Betahistine prevents development of endolymphatic hydrops in a mouse model of insulin resistance and diabetes.

BACKGROUND: Diabetes is associated with inner ear dysfunction. Furthermore, C57BL/6J mice fed high fat diet (HFD), a model for insulin resistance and diabetes, develop endolymphatic hydrops (EH). AIM: Evaluate if betahistine, spironolactone (aldosterone antagonist) and empagliflozin (sodium -glucose cotransporter2 inhibitor) can prevent EH induced by HFD and explore potential mechanisms. METHODS: C57BL/6J mice fed HFD were treated with respective drug. The size of the endolymphatic fluid compartment was measured using contrast enhanced MRI. Secondarily, mice treated with cilostamide, a phosphodiesterase3 inhibitor, to induce EH and HEI-OC1 auditory cells were used to study potential cellular mechanisms of betahistine. RESULTS: HFD-induced EH was prevented by betahistine but not by spironolactone and empagliflozin. Betahistine induced phosphorylation of protein kinaseA substrates but did not prevent cilostamide-induced EH. CONCLUSIONS: Betahistine prevents the development of EH in mice fed HFD, most likely not involving pathways downstream of phosphodiesterase3, an enzyme with implications for dysfunction in diabetes. The finding that spironolactone did not prevent HFD-induced EH suggests different mechanisms for EH induction/treatment since spironolactone prevents EH induced by vasopressin, as previously observed. SIGNIFICANCE: This further demonstrates that independent mechanisms can cause hydropic inner ear diseases which suggests different therapeutic approaches and emphazises the need for personalized medicine.

Insulin regulates Nedd4-2 via a PKB-dependent mechanism in HEI-OC1 auditory cells-crosstalks with sphingolipid and cAMP signaling.

BACKGROUND: The mechanisms of association between diabetes and inner ear dysfunction are unknown, although endolymphatic hydrops may be involved. We have previously shown that insulin signaling components are expressed in human saccule and that insulin signaling takes place in HEI-OC1 auditory cells. AIM: To explore Nedd4-2 as a target for insulin signaling. MATERIALS AND METHODS: Effects of insulin were analyzed using western blot and confocal microscopy in HEI-OC1 auditory cells. RESULTS: Insulin induced phosphorylation of Nedd4-2 and increased the amount of ENaC at the plasma membrane. Also, protein kinase B (PKB) and NDRG1, a substrate for SGK1 (serum and glucocorticoid stimulated kinase), were phosphorylated in response to insulin. The SGK1 inhibitor GSK650394 prevented insulin-induced phosphorylation of NRDG1, but not of PKB and Nedd4-2, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and the PKB inhibitor MK2206 inhibited phosphorylation of all components. Ceramides prevented insulin-induced phosphorylation of PKB and NDRG1, but not of Nedd4-2. The ceramide metabolite sphingosine 1-phosphate induced phosphorylation of Nedd4-2. CONCLUSIONS: Insulin induces phosphorylation of Nedd4-2, most likely involving PI3K/PKB signaling. Sphingosine 1-phosphate might protect Nedd4-2 against ceramide-induced insulin resistance. SIGNIFICANCE: Insulin-mediated regulation of Nedd4-2 might impact on inner ear sodium homeostasis with implications for diabetes-induced inner ear damage.

Inner ear is a target for insulin signaling and insulin resistance: evidence from mice and auditory HEI-OC1 cells.

OBJECTIVE: The mechanisms underlying the association between diabetes and inner ear dysfunction are not known yet. The aim of the present study is to evaluate the impact of obesity/insulin resistance on inner ear fluid homeostasis in vivo, and to investigate whether the organ of Corti could be a target tissue for insulin signaling using auditory House Ear Institute-Organ of Corti 1 (HEI-OC1) cells as an in vitro model. METHODS: High fat diet (HFD) fed C57BL/6J mice were used as a model to study the impact of insulin resistance on the inner ear. In one study, 12 C57BL/6J mice were fed either control diet or HFD and the size of the inner ear endolymphatic fluid compartment (EFC) was measured after 30 days using MRI and gadolinium contrast as a read-out. In another study, the size of the inner ear EFC was evaluated in eight C57BL/6J mice both before and after HFD feeding, with the same techniques. HEI-OC1 auditory cells were used as a model to investigate insulin signaling in organ of Corti cells. RESULTS: HFD feeding induced an expansion of the EFC in C57BL/6J mice, a hallmark of inner ear dysfunction. Insulin also induced phosphorylation of protein kinase B (PKB/Akt) at Ser473, in a PI3-kinase-dependent manner. The phosphorylation of PKB was inhibited by isoproterenol and IBMX, a general phosphodiesterase (PDE) inhibitor. PDE1B, PDE4D and the insulin-sensitive PDE3B were found expressed and catalytically active in HEI-OC1 cells. Insulin decreased and AICAR, an activator of AMP-activated protein kinase, increased the phosphorylation at the inhibitory Ser79 of acetyl-CoA carboxylase, the rate-limiting enzyme in de novo lipogenesis. Furthermore, the activity of hormone-sensitive lipase, the rate-limiting enzyme in lipolysis, was detected in HEI-OC1 cells. CONCLUSIONS: The organ of Corti could be a target tissue for insulin action, and inner ear insulin resistance might contribute to the association between diabetes and inner ear dysfunction.

Endolymphatic hydrops induced by different mechanisms responds differentially to spironolactone: a rationale for understanding the diversity of treatment responses in hydropic inner ear disease.

Background: The exact pathophysiological mechanism(s) underlying endolymphatic hydrops (EH) remain elusive. We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes (PDE3, PDE4, PDE5) results in the development of EH to mice. Aims/objectives: Evaluate the ability of spironolactone, an aldosterone antagonist, to prevent EH, when induced by different pathways. Material and methods: Mice were treated for 4 weeks with vasopressin, the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram in the presence or absence of spironolactone. EH was assessed using high resolution 9.4T MRI. The expression of proteins in human saccule sensory epithelium was studied with immunohistochemistry. Results: Spironolactone prevents EH induced by vasopressin and rolipram, but not hydrops induced by cilostamide. The aldosterone target ENaC and the mineralocorticoid receptor were expressed in the human saccule sensory epithelium. Conclusions: The effect of spironolactone on EH appears to be pathway-dependent and may provide explanations why certain drugs may be effective in some patients with hydropic ear disease while not in others. Significance: Extrapolating this finding to the clinic supports that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, as different pathways may be needed to be targeted for treatment.

Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI.

CONCLUSION: The data indicate important roles for phosphodiesterase (PDE) 3, 4, 5, and related cAMP and cGMP pools in the regulation of inner ear fluid homeostasis. Thus, dysfunction of these enzymes might contribute to pathologies of the inner ear. OBJECTIVE: The mechanisms underlying endolymphatic hydrops, a hallmark of inner ear dysfunction, are not known in detail; however, altered balance in cAMP and cGMP signaling systems appears to be involved. Key components of these systems are PDEs, enzymes that modulate the amplitude, duration, termination, and specificity of cAMP and cGMP signaling. METHOD: To evaluate the role of PDE3, 4, and 5 and associated cAMP and cGMP pools in inner ear function, the effect of cilostamide (PDE3 inhibitor), rolipram (PDE4 inhibitor), and sildenafil (PDE5 inhibitor), administrated via mini-osmotic pumps, on mouse inner ear fluid homeostasis was evaluated using 9.4T in vivo MRI in combination with intraperitoneally administered Gadolinium contrast. Also, using human saccule as a model, the expression of PDEs and related signaling molecules and targets was studied using immunohistochemistry. RESULTS: PDE3, PDE4, as well as PDE5 inhibitors resulted in the development of endolymphatic hydrops. Furthermore, PDE3B, PDE4D, and some related signaling components were shown to be expressed in the human saccule.

Branched short-chain fatty acids modulate glucose and lipid metabolism in primary adipocytes.

Short-chain fatty acids (SCFAs), e.g. acetic acid, propionic acid and butyric acid, generated through colonic fermentation of dietary fibers, have been shown to reach the systemic circulation at micromolar concentrations. Moreover, SCFAs have been conferred anti-obesity properties in both animal models and human subjects. Branched SCFAs (BSCFAs), e.g., isobutyric and isovaleric acid, are generated by fermentation of branched amino acids, generated from undigested protein reaching colon. However, BSCFAs have been sparsely investigated when referring to effects on energy metabolism. Here we primarily investigate the effects of isobutyric acid and isovaleric acid on glucose and lipid metabolism in primary rat and human adipocytes. BSCFAs inhibited both cAMP-mediated lipolysis and insulin-stimulated de novo lipogenesis at 10 mM, whereas isobutyric acid potentiated insulin-stimulated glucose uptake by all concentrations (1, 3 and 10 mM) in rat adipocytes. For human adipocytes, only SCFAs inhibited lipolysis at 10 mM. In both in vitro models, BSCFAs and SCFAs reduced phosphorylation of hormone sensitive lipase, a rate limiting enzyme in lipolysis. In addition, BSCFAs and SCFAs, in contrast to insulin, inhibited lipolysis in the presence of wortmannin, a phosphatidylinositide 3-kinase inhibitor and OPC3911, a phosphodiesterase 3 inhibitor in rat adipocytes. Furthermore, BSCFAs and SCFAs reduced insulin-mediated phosphorylation of protein kinase B. To conclude, BSCFAs have effects on adipocyte lipid and glucose metabolism that can contribute to improved insulin sensitivity in individuals with disturbed metabolism.

Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI.

From histopathological specimens, endolymphatic hydrops has been demonstrated in association with inner ear disorders. Recent studies have observed findings suggestive of hydrops using MRI in humans. Previous studies suggest that vasopressin may play a critical role in endolymph homeostasis and may be involved in the development of Ménière's disease. In this study we evaluate the effect of vasopressin administration in vivo in longitudinal studies using two mouse strains. High resolution MRI at 9.4 T in combination with intraperitoneally delivered Gadolinium contrast, was performed before and after chronic subcutaneous administration of vasopressin via mini-osmotic pumps in the same mouse. A development of endolymphatic hydrops over time could be demonstrated in C57BL6 mice (5 mice, 2 and 4 weeks of administration) as well as in CBA/J mice (4 mice, 2 weeks of administration; 6 mice, 3 and 4 weeks of administration). In most C57BL6 mice hydrops developed first after more than 2 weeks while CBA/J mice had an earlier response. These results may suggest an in vivo model for studying endolymphatic hydrops and corroborates the future use of MRI as a tool in the diagnosis and treatment of inner ear diseases, such as Ménière's disease. MRI may also be developed as a critical tool in evaluating inner ear homeostasis in genetically modified mice, to augment the understanding of human disease.