Niosome-carvedilol protects DNA damage of supraphysiologic concentrations of insulin using comet assay: An in vitro study.

Introduction: Hyperinsulinemia occurs in type 2 diabetic patients with insulin resistance. This increase in insulin levels in the blood increases reactive oxygen species production and oxidative stress, resulting in DNA damage. Carvedilol (CRV) is a non-selective beta-blocker, and research has shown that this compound and its metabolites have anti-oxidative properties. Carvedilol can, directly and indirectly, reduce reactive oxygen species (ROS) and has a protective effect on DNA damage from oxidative stress. Given the insolubility of CRV in water, finding new methods to increase its solubility can be an essential step in research. This study aimed to determine whether carvedilol could have a protective effect on insulin-induced genomic damage. Methods: We treated cells with insulin alone, amorphous-CRV alone, and amorphous-CRV and niosomal-CRV with insulin and DNA damage were investigated using the comet method to achieve this goal. Results: Our results showed that insulin in the studied concentration has a significant genotoxic effect and non-cytotoxic at higher concentrations. CRV, both in amorphous and niosome form, reduced insulin-induced DNA damage by reducing ROS production. The comet assay results demonstrate that treating HUVEC cells in pretreatment condition with amorphous-CRV and niosome-CRV significantly reduces DNA damage of insulin.

Treatment-Induced Neuropathy in Diabetes (TIND)-Developing a Disease Model in Type 1 Diabetic Rats.

Treatment-induced neuropathy in diabetes (TIND) is defined by the occurrence of an acute neuropathy within 8 weeks of an abrupt decrease in glycated hemoglobin-A1c (HbA1c). The underlying pathogenic mechanisms are still incompletely understood with only one mouse model being explored to date. The aim of this study was to further explore the hypothesis that an abrupt insulin-induced fall in HbA1c may be the prime causal factor of developing TIND. BB/OKL (bio breeding/OKL, Ottawa Karlsburg Leipzig) diabetic rats were randomized in three groups, receiving insulin treatment by implanted subcutaneous osmotic insulin pumps for 3 months, as follows: Group one received 2 units per day; group two 1 unit per day: and group three 1 unit per day in the first month, followed by 2 units per day in the last two months. We serially examined blood glucose and HbA1c levels, motor- and sensory/mixed afferent conduction velocities (mNCV and csNCV) and peripheral nerve morphology, including intraepidermal nerve fiber density and numbers of Iba-1 (ionized calcium binding adaptor molecule 1) positive macrophages in the sciatic nerve. Only in BB/OKL rats of group three, with a rapid decrease in HbA1c of more than 2%, did we find a significant decrease in mNCV in sciatic nerves (81% of initial values) after three months of treatment as compared to those group three rats with a less marked decrease in HbA1c <2% (mNCV 106% of initial values, p ≤ 0.01). A similar trend was observed for sensory/mixed afferent nerve conduction velocities: csNCV were reduced in BB/OKL rats with a rapid decrease in HbA1c >2% (csNCV 90% of initial values), compared to those rats with a mild decrease <2% (csNCV 112% of initial values, p ≤ 0.01). Moreover, BB/OKL rats of group three with a decrease in HbA1c >2% showed significantly greater infiltration of macrophages by about 50% (p ≤ 0.01) and a decreased amount of calcitonin gene related peptide (CGRP) positive nerve fibers as compared to the animals with a milder decrease in HbA1c. We conclude that a mild acute neuropathy with inflammatory components was induced in BB/OKL rats as a consequence of an abrupt decrease in HbA1c caused by high-dose insulin treatment. This experimentally induced neuropathy shares some features with TIND in humans and may be further explored in studies into the pathogenesis and treatment of TIND.

Long-Term Nonclinical Pulmonary Safety Assessment of Afrezza, a Novel Insulin Inhalation Powder.

Afrezza delivers inhaled insulin using the Gen2 inhaler for the treatment of patients with type 1 and type 2 Diabetes. Afrezza was evaluated in long-term nonclinical pulmonary safety studies in 2 toxicology species. Chronic inhalation toxicology studies in rat (26 weeks) and dog (39 weeks) and an inhalation carcinogenicity study in rats were conducted with Technosphere insulin (Afrezza) and with Technosphere alone as a vehicle control. Respiratory tract tissues were evaluated by histopathology and cells expressing proliferating cell nuclear antigen (PCNA) were quantified in lungs of rats. Microscopic findings in rats exposed to Afrezza were attributed to the Technosphere particle component, were confined to nasal epithelia, and consisted of eosinophilic globules and nasal epithelial degeneration. There were no Afrezza-related changes in pulmonary PCNA labeling indices in alveoli, large bronchioles, or terminal bronchioles. Microscopic findings in rats exposed to Technosphere particles included eosinophilic globules, mucus cell hyperplasia, and epithelial degeneration in the nasal cavities. PCNA labeling indices were increased in large bronchioles and terminal bronchioles but not in alveoli. There were no Technosphere particle-related findings in the dog study. Afrezza did not exhibit carcinogenic potential in the 2-year study in rats. These nonclinical inhalation studies support the use of Afrezza in humans over extended periods.

Adipose stem cell secretome markedly improves rodent heart and human induced pluripotent stem cell-derived cardiomyocyte recovery from cardioplegic transport solution exposure.

Heart transplantation is a life-saving therapy for end-stage organ failure. Organ deterioration during transportation limits storage to 4 hours, limiting hearts available. Approaches ameliorating organ damage could increase the number of hearts acceptable for transplantation. Prior studies show that adipose-derived stem/stromal cell secretome (ASC-S) rescues tissues from postischemic damage in vivo. This study tested whether ASC-S preserved the function of mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iCM) exposed to organ transportation and transplantation conditions. Hearts were subjected to cold University of Wisconsin (UW) cardioplegic solution ± ASC-S for 6 hours followed by analysis using the Langendorff technique. In parallel, the effects of ASC-S on the recovery of iCM from UW solution were examined when provided either during or after cold cardioplegia. Exposure of hearts and iCM to UW deteriorated contractile activity and caused cell apoptosis, worsening in iCM as a function of exposure time; these were ameliorated by augmenting with ASC-S. Silencing of superoxide dismutase 3 and catalase expression prior to secretome generation compromised the ASC-S cardiomyocyte-protective effects. In this study, a novel in vitro iCM model was developed to complement a rodent heart model in assessing efficacy of approaches to improve cardiac preservation. ASC-S displays strong cardioprotective activity on iCM either with or following cold cardioplegia. This effect is associated with ASC-S-mediated cellular clearance of reactive oxygen species. The effect of ASC-S on the temporal recovery of iCM function supports the possibility of lengthening heart storage by augmenting cardioplegic transport solution with ASC-S, expanding the pool of hearts for transplantation.

Alternatives to Insulin for the Regulation of Blood Sugar Levels in Type 2 Diabetes.

This short overview focuses on the causation and treatment of type 2 diabetes (T2D). Emphasis is given to the historical basis of understanding this disease and the background leading to emergence of the central role of insulin. The strengths of insulin administration in the treatment of diabetes are profound, but these need to be balanced against several serious shortcomings of its extended use. Some alternative approaches to T2D management are considered. Insulin is no longer considered as the first choice for type 2 diabetes, and an expanding range of new therapeutic possibilities is emerging. While these may lack the potency of insulin, at a minimum, they allow a major reduction in the intensity of insulin use. In view of the rising worldwide incidence of this disease, it is imperative to develop safe and inexpensive means of limiting its potential for impairment of normal functioning.

Fermentable fiber-induced hepatocellular carcinoma in mice recapitulates gene signatures found in human liver cancer.

Hepatocellular carcinoma (HCC), the most malignant form of primary liver cancer, is the fourth most prevalent cause of cancer mortality globally. It was recently discovered that the dietary fermentable fiber, inulin, can reprogram the murine liver to favor HCC development in a gut microbiota-dependent manner. Determining the molecular pathways that are either over expressed or repressed during inulin-induced HCC would provide a platform of potential therapeutic targets. In the present study, we have combined analysis of the novel inulin-induced HCC murine model and human HCC samples to identify differentially expressed genes (DEGs) in hepatocarcinogenesis. Hepatic transcriptome profiling revealed that there were 674 DEGs in HCC mice compared to mice safeguarded from HCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered enrichment in ECM-receptor interaction, steroid hormone biosynthesis, PPAR signaling pathway, focal adhesion and protein digestion and absorption during inulin-induced HCC. Tandem mass tag based quantitative, multiplexed proteomic analysis delineated 57 differentially expressed proteins, where the over-expressed proteins were associated with cell adhesion molecules, valine, leucine and isoleucine degradation and ECM-receptor interaction. After obtaining the human orthologs of the mouse genes, we did a comparison analysis to level 3 RNA-seq data found in the Cancer Genome Atlas (TCGA) database, corresponding to human HCC (n = 361) and healthy liver (n = 50) samples. Out of the 549 up-regulated and 68 down-regulated human orthologs identified, 142 genes (137 significantly over-expressed and 5 significantly under-expressed) were associated with human HCC. Using univariate survival analysis, we found 27 over-expressed genes involved in cell-cell adhesion and cell division that were associated with poor HCC patient survival. Overall, the genetic and proteomics signatures highlight potential underlying mechanisms in inulin-induced HCC and support that this murine HCC model is human relevant.

Overlapping mechanism of the induction of genomic damage by insulin and adrenaline in human promyelocytic HL-60 cells.

Endogenous hormones systemically regulate the growth and metabolism and some prior studies have shown that their imbalance can have a potential to induce genomic damage in in vitro and animal models. Some conditions that are associated with elevated levels of endogenous hormones are hyperinsulinemia and intense exercise-induced stress causing increased adrenaline. In this study we test whether these two hormones, could cause an additive increase in genomic damage and whether they have an overlapping mechanism of action. For this, we use the human promyelocytic HL60 cells, as they express the receptors for both hormones. At doses taken from the saturation level of the individual dose response curves, no additivity in genomic damage was detected through micronucleus induction. This hints towards a common step in the pathway, which is under these conditions fully activated by each of the individual hormone. To investigate this further, individual and common parts in insulin and adrenaline signalling such as their respective hormone receptors, the downstream protein AKT and the involvement of mitochondria and NADPH oxidase (NOX) enzymes were studied. The results indicate no additive effect of high hormone concentrations in genomic damage in the in vitro model, which may be due to exhaustion of the NOX 2-mediated reactive oxygen production. It remains to be determined whether a similar situation may occur in in vivo situations.

Temporal analysis of histopathology and cytokine expression in the rat cerebral cortex after insulin-induced hypoglycemia.

Hypoglycemic coma causes neuronal death in the cerebral neocortex; however, its unclear pathogenesis prevents the establishment of preventive measures. Inflammation plays a pivotal role in neuronal damage in the hypoglycemic state; however, the dynamics of glial cell activation or cytokine expression remain unknown. Here, we aimed to elucidate the spatiotemporal morphological changes of microglia and time-course cytokine expression profiles in the rat cerebral cortex after hypoglycemic coma. We performed histopathological and immunohistochemical (Iba1, neuronal nuclei, glial fibrillary acidic protein) analyses in the cingulate cortex and four areas of the neocortex: hindlimb area (HL), parietal cortex area 1 (Par1), parietal cortex area 2 (Par2), and perirhinal cortex (PRh). We measured tumor necrosis factor alpha (TNFα) and interleukin-6 messenger RNA (mRNA) expression by real-time reverse transcriptase-polymerase chain reaction. Necrotic neurons appeared in the neocortex as early as 3 h after hypoglycemic coma, while they were absent in the cingulate cortex. Neuronal nuclei-immunopositive neurons in the HL, Par2, and PRh were significantly less abundant than in the control at day 1. In Iba1 immunostaining, large rod-shaped cells were detected at 3-6 h after hypoglycemia, and commonly observed in the HL, Par2, and PRh. After 6 h, rod-shaped cells were rarely observed; instead, there was a prominent infiltration of hypertrophic and ameboid-shaped cells until day 7. The mRNA expression of TNFα was significantly higher than the control at 3-6 h after hypoglycemia in the neocortex, while it was significantly higher only at 3 h in the cingulate cortex. Our results indicate that early and transient appearance of rod-shaped microglia and persisting high TNFα expression levels characterize inflammatory responses to hypoglycemic neuronal damage in the cerebral neocortex, which might contribute to neuronal necrosis in response to transient hypoglycemic coma.

Insulin Exacerbates Inflammation in Fibroblast-Like Synoviocytes.

Osteoarthritis (OA) is considered the most frequent degenerative disease and is characterized by cartilage degradation and synovial inflammation. Fibroblast-like synoviocytes (FLSs) are vital to synovial inflammation in OA. Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and hyperinsulinemia (HINS) and has been demonstrated to be an independent risk factor for OA. Autophagy is involved in the processes of various inflammatory diseases, and autophagy inhibition can stimulate OA development. Thus, we aimed to investigate the role of insulin in the inflammatory phenotype and autophagy of FLSs in OA. The data showed that cell viability and proinflammatory cytokine production in FLSs were both increased after insulin stimulation. We also found that high insulin could promote macrophage infiltration and chemokine production but inhibited autophagy in FLSs. To further explore the potential mechanisms, the effects of insulin on PI3K/Akt/mTOR and NF-ĸB signaling activation were evaluated. The results indicated that insulin activated PI3K/Akt/mTOR/NF-ĸB signaling, and the above-mentioned inflammatory responses, including autophagy inhibition, were notably attenuated by specific signaling inhibitors in the presence of high insulin. Moreover, the data showed that a positive feedback loop existed between proinflammatory cytokines (e.g., IL-1ß, IL-6, and TNF-α) and PI3K/mTOR/Akt/NF-ĸB signaling in FLSs, and insulin enhanced this feedback loop to accelerate OA progression. Our study suggests that insulin may be a novel therapeutic strategy for OA prevention and treatment in the future.

Acyl-ghrelin Is Permissive for the Normal Counterregulatory Response to Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia leads to far-ranging negative consequences in patients with diabetes. Components of the counterregulatory response (CRR) system that help minimize and reverse hypoglycemia and coordination between those components are well studied but not yet fully characterized. Here, we tested the hypothesis that acyl-ghrelin, a hormone that defends against hypoglycemia in a preclinical starvation model, is permissive for the normal CRR to insulin-induced hypoglycemia. Ghrelin knockout (KO) mice and wild-type (WT) littermates underwent an insulin bolus-induced hypoglycemia test and a low-dose hyperinsulinemic-hypoglycemic clamp procedure. Clamps also were performed in ghrelin-KO mice and C57BL/6N mice administered the growth hormone secretagogue receptor agonist HM01 or vehicle. Results show that hypoglycemia, as induced by an insulin bolus, was more pronounced and prolonged in ghrelin-KO mice, supporting previous studies suggesting increased insulin sensitivity upon ghrelin deletion. Furthermore, during hyperinsulinemic-hypoglycemic clamps, ghrelin-KO mice required a 10-fold higher glucose infusion rate (GIR) and exhibited less robust corticosterone and growth hormone responses. Conversely, HM01 administration, which reduced the GIR required by ghrelin-KO mice during the clamps, increased plasma corticosterone and growth hormone. Thus, our data suggest that endogenously produced acyl-ghrelin not only influences insulin sensitivity but also is permissive for the normal CRR to insulin-induced hypoglycemia.

Development and Validation of an Image Analysis System for the Measurement of Cell Proliferation in Mammary Glands of Rats.

Reliable detection and measurement of cell proliferation are essential in the preclinical assessment of carcinogenic risk of therapeutics. In this context, the assessment of mitogenic potential on mammary glands is crucial in the preclinical safety evaluation of novel insulins. The existing manual counting is time-consuming and subject to operator bias. To standardize the processes, make it faster, and resistant to errors, we developed a semiautomated image analysis system (CEPA software, which is open-source) for counting of proliferating cells in photomicrographs of mammary gland sections of rats labeled with Ki-67. We validated the software and met the predefined targets for specificity, accuracy, and reproducibility. In comparison to manual counting, the respective mean differences in absolute labeling indices (LIs) for CEPA software were 3.12% for user 1 and 3.05% for user 2. The respective regression analysis revealed a good correlation between the CEPA software user and manual counting. Moreover, the CEPA software showed enhanced reproducibility between independent users. The interuser variability is centered around 0 and the absolute difference was about 0.53% LI. Based on validation data, our software has superiority to the manual counting and is a valid and reliable tool for the routine analysis of cell proliferation in mammary glands from rats exposed to insulin analogs.

Ultrastructural examination of basement membrane pathology in horses with insulin-induced laminitis.

The third phalanx of the equine digit is suspended within the hoof capsule by a specialized interdigitating dermoepidermal layer called the lamellae, which fails during laminitis. Pathology of the basement membrane (BM), which interfaces epidermis and dermis, is evident during acute laminitis. However, BM damage appears to be less prevalent in ponies with the insulin-associated form of laminitis. The aim of the present study was to investigate changes to the ultrastructure and morphometry of the lamellar BM in the acute phase of insulin-induced laminitis in horses. Lamellar tissue from the left forefoot of 3 horses with acute hyperinsulinemic laminitis was examined with transmission electron microscopy and compared with tissue from normal horses. Lamellar BM width and hemidesmosome (HD) density were assessed every 5 µm along ∼200 µm of secondary epidermal lamellar BM. The BM zone of treated horses was extensively disorganized with loss of uniformity of the lamina lucida and lamina densa, fragmentation and disorientation of HDs, and cytoskeletal disengagement of the HDs. The mean (±SD) lamellar BM was twice as wide in treated (0.25 ± 0.05 µm), compared with control (0.14 ± 0.02 µm), horses. The HD density (HDs/µm) was reduced by half in the treatment group (1.88 ± 0.37), compared with controls (3.6 ± 0.13). The reduced number of HDs in horses with laminitis may contribute to the weakening of the dermoepidermal junction and lamellar failure. Disassembly of HDs during excessive cellular proliferation, secondary to hyperinsulinemia, may account for HD loss. Further investigation of the underlying etiopathogenesis of BM dysfunction during hyperinsulinemic laminitis in horses may facilitate an improved understanding of the disease.

Blockade of Acid-Sensing Ion Channels Attenuates Recurrent Hypoglycemia-Induced Potentiation of Ischemic Brain Damage in Treated Diabetic Rats.

Diabetes is a chronic metabolic disease and cerebral ischemia is a serious complication of diabetes. Anti-diabetic therapy mitigates this complication but increases the risk of exposure to recurrent hypoglycemia (RH). We showed previously that RH exposure increases ischemic brain damage in insulin-treated diabetic (ITD) rats. The present study evaluated the hypothesis that increased intra-ischemic acidosis in RH-exposed ITD rats leads to pronounced post-ischemic hypoperfusion via activation of acid-sensing (proton-gated) ion channels (ASICs). Streptozotocin-diabetic rats treated with insulin were considered ITD rats. ITD rats were exposed to RH for 5 days and were randomized into Psalmotoxin1 (PcTx1, ASIC1a inhibitor), APETx2 (ASIC3 inhibitor), or vehicle groups. Transient global cerebral ischemia was induced overnight after RH. Cerebral blood flow was measured using laser Doppler flowmetry. Ischemic brain injury in hippocampus was evaluated using histopathology. Post-ischemic hypoperfusion in RH-exposed rats was of greater extent than that in control rats. Inhibition of ASICs prevented RH-induced increase in the extent of post-ischemic hypoperfusion and ischemic brain injury. Since ASIC activation-induced store-operated calcium entry (SOCE) plays a role in vascular tone, next we tested if acidosis activates SOCE via activating ASICs in vascular smooth muscle cells (VSMCs). We observed that SOCE in VSMCs at lower pH is ASIC3 dependent. The results show the role of ASIC in post-ischemic hypoperfusion and increased ischemic damage in RH-exposed ITD rats. Understanding the pathways mediating exacerbated ischemic brain injury in RH-exposed ITD rats may help lower diabetic aggravation of ischemic brain damage.

Direct toxicity of insulin on the human placenta and protection by metformin.

OBJECTIVE: To study the effects of insulin and metformin on primary trophoblasts from early pregnancies. DESIGN: Experimental in vitro study. SETTING: Academic research institute. PATIENT(S): Trophoblasts from healthy patients undergoing first trimester elective termination of pregnancy and primary lung fibroblasts (IMR-90). INTERVENTION(S): Culture and treatment with insulin and metformin of primary trophoblasts and primary lung fibroblasts (IMR-90). MAIN OUTCOME MEASURE(S): DNA damage measured by expression of γ-H2AX with immunofluorescence and Western blot. Apoptosis measured by expression of cleaved caspase-3 by Western blot. Cell survival measured by cell proliferation assay. RESULT(S): Culture of purified primary trophoblast cells in the presence of insulin at levels as low as 1 nM resulted in a 386% increase in the number of cell with elevated γ-H2AX expression, a 66% reduction in cell survival and a marked increase of cleaved caspase-3 expression. Pretreatment of trophoblasts with therapeutic doses of metformin prevented the detrimental effects of insulin. Treatment with insulin and/or metformin had no effects on primary fibroblasts. CONCLUSION(S): Elevated insulin levels are directly toxic to first trimester trophoblasts and result in increased DNA damage, apoptosis, and decreased cell survival. These effects are prevented by metformin. Trophoblast cells from early pregnancy are uniquely vulnerable to elevated levels of insulin. These findings, if confirmed in vivo, suggest that there may be a role for insulin resistance screening before attempting pregnancy and for focusing on prevention of hyperinsulinemia during early pregnancy.

Protective effect of extractive and biotechnological chondroitin in insulin amyloid and advanced glycation end product-induced toxicity.

Glycosaminoglycans are extracellular matrix components related to several biological functions and diseases. Chondroitin sulfate is a sulphated glycosaminoglycan synthesized as part of proteoglycan molecules. They are frequently associated with amyloid deposits and possess an active role in amyloid fibril formation. Recently, a neuroprotective effect of extracellular matrix components against amyloid toxicity and oxidative stress has been reported. Advanced glycation end products (AGEs), the end products of the glycation reaction, have been linked to amyloid-based neurodegenerative disease as associated with oxidative stress and inflammation. In this study we have analyzed the effect of chondroitin sulfate isolated from different species, in comparison with a new biotechnological unsulfated chondroitin, in the amyloid aggregation process of insulin, as well as the ability to prevent the formation of AGEs and related toxicity. The results have showed a determining role of chondroitin sulfate groups in modulating insulin amyloid aggregation. In addition, both sulfated and unsulfated chondroitins have shown protective properties against amyloid and AGEs-induced toxicity. These data are very relevant as a protective effect of these glycosaminoglycans in the AGE-induced toxicity was never observed before. Moreover, considering the issues related to the purity and safety of chondroitin from natural sources, this study suggests a new potential application for the biotechnological chondroitin.

Protective effects of silibinin on insulin amyloid fibrillation, cytotoxicity and mitochondrial membrane damage.

A growing body of evidence suggests that secretion and assembly of insulin to amyloid fibrils reduce its efficacy in treating type II diabetes and may lead to dysfunctioning of several organs. The research presented here explores the effects of silibinin on the in vitro amyloid fibrillation and cytotoxicity of bovine insulin fibrils on SH-SY5Y human neuroblastoma cells. Interaction of the resulting structures with rat brain mitochondria was also investigated. Using a range of methods for amyloid detection we showed that insulin fibrillation was significantly inhibited by silibinin in a dose-dependent fashion. Moreover, we found that silibinin was very effective in attenuating insulin fibril-induced neuronal toxicity characterized by decrease of cell viability, the release of lactate dehydrogenase, intracellular reactive oxygen species enhancement, morphological alterations, and apoptotic cell death induction. While insulin fibrillation products showed the capacity to damage mitochondria, the resultant structures produced in the presence of silibinin were totally ineffective. Together, results demonstrate the capacity of insulin fibrils to cause SH-SY5Y cell death by inducing necrosis/apoptosis changes and suggest how silibinin may afford protection. It is concluded that elucidation of such protection may provide important insights into the development of preventive and therapeutic agents for amyloid-related diseases.

Aquaporin-4 deletion ameliorates hypoglycemia-induced BBB permeability by inhibiting inflammatory responses.

BACKGROUND: Severe hypoglycemia induces brain edema by upregulating aquaporin-4 (AQP4) expression and by degrading tight junctions. Acute severe hypoglycemia induces a proinflammatory environment that may contribute to a disruption in the epithelial barrier by decreasing tight junction protein expression. Interestingly, the altered AQP4 expression has been considered to play a critical role in neuroinflammation during acute brain injury. It has been shown that AQP4 deletion reduces brain inflammation in AQP4-null mice after intracerebral LPS injection. However, the effect of AQP4 deletion regarding protection against hypoglycemia-induced blood-brain barrier (BBB) breakdown is unknown. METHODS: An acute severe hypoglycemic stress model was established via injection of 4 unit/kg body weight of insulin. Evans blue (EB) staining and water measurement were used to assess BBB permeability. Western blot, reverse transcription polymerase chain reaction, and immunofluorescence were used to detect the expression of related proteins. The production of cytokines was assessed via enzyme-linked immunosorbent assay. RESULTS: Hypoglycemia-induced brain edema and BBB leakage were reduced in AQP4-/- mice. AQP4 deletion upregulated PPAR-γ and inhibited proinflammatory responses. Moreover, knockdown of aquaporin-4 by small interfering RNA in astrocytes co-cultured with endothelial cells effectively reduced transendothelial permeability and degradation of tight junctions. Treatment with PPAR-γ inhibitors showed that upregulation of PPAR-γ was responsible for the protective effect of AQP4 deletion under hypoglycemic conditions. CONCLUSIONS: Our data suggest that AQP4 deletion protects BBB integrity by reducing inflammatory responses due to the upregulation of PPAR-γ expression and attenuation of proinflammatory cytokine release. Reduction in AQP4 may be protective in acute severe hypoglycemia.

Murine model and mechanisms of treatment-induced painful diabetic neuropathy.

Diabetes mellitus represents a group of metabolic diseases that are characterized by hyperglycemia caused by either lack of insulin production or a reduced ability to respond to insulin. It is estimated that there were 347 million people worldwide who suffered from diabetes in 2008 and incidence is predicted to double by 2050. Neuropathy is the most common complication of long-term diabetes and approximately 30% of these subjects develop chronic neuropathic pain. A distinct acute, severe form of neuropathic pain, called insulin neuritis or treatment-induced painful neuropathy of diabetes (TIND), may also occur shortly after initiation of intensive glycemic control, with an incidence rate of up to 10.9%. The pathological mechanisms leading to TIND, which is mostly unresponsive to analgesics, are not yet understood, impeding the development of therapies. Studies to date have been clinical and with limited cohorts of patients. In the current study, we developed chronic and acute insulin-induced neuropathic pain in mice with type 2 insulin-resistant diabetes. Furthermore, we determined that insulin-induced acute allodynia is independent of glycemia levels, can also be induced with Insulin-like Growth Factor 1 (IGF1) and be prevented by inhibition of AKT, providing evidence of an insulin/IGF1 signaling pathway-based mechanism for TIND. This mouse model is useful for the elucidation of mechanisms contributing to TIND and for the testing of new therapeutic approaches to treat TIND.

Glycemia and the cardioprotective effects of insulin pre-conditioning in the isolated rat heart.

BACKGROUND: While acute hyperglycemia has been shown to mitigate the beneficial effects of ischemic preconditioning, its effect on insulin-induced preconditioning remains unclear. METHODS: The study was designed to test the hypothesis that acute hyperglycemia diminishes the cardioprotective effects following a 20-min pre-ischemic pre-conditioning with insulin in the isolated rat heart using the Langendorff system. Forty hearts were assigned to receive modified Krebs-Henseleit (KH) buffer containing 0.5 U/L insulin and 100 mg/dL glucose (InsG100, n = 10), KH buffer with 100 mg/dL glucose (G100, n = 10), KH buffer supplemented with 0.5 U/L insulin and 600 mg/dL glucose (InsG600, n = 10), or with 600 mg/dL glucose (G600, n = 10). To match the osmotic pressure of the InsG600 group, 27.5 mmol/L of mannitol was added to KH solution in the InsG100 and G100 group. The four groups were perfused with each solution for 20 min prior to 15 min of no-flow ischemia, and during 20 min of reperfusion. Only during the ischemic period the heart was paced at 222 beats/min. Measurements of heart rate, coronary flow and maximum of LV derivative of pressure development (dP/dt max) were recorded. Myocardial phospho-protein kinase B (p-Akt) and tumor necrosis factor-α (TNF-α) levels were assayed by enzyme-linked immunosorbent assay and sandwich ELISA, respectively following reperfusion. RESULTS: After reperfusion, LV dP/dt max and heart rate in the InsG100 group was significantly higher than that in the other three groups. The myocardial p-Akt level in the InsG100 group was significantly elevated when compared to the InsG600 group at the end of reperfusion. The p-Akt levels in the InsG600 and InsG100 group were significantly higher than in the corresponding non-insulin groups. CONCLUSIONS: Acute hyperglycemia diminishes the cardioprotective effects of insulin preconditioning in the isolated rat heart, possibly mediated through the suppression of myocardial Akt phosphorylation.