to show corresponding results [24]. Results: We showed that while subconjunctival insulin administration only resulted in a reduction of the intensity of the GFAP staining in Müller cells, phloridzin treatment totally restored the original expression pattern of GFAP with the sole expression in the astrocytes. Following incubation and washes, the colorimetric solution was added and incubated until the colorimetric reaction developed. Retinas were homogenized by sonication in lysis buffer (see above) and 500 µg of tissue lysates were immuno-precipitated using anti-IR or anti-IGF1R antibodies (Santa Cruz), as described previously [13]. Affiliation Vitreous glucose was measured at time of harvest to monitor the effect of the therapeutic treatments on ocular glucose levels using a glucometer as previously described by Kirwin et al. As a result, your blood glucose levels remain high, increasing the risk of diabetic ketoacidosis , which exacerbates hyperglycemia. Competing interests: The authors have read the journal's policy and have the following conflicts: Penn State University holds intellectual property related to the use of periocular insulin to treat diabetic retinopathy. One study showed that insulin deficiency is the major driver of the defects in the basal hypothalamo-pituitary-adrenal function in diabetes [35] while another study recently used intraventricular insulin versus systemic phloridzin treatment to compare the respective roles of insulin and glycemia in the control of lipid in the brain during diabetes [7]. Further analysis of lower doses of insulin showed that administration of 20–40mIU of insulin had no systemic effect on serum glucose (B) or insulin (C) levels, while significantly increasing retinal insulin levels (D). Microarray data were analyzed using the MetaCore™ (Genego Inc.) software suite for pathway analysis to identify the most significant pathways affected by diabetes and reversed by the independent treatments. demonstrated that brain insulin deficiency, rather than hyperglycemia, was directly responsible for the reduced cholesterol biosynthesis observed during diabetes. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, https://doi.org/10.1371/journal.pone.0026498. These findings are important for understanding the contributions of factors in the diabetic milieu that contribute to diabetic retinopathy and may be modifiable to improve visual outcomes in patients. Since the two treatments have different effects on hyperglycemia and insulin levels, they were then used in combination to test for a potential additive effect. Epub 2013 Aug 28. Epub 2019 May 15. Department of Ophthalmology, Penn State College of Medicine, Hershey, Pennsylvania, United States of America, Current address: Division of Ophthalmology, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Japan. This condition is more common in Type 2, or non-insulin-dependent diabetics. Glycemia is the presence of sugar (glucose) in the blood - hyperglycemia indicates excess glucose in the blood while hypoglycemia refers to abnormally low presence of glucose in the blood. These data suggests that phloridzin could directly reverse the reactive gliosis induced by diabetes through the control of the expression of mDIA2 and potentially other proteins that regulate cytoskeleton remodeling [39]. After adding the stop solution, the colorimetric signal was measured with a fluorescence plate reader (SpectraMax Gemini EM; Molecular Devices) with excitation at 405 and 490 nm. There has been increasing interest in dissecting the relative contributions of hyperglycemia and hypoinsulinemia to the pathophysiology of diabetes and diabetes complications because current treatments for diabetes fail to normalize metabolism or eliminate the risk of complications, and hypoglycemia limits the use of intensive insulin therapy [1]. https://doi.org/10.1371/journal.pone.0026498.g005. No, Is the Subject Area "Cell death" applicable to this article? 1. Activation of the complement pathway has been demonstrated by increased protein expression of several factors such as C3, C4b, C9 and factor B in the vitreous of diabetic patients with proliferative diabetic retinopathy [34]. Retinal Akt serine 473 phosphorylation was significantly elevated in eyes receiving insulin (*p<0.05; A). The beneficial effects of local insulin on the viability of sensory neurons in the retina in this study parallel the improved nerve conduction velocity observed in diabetic rats following insulin injection adjacent to the sciatic nerve or intrathecally [31], [32]. We then studied the effects of both treatments on the insulin signaling pathway and its disruption during diabetes. Representative immunoblots and quantification of Akt phosphorylation in retinas of non-diabetic rats receiving subconjunctival insulin. Protein concentrations of tissue lysates were determined using a DC protein assay kit (Bio-Rad, Hercules, CA) and retinal insulin levels were normalized to total retinal protein concentration. Controls were prepared by omitting the primary antibody during the incubation; no specific staining could be detected in these controls. This is particularly noted in infants born to mothers who have uncontrolled diabetes. The first two pathways highlighted by the pathway analysis, G-protein signaling and IL-1 beta-dependent CFTR expression, along with the effect on ATP metabolism suggested an impact on retinal glial cells. Conceived and designed the experiments: PEF SFA AJB TWG. In keeping with these observations, transcriptomic analysis demonstrated that these treatments normalized partially overlapping sets of retinal genes that were altered by diabetes. Our pathway analysis of the effects of phloridzin and local insulin on the retinal transcriptome changes induced by diabetes clearly demonstrated that only local insulin specifically repressed retinal inflammation, particularly the complement activation. Untreated type 1 diabetes presents with hypoinsulinemia, due to the immune system attacking the body such that this produces beta cell dysfunction. Briefly, supernatants (500 µg of protein) of retinal tissue homogenates prepared as for the IR or IGF1R kinase assay were subjected to immunoprecipitation (1 h at 4°C) with 2 µg of anti-Akt-1 or Akt-3 primary antibody (Millipore). A major difference between the brain and the retina and the peripheral insulin-sensitive tissues is the blood-brain and blood-retinal barriers, which control nutrient exchange between the systemic circulation and these tissues. Also, while retinal insulin levels after subconjunctival injections are supra-physiologic, they did not cause any increase in retinal cell death (Figure 3). Most but not all patients with FBS have impaired glucose tolerance/diabetes range hyperglycemia after OGTT while maintaining normal HbA1c. These properties, which could be involved in the protective role of phloridzin against diabetes in retinal cells will be further investigated in the future. Specificity of the treatments was also observed when rats with longer duration of diabetes (12 weeks) were treated (Figure 2C and 2D). This observation again confirms the differential mechanisms and responses involved in the beneficial effects of the two treatments. Mutations in GLUT2, the gene for the liver-type glucose transporter, in patients with Fanconi-Bickel syndrome. A Cell Death Detection ELISA (Roche Diagnostics) was used according to the manufacturer's instructions with minor modifications. Clipboard, Search History, and several other advanced features are temporarily unavailable. We previously showed that systemic insulin therapy could restore retinal insulin signaling and prevent retinal cell death induced by diabetes [14], [18]. The vicious cycle of stress-induced hyperglycemia and hypoinsulinemia subsequently causes maladaptive responses in immune function, fuel production, and synthesis of mediators that cause further tissue and organ dysfunction . [Usefulness of oral glucose tolerance test (OGTT) in output patients with advanced chronic renal failure (CRF)]. The same study also showed increased mRNA expression of C3 and factor B in the retina from other diabetic patients with proliferative diabetic retinopathy (PDR). The systemic effects of both local insulin and phloridzin treatments were analyzed by monitoring glucose and insulin serum levels. Is the Subject Area "Insulin" applicable to this article? The authors found brain Srebf2 and Srebf1a decreased in diabetic mice and reversed by systemic insulin. Mutations in SLC2A2 gene reveal hGLUT2 function in pancreatic β cell development. The reversal observed when retinal insulin levels were increased suggests that this is also due to loss of ligand or ligand sensitivity. This prompted us to assess the effect of both treatments on gliosis, as indicated by Müller cell expression of glial fibrillary acid protein (GFAP) in the retinas of diabetic animals. Dermatophytes are fungi responsible for causing superficial infections. Pathway analysis of the specific effects of phloridzin treatment highlighted its impact on the reversal of the deregulation of G-protein signaling. Immunohistochemistry was performed as previously described [22]. In the present study, subconjunctival administration of low-dose insulin partially prevented this increased cell death as demonstrated by both reduction in the number of TUNEL-positive retinal cells and DNA fragmentation in the treated rats when compared to the untreated diabetic animals (Figure 3A and 3B). No, Is the Subject Area "Glucose" applicable to this article? Hyperglycemia (high blood glucose) means there is too much sugar in the blood because the body lacks enough insulin. Sowers JR(1), Standley PR, Ram JL, Jacober S, Simpson L, Rose K. Author information: (1)Department of Internal Medicine, Wayne State University School of Medicine, Detroit, Michigan 48236. Briefly, eyecups were embedded in OCT and snap-frozen in dry ice, cooled with 2-methylbutane, directly after enucleation. Interestingly over 200 of them were reversed by both treatment, but only a third of them were reversed to a similar extent. In patients with diabetes mellitus (DM), dermatophytosis is usually more severe and recurrent. In this study, we used this characteristic to independently study the effects of insulin-independent glycemic normalization and locally-restricted insulin signaling stimulation. Also of note, our previous study found that changes in13 of these 15 same mRNA biomarkers were significantly reversed by systemic insulin treatment [28]. Hyperinsulinemia-the … Total and LDL cholesterol as well as triglyceride concentrations were elevated. Fanconi-Bickel syndrome (FBS) is a rare autosomal recessive disorder characterized by the combination of hepatorenal glycogen accumulation and Fanconi-type nephropathy. These results suggest that hyperglycemia induces resistance to growth factor action in the retina and clearly demonstrate that both restoration of glycemic control and retinal insulin signaling can act through different pathways to both normalize diabetes-induced retinal abnormality and prevent vision loss. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Hyperglycemia has a slower onset and may occur over hours to days. Both fasting hypoglycemia and post-OGTT hyperglycemia seem to improve with age. First, we demonstrated that the increased retinal insulin levels after subconjunctival injection of insulin in control rats correlated with increased Akt phosphorylation when compared to basal levels, indicating the activation of the insulin signaling pathway and confirming the biological activity of the exogenous insulin reaching the retina (Figure 4A). This could be demonstrated by further studying the effect of local insulin on the reduction of fatty acid elongases observed in rats with untreated short-term diabetes [37]. It can link to insulin resistance and diabetes. n≥8/group; *significantly different from control the same day (P<0.05); #significantly different from untreated diabetic the same day (P<0.05). This hypothesis is supported by the fact that systemic insulin similarly restores Hs3st2 expression [44] and could explain why Müller cells remain partially activated (gliosis) despite local insulin-treatment while activation is totally reversed by phloridzin treatment and systemic insulin therapy [45], confirming that local insulin administration could complement systemic insulin treatment. Abstract. We have previously demonstrated that diabetes significantly increases retinal cell death within 4 weeks after the onset of hyperglycemia, systemic insulin administration restores depressed insulin receptor signaling and cell death, and intravitreal insulin injection restores retinal insulin receptor activity [14]. Briefly, quantitative PCR was performed using the 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), 384-well optical plates, and Assay-On-Demand (Applied Biosystems) gene specific primers and probes. https://doi.org/10.1371/journal.pone.0026498.g006. Chips were dried and subsequently scanned using a BeadArray Reader and images were imported into GenomeStudio software v2010.1 (Illumina Inc, San Diego, CA). One of the effects of elevated glucose is increased activation of the hexosamine biosynthesis pathway (HBP), which leads to increased addition of O-GlcNAc modifications which is known to be a mechanism of inhibition of protein action, including the insulin signaling pathway, by competition with activating phosphosites [43]. Would you like email updates of new search results? In all kinase assays equivalent efficiency of the immunoprecipitation was verified by immunoblot analysis of the immune complexes after sufficient decay of the radioactivity. Lo, The University of Hong Kong, Hong Kong, Received: August 18, 2011; Accepted: September 27, 2011; Published: October 26, 2011. here. Curr Diab Rep. 2018 Jun 13;18(7):46. doi: 10.1007/s11892-018-1016-2. Yes Basically, it’s occurs when you have too much insulin in your blood. No, Is the Subject Area "Blood sugar" applicable to this article? Untreated hyperglycemia can lead to serious health problems. Likewise, we demonstrated that twice daily subcutaneous administration of the SGLT1 and SGLT2 glucose transporter inhibitor phloridzin, restored normal glycemia without affecting systemic or ocular levels of insulin (Figure 2). Systemic insulin administration both restores depressed insulin signaling through increased ligand availability, and normalizes blood glucose levels through increased glucose uptake, storage and utilization in insulin sensitive tissues. Sections (10 µm) from each experimental group were mounted on the same slide. Remarkably, local insulin and phloridzin each reversed one of the 2 biomarkers not affected by systemic insulin (Figure 5E), QRT-PCR analysis of this panel of genes also allowed for the validation of the results of the microarray analysis as demonstrated by the good accordance between the levels of expression detected by both methods (Figure 5F). Phloridzin inhibits the sodium-linked glucose transporters SGLT1 and SGLT2 in kidney tubular and small intestinal epithelium [2]. Wrote the paper: PEF TWG. STZ-injected rats were considered diabetic when exhibiting blood glucose levels >13.9 mmol/l (250 mg/dl) within 5 days after diabetes induction (One-Touch meter; Lifescan, Milpitas, CA). [6] observed increased insulin secretion following phloridzin injections in a partially pancreatectomized rat model, while Lisato et al [5], similarly to us, showed no effect of phloridzin on serum insulin levels in a streptozotocin-induced diabetic rat model. Privacy, Help The retinal sections were counterstained with Hoecht to visualize nuclei layers (blue). We showed that phloridzin but not local insulin administration reduced ocular glucose levels. As previously demonstrated, Akt1 and Akt3 activities in the retina were reduced by diabetes (Figure 4D and 4F). https://doi.org/10.1371/journal.pone.0026498.g003. Briefly, Chips were incubated in a hybridization oven for 20 h at 58°C before being disassembled, washed and Streptavadin-Cy3 stained. Accessibility https://doi.org/10.1371/journal.pone.0026498.g004. Total RNA from retinal tissues was isolated with Tri-Reagent/BCP (Molecular Research Center, Cincinnati, OH) following standard methods and quality and quantity was assessed using the RNA 6000 Nano LabChip with an Agilent 2100 Expert Bioanalyzer (Agilent, Palo Alto, CA). broad scope, and wide readership – a perfect fit for your research every time. After stopping the kinase reaction by brief centrifugation, the supernatant was spotted onto filter papers that then underwent several washes before being analyzed for radioactive counts on a scintillation counter. Revised the manuscript: PEF MKL CENR RSJS MN SFA AJB TWG. Patients with FBS are relatively hypoinsulinemic. Surprisingly, no additive effect was observed, suggesting that both treatments use similar or converging parallel intracellular signaling pathways that are individually sufficient, at least for short-term cell survival. The means ± SEM and statistically significant differences are reported. Our analysis also showed that phloridzin specifically restored pathways regulated by interleukin-1 beta and nerve growth factor, the receptors for which and themselves are expressed by retinal glial cells [40], [41], [42], once more pointing out the role of Müller cells in the effects of phloridzin on diabetes. The array data are MIAME compliant and have been deposited in the ArrayExpress MIAME compliant database (accession # E-MTAB-771). Hyperglycemia Symptoms (High Blood Sugar) Hyperglycemia is defined as having an abnormally high blood glucose. Diabetic retinopathy is one of the major complications of diabetes but the selective impact of hyperglycemia and hypoinsulinemia on retinal cell response have not been determined in vivo. [4] used phloridzin to examine the insulin-independent effects of glycemic normalization on peripheral and hepatic insulin resistance.

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