Hyperglycemic Fish Research Articles

The Effects of Lavendustin‐A on Peripheral Nerve Degeneration in Hyperglycemic Danio rerio

Diabetic peripheral neuropathy (DPN) results in degenerating peripheral nerves and affects over 50% of people with diabetes. Despite its high prevalence, DPN’s molecular pathways remain unknown, limiting treatment to pain management. The Clark lab has characterized two novel models of hyperglycemia in zebrafish in which DPN‐like phenotypes are recapitulated, including demyelination. In an attempt to elucidate the unknown molecular mechanisms of DPN, candidate small molecule drugs are currently being tested to accentuate or attenuate neurodegeneration. Lavendustin‐A, an EGFR tyrosine kinase inhibitor (TKI), was hypothesized to have a neuroprotective role, as overactivation of EGFR in hyperglycemic environments is associated with fibrosis and the inhibition of regeneration in various tissue types. Tg(mbp:gfpcaax) larval zebrafish were incubated in egg water (control 1) or 120mM glucose solution (control 2 and treatment) starting five days post‐fertilization (dpf) until 10dpf. From 10‐12dpf, control 1 remained in egg water + 1% DMSO, control 2 remained in glucose solution + 1% DMSO, and the treatment group remained in glucose solution + 1% DMSO + 65.5µM Lavendustin‐A. 12dpf, lateral images of myelinated (MBP+) peripheral motor nerves were taken on an EVOS imaging system. Fiji (NIH) software was used to measure the length and fluorescent intensity of the myelinated nerves. Both nerve length (*p=0.025) and MBP fluorescent intensity (**p=0.0013) were significantly greater in the treatment group compared to the hyperglycemic fish (control 2), and no significant difference was observed between the treatment group and control 1. To determine if function was impacted, a touch‐response assay was conducted before (10dpf) and after (12dpf) drug treatment. Escape response was significantly impaired in hyperglycemic fish (treatment and control 2) at 10dpf (***p=0.0007) and significantly improved in the treatment group by 12dpf (***p=0.0003), yet did not fully recover, as there was still a significant difference between the treatment group and control 1 (**p=0.0017). Our data suggests a possible role of EGFR in hyperglycemia‐induced neurodegeneration, getting us one step closer to identifying potential therapeutics for clinical application.

Optimization of ultrasonic-assisted extraction of polyphenols from the polyherbal formulation of Cinnamomum verum, Origanum majorana, and Origanum vulgare and their anti-diabetic capacity in zebrafish (Danio rerio)

The Cinnamomum verum (CV), Origanum majorana (CM), and Origanum vulgare (OV) have been used in traditional medicine in several regions of México for their anti-diabetic properties. In this study investigated the variables of ultrasound-assisted extraction for the polyphenolic compounds from the combination of these plants and explore their potential antidiabetic activities on glucose-induced-diabetic zebrafish. Determined the optimum conditions for ultrasonic-assisted extraction (UAE) to maximum recovery amounts of phenolic compounds from the extract of these plants. Polyphenols were detected in the extracts using HPLC-DAD-analysis. Extracts were evaluated on zebrafish exposed to high glucose concentration (110 mM) for two weeks. Results showed second-order polynomial mathematical models with a high coefficient of determination (R2 > 0.9564). Optimized extraction conditions for UAE from the combination of the 3 plants (COV) were as follows: 66.03%, ethanol, 28.87 min, and 21.51 mL/g for maximal flavonoids extraction. Used the same optimal extraction conditions for CV, CM, and OV. Results from LC-MS/MS indicated 9 polyphenolic compounds in CV, 12 in CM, and 6 in OV, the content of total polyphenols was 310.28, 90.42, and 126.74 mg GAE 100 g−1 dry weight, respectively. However, hyperglycemic fish showed an increase in cholesterol and triglyceride levels whereas extracts completely prevented these metabolic alterations. COV showed higher anti-diabetic ability than CV, CM, and OV, suggesting a synergistic effect between them. Our investigation developed a new herbal formulation of Cinnamomum verum; Origanum majorana; Origanum vulgare that has proven effective in animals with type 2 diabetes will form a new class of supplements to treat diabetic complications.

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

Modulation of redox and insulin signaling underlie the anti-hyperglycemic and antioxidant effects of diphenyl diselenide in zebrafish

The organic selenium compound diphenyl diselenide (DD) has been recognized as an antioxidant and neuroprotective agent, exerting an anti-hyperglycemic effect in experimental models of diabetes. However, the precise mechanisms involved in the protection are unclear. Using the zebrafish (Danio rerio) as a model organism, here we investigated biomarkers underlying the protective effects of DD against hyperglycemia, targeting in a transcriptional approach the redox and insulin-signaling pathway. Fish were fed on a diet containing DD (3 mg/kg) for 74 days. In the last 14 days, they were exposed to a 111 mM glucose solution to induce a hyperglycemic state. DD reduced blood glucose levels as well as normalized the brain mRNA transcription of four insulin receptors-coding genes (Insra1, Insra2, Insrb1, Insrb2), which were down-regulated by glucose. DD alone caused an up-regulation of relative mRNA transcription in both Insra receptors and glucose transporter 3 genes. DD counteracted hyperglycemia-induced lipid peroxidation, protein and thiol depletion. Along with the decreased activity of antioxidant enzymes SOD and GPx, the brain of hyperglycemic fish presented a reduction in mRNA transcription of FoxO3A, FoxO3B, Nrf2, GPx3A, SOD1, and SOD2 genes. Besides normalizing the transcriptional levels, DD caused an up-regulation of relative mRNAs that encode Nrf2, FoxO1A, FOXO3A, GPx4A, PTP1B, AKT and SelP. Collectively, our findings suggest that the antioxidant and anti-hyperglycemic actions of DD in a zebrafish diabetes model are likely associated with the regulation of the oxidative stress resistance and the insulin-signaling pathway and that could be related to the modulation at mRNA level of two important transcription factors, Nrf2 and FoxO.

Liquiritigenin Reduces Blood Glucose Level and Bone Adverse Effects in Hyperglycemic Adult Zebrafish.

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia that induces other pathologies including diabetic retinopathy and bone disease. Adult Danio rerio (zebrafish) represents a powerful model to study both glucose and bone metabolism. Then, the aim of this study was to evaluate the effects of liquiritigenin (LTG) on blood glucose level and diabetes complications in hyperglycemic adult zebrafish. LTG is a flavonoid extracted from Glycyrrhiza glabra roots which possess important antioxidant, anti-inflammatory, and anti-diabetic properties. During four weeks of glucose treatment, LTG significantly prevented the onset of the hyperglycemia in adult zebrafish. Moreover, hyperglycemic fish showed increased advanced glycation end-products (AGEs) and parathormone levels whereas LTG completely prevented both of these metabolic alterations. Large bone-loss areas were found in the scales of glucose-treated fish whereas only small resorption lacunae were detected after glucose/LTG treatment. Biochemical and histological tartrate resistant acid phosphatase (TRAP) assays performed on explanted scales confirmed that LTG prevented the increase of osteoclastic activity in hyperglycemic fish. The osteoblastic alkaline phosphatase (ALP) activity was clearly lost in scales of glucose-treated fish whereas the co-treatment with LTG completely prevented such alteration. Gene expression analysis showed that LTG prevents the alteration in crucial bone regulatory genes. Our study confirmed that LTG is a very promising natural therapeutic approach for blood glucose lowering and to contrast the development of bone complications correlated to chronic hyperglycemia.

Hyperglycemia elicits anxiety-like behaviors in zebrafish: Protective role of dietary diphenyl diselenide

Diabetes mellitus (DM) is a chronic metabolic disease that may comorbid with various psychiatric disorders, such as anxiety and depression. The search for effective therapeutics to alleviate hyperglycemia and complications resulting from DM is continuous. Here we investigate the effects of diphenyl diselenide (DD), an organoselenium compound with several pharmacological properties, in a zebrafish model of hyperglycemia. Fish were fed for 74 days with a diet containing 3 mg/Kg DD, a concentration chosen after experiments based in a dose-response curve (DD 1, 2 and 3 mg/Kg) that did not cause overt toxicity (mortality, weight loss and neurobehavioral deficits). In the last 14 days of the experimental period, fish were concomitantly exposed to a glucose solution (111 mM). Afterwards, blood glucose levels, brain selenium (Se) content, and behavioral analysis aiming to assess anxiety-like behaviors and locomotor/exploratory activities were performed. In the novel tank diving test, glucose decreased vertical exploration and fish spent less time in the lit area when tested in the light-dark test, suggesting increased anxiety-like behavior. Moreover, DD decreased blood glucose levels in hyperglycemic fish as well as prevented the development of anxiety-related symptoms. DD diet alone did not change glycemia and behavioral parameters, but increased Se levels in the brain without affecting the cellular viability. Collectively, our findings highlight the growing utility of this zebrafish hyperglycemia model as a valuable strategy for further research in DM field and neuroprotective approaches.

Blind fish: An eye opener.

Lay Summary: Different species of vertebrates have conditions similar to human obesity, insulin resistance and type 2 diabetes. Increasing number of studies are now revealing that the causes and interrelationships between these states are substantially different in different species. Comparative physiology may turn out to be an eye opener for evolutionary theories of diabetes. Obesity induced insulin resistance is believed to be central to type 2 diabetes. Recent work on Mexican cavefish, Astyanax mexicanus, has revealed a hyperglycemic phenotype similar to human type 2 diabetes but here insulin resistance is the cause of obesity rather than an effect. Instead of developing diabetic complications, the hyperglycemic fish lead a healthy and long life. In addition to fish, insulin resistance in hibernating bears, dolphins, horses, bonnet macaques and chimpanzees demonstrate that the relationship between diet, obesity, insulin sensitivity and diabetes is widely different in different species. Evolutionary hypotheses about type 2 diabetes should explain these differences.

Activation of the Nkx2.5-Calr-p53 signaling pathway by hyperglycemia induces cardiac remodeling and dysfunction in adult zebrafish.

ABSTRACTHyperglycemia is an independent risk factor for diabetic cardiomyopathy in humans; however, the underlying mechanisms have not been thoroughly elucidated. Zebrafish (Danio rerio) was used in this study as a novel vertebrate model to explore the signaling pathways of human adult cardiomyopathy. Hyperglycemia was induced by alternately immersing adult zebrafish in a glucose solution or water. The hyperglycemic fish gradually exhibited some hallmarks of cardiomyopathy such as myocardial hypertrophy and apoptosis, myofibril loss, fetal gene reactivation, and severe arrhythmia. Echocardiography of the glucose-treated fish demonstrated diastolic dysfunction at an early stage and systolic dysfunction at a later stage, consistent with what is observed in diabetic patients. Enlarged hearts with decreased myocardial density, accompanied by decompensated cardiac function, indicated that apoptosis was critical in the pathological process. Significant upregulation of the expression of Nkx2.5 and its downstream targets calreticulin (Calr) and p53 was noted in the glucose-treated fish. High-glucose stimulation in vitro evoked marked apoptosis of primary cardiomyocytes, which was rescued by the p53 inhibitor pifithrin-μ. In vitro experiments were performed using compound treatment and genetically via cell infection. Genetically, knockout of Nkx2.5 induced decreased expression of Nkx2.5, Calr and p53. Upregulation of Calr resulted in increased p53 expression, whereas the level of Nkx2.5 remained unchanged. An adult zebrafish model of hyperglycemia-induced cardiomyopathy was successfully established. Hyperglycemia-induced myocardial apoptosis was mediated, at least in part, by activation of the Nkx2.5–Calr–p53 pathway in vivo, resulting in cardiac dysfunction and hyperglycemia-induced cardiomyopathy.

Chronic hyperglycemia affects bone metabolism in adult zebrafish scale model.

Type II diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia that induce other pathologies including diabetic retinopathy and bone disease. The mechanisms implicated in bone alterations induced by type II diabetes mellitus have been debated for years and are not yet clear because there are other factors involved that hide bone mineral density alterations. Despite this, it is well known that chronic hyperglycemia affects bone health causing fragility, mechanical strength reduction and increased propensity of fractures because of impaired bone matrix microstructure and aberrant bone cells function. Adult Danio rerio (zebrafish) represents a powerful model to study glucose and bone metabolism. Then, the aim of this study was to evaluate bone effects of chronic hyperglycemia in a new type II diabetes mellitus zebrafish model created by glucose administration in the water. Fish blood glucose levels have been monitored in time course experiments and basal glycemia was found increased. After 1 month treatment, the morphology of the retinal blood vessels showed abnormalities resembling to the human diabetic retinopathy. The adult bone metabolism has been evaluated in fish using the scales as read-out system. The scales of glucose-treated fish didn't depose new mineralized matrix and shown bone resorption lacunae associated with an intense osteoclast activity. In addition, hyperglycemic fish scales have shown a significant decrease of alkaline phosphatase activity and increase of tartrate-resistant acid phosphatase activity, in association with alterations in other bone-specific markers. These data indicates an imbalance in bone metabolism, which leads to the osteoporotic-like phenotype visualized through scale mineral matrix staining. The zebrafish model of hyperglycemic damage can contribute to elucidate in vivo the molecular mechanisms of metabolic changes, which influence the bone tissues regulation in human diabetic patients.

Pushing the limits of glucose kinetics: how rainbow trout cope with a carbohydrate overload.

Rainbow trout are generally considered to be poor glucoregulators. To evaluate this, exogenous glucose was administered to chronically hyperglycemic fish at twice the endogenous rate of hepatic production, and their ability to modulate glucose fluxes was tested. Our goals were to determine: (1) whether hyperglycemic fish maintain higher glucose fluxes than normal; (2) whether they can lower hepatic production (Ra glucose) or stimulate disposal (Rd glucose) to cope with a carbohydrate overload; and (3) an estimate of the relative importance of glucose as an oxidative fuel. Results show that hyperglycemic trout sustain elevated baseline Ra and Rd glucose of 10.6 ± 0.1 µmol kg(-1) min(-1) (or 30% above normal). If 50% of Rd glucose was oxidized as in mammals, glucose could account for 36 to 100% of metabolic rate when exogenous glucose is supplied. In response to exogenous glucose, rainbow trout can completely suppress hepatic glucose production and increase disposal 2.6-fold, even with chronically elevated baseline fluxes. Such large changes in fluxes limit the increase in blood glucose to 2.5-fold and are probably mediated by the effects of insulin on glucose transporters 2 and 4 and on key enzymes of carbohydrate metabolism. Without this strong and rapid modulation of glucose kinetics, glycemia would rise four times faster to reach dangerous levels, exceeding 100 mmol l(-1). Such responses are typical of mammals, but rather unexpected for an ectotherm. The impressive plasticity of glucose kinetics demonstrated here suggests that trout have a much better glucoregulatory capacity than is usually portrayed in the literature.

Serotonin-induced brain glycogenolysis in rainbow trout (Oncorhynchus mykiss)

In this study, we evaluated the serotonin-mediated control of cerebral glycogen levels in the rainbow trout, Oncorhynchus mykiss. Intracerebroventricular (i.c.v.) administration of serotonin (5-HT) to normoglycemic trout (time and dose response) decreased glycogen levels in the brain and increased brain glycogen phosphorylase activity (time response). In hypoglycemic fish (that had been fasted for 5 and 10 days), there was a time-dependent decrease in brain glycogen levels; under these conditions, i.c.v. administration of 5-HT also reduced the brain glycogen content in fish that had been fasted for 5 days. In fish with local cerebral hypoglycemia (induced by 2-DG administration), the glycogen levels decreased and, as above, i.c.v. administration of 5-HT also lowered the glycogen content. In hyperglycemic fish, 5-HT did not affect glycogen levels. Administration of receptor agonists 5-HT1A (8-OH-DPAT), 5-HT1B (anpirtoline and CP93129) or 5-HT2 (α-m-5-HT) decreased the brain glycogen levels. This effect was antagonized by the administration of receptor antagonists 5-HT1A (WAY100135 and NAN190), 5-HT1B (NAS181) and 5-HT2B/C (SB206553). Administration of the receptor agonists (±)-DOI (5-HT2A/2C), m-CPP (5-HT2B/2C), BW723C86 (5-HT2B) and WAY 161503 (5-HT2C) led to decreases in the levels of brain glycogen. We found that 5-HT is involved in the modulation of brain glycogen homeostasis in the rainbow trout, causing a glycogenolytic effect when fish are in a normoglycemic or hypoglycemic state, but not when they are in a hyperglycemic state. 5-HT1A, 5-HT1B, 5HT2B and 5-HT2C-like receptors appeared to be involved in the glycogenolytic action of 5-HT, although the effect mediated by 5-HT1A or 5-HT1B was apparently stronger.

Glucosensing capacity in liver of rainbow trout displays day-night variations possibly related to melatonin action

To assess whether the glucosensing capacity in peripheral (liver and Brockmann bodies) and central (hypothalamus and hindbrain) locations of rainbow trout displays day-night variations in its response to changes in circulating glucose levels, we evaluated the response of parameters related to glucosensing [glucose, glycogen and glucose 6-phosphate levels, activities of glucokinase (GK), glycogen synthetase (GSase) and pyruvate kinase (PK), and mRNA abundance of GK, glucose transporter 2 (GLUT2), and K(ATP) channel subunits Kir6.x-like and sulfonylurea receptor (SUR)-like] in fish subjected to hyperglycemic treatment under night or day conditions. No day-night significant variations were noticed in the glucosensing capacity of the hypothalamus, hindbrain and Brockmann bodies. In contrast, a clear differential response was noticed in the liver, where glucose levels, GK activity (and mRNA levels) and GSase activity displayed increased values during the day in hyperglycemic fish compared with controls, and lower (GK mRNA levels) or non-existent (glucose, GK and GSase activities, and Kir6.x-like mRNA levels) values during the night. A similar decrease in parameters related to glucosensing in the liver was observed when fish under day conditions were treated with melatonin, suggesting a modulatory role of melatonin in day-night changes of the glucosensing response in the same tissue.

Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus

The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background.

Histopathology of the endocrine pancreas of a fresh-water fish, <i>Clarias batrachus </i>(L.)

Intramuscular administration of epinephrine in 2 mg/kg and 5 mg/kg body weight doses to Clarias batrachus caused significant hyperglycemia between 12 and 18 h after treatment. The hormone produced a small decrease in the liver glycogen, but induced a notable fall in the glycogen of muscles and a transient increase in the brain glycogen. Light-microscopical examination of the pancreatic islets revealed degranulation and vacuolization of the beta-cells accompanied by acute necrosis. Damage to the alpha-cells was also noticed in hyperglycemic fish. The changes in the cellular configuration of the islets, blood glucose and on the tissue glycogen contents were dose-dependent. It is suggested that epinephrine alters the carbohydrate metabolism through the alpha-cells of the islets.