This thesis examined how and why TRPV1 function is being modulated in sensory neuropathy and explored the potential of its rescue in the urinary bladder of STZ-­‐induced diabetic rats. Diabetes induced a rapid decline in TRPV1 function and changes in neurogenically mediated electrically-­‐evoked responses together with a gradual decline in muscarinic function. Diabetic bladder was also deficient in muscarinic and TRPV1 organ bath temperature-­‐induced changes but not in those affecting spontaneous contractile activity. Exposure to a potential neuropathy causative agent, methylglyoxal was studied and its mechanism of action explored through the use of TRPA1 ligands. Methylglyoxal exposure mimicked some of the effects of diabetes on TRPV1, neurogenic electrically evoked responses and muscarinic function. Methylglyoxal effects were seen to be partly through TRPA1 receptor activation but other as yet undefined pathways were also involved. Use of TRPA1 ligands revealed an unexpected complexity of the interaction of the TRPA1 receptor with TRPV1. Finally the potential of reversing the diminished TRPV1 response was examined through the use of three known sensitising agents, bradykinin, NGF and insulin. Bradykinin was the only agent seen to reverse the TRPV1 diminished response back up to to control equivalent levels and through the use of bradykinin selective ligands, it was seen that the dual activation of BK-­‐1 and BK-­‐2 receptor was necessary to rescue the TRPV1 response. The likely mechanism of action of bradykinin was through prostaglandin production as indomethacin blocked TRPV1 rescue. In the acute stage of diabetes, TRPV1 function is downregulated and may be caused by exposure to a neuropathy-­‐causing metabolite such as methylglyoxal. The TRPV1 function still retains plasticity at this acute stage because function could be enhanced back to control levels by bradykinin receptor activation : a potential for early therapeutic intervention.

It is currently accepted that the most appropriate diet in the treatment of non-insulin-dependent diabetes mellitus &quoteNIDDM&quote is high in carbohydrates, high in fibre and low in fat. Dietary fibre reduces the rate of carbohydrate absorption, which may have a beneficial effect on insulin action. Furthermore, high fibre diets also increase the amount of carbohydrates which are not absorbed from the small intestine. These malabsorbed carbohydrates are fermented by the bacterial population in the large intestine, producing short chain fatty acids &quoteSCFA&quote, including propionate, which has been shown to alter liver carbohydrate metabolism. This thesis investigated the actions of slowed carbohydrate absorption and carbohydrate malabsorption in streptozotocin-induced &quoteSTZ&quote diabetic rats. High carbohydrate diet supplemented with guar gum, a soluble dietary fibre, fed to STZ diabetic rats improved insulin sensitivity. investigation of the alterations in the stomach and small intestine demonstrated that guar increased the viscosity of the meal in the intestine. The action of increased fermentation, producing more propionate, was investigated by supplementing propionate into the diets of STZ diabetic rats or when perfused into isolated rat livers. No changes in insulin action or liver glucose metabolism were measured. in addition, it was shown that guar gum reduces food intake in STZ diabetic rats. Mild reductions in food intake in STZ diabetic rats were shown to increase insulin action. In summary, STZ diabetic rats fed high carbohydrate, high fibre diets reductions in food consumption and slowed carbohydrate absorption are important factors which may lower blood glucose concentrations and increase insulin action. increased SCFA production is unlikely to contribute significantly to the improvements in insulin action.

One of the most common risk factors for stroke is diabetes. Diabetics are 2 to 4 times more likely to have a stroke and are also significantly more likely to show poor functional recovery. In order to determine why diabetes is associated with poor stroke recovery, we tested the hypotheses that diabetes either exacerbates initial stroke damage, or inhibits neuronal circuit plasticity in surviving brain regions that is crucial for successful recovery. Type 1 diabetes was chemically induced in mice four weeks before receiving a targeted photothrombotic stroke in the right forelimb somatosensory cortex to model a chronic diabetic condition. Following stroke, a subset of diabetic mice were treated with insulin to determine if controlling blood glucose levels could improve stroke recovery. Consistent with previous studies, one behavioural test revealed a progressive improvement in sensory function of the forepaw in non-diabetic mice after stroke. By contrast, diabetic mice treated with and without insulin showed persistent deficits in sensori-motor forepaw function. To determine whether these different patterns of stroke recovery correlated with changes in functional brain activation, forepaw evoked responses in the somatosensory cortex were imaged using voltage sensitive dyes at 1 and 14 weeks after stroke. In both diabetic and non-diabetic mice that did not have a stroke, brief mechanical stimulation of the forepaw evoked a robust and near simultaneous depolarization in the primary (FLS1) and secondary somatosensory (FLS2) cortex. One week after stroke, forepaw-evoked responses had not been remapped in the peri-infarct cortex in both diabetic and non-diabetic mice. Fourteen weeks after stroke, forepaw evoked responses in non-diabetic mice re-emerged in the peri-infarct cortex whereas diabetic mice showed very little activation, reminiscent of the 1 week recovery group. Moreover, controlling hyperglycemia using insulin therapy failed to restore sensory evoked responses in the peri-infarct cortex. In addition to these differences in peri-infarct responsiveness, we discovered that stroke was associated with increased responsiveness in FLS2 of non-diabetic, but not diabetic or insulin treated mice. To determine the importance of FLS2 in stroke recovery, we silenced the FLS2 cortex and found that it re-instated behavioural impairments in stroke recovered mice, significantly more so than naïve mice that still had a functioning FLS1. Collectively, these results indicate that both diabetes and the secondary somatosensory cortex play an important role in determining the extent of functional recovery after ischemic cortical stroke. Furthermore, the fact that insulin therapy after stroke did not normalize functional recovery, suggests that prolonged hyperglycemia (before stroke) may induce pathological changes in the brain’s circulation or nervous system that cannot be easily reversed. / Graduate

Zusammenfassung Thure Adler Der Streptozotozin-induzierte Diabetes in der transgenen CD4/DR17-Maus Aus dem Institut für Immunologie der Veterinärmedizinischen Fakultät und dem Institut für Klinische Immunologie und Transfusionsmedizin der Medizinischen Fakultät der Universität Leipzig, 80 Seiten, 24 Abbildungen, 20 Tabellen, 217 Literaturangaben Die Verwendung transgener Tiere, die humane Moleküle exprimieren, gewinnt zunehmend an Bedeutung bei der Erforschung der Funktionen solcher Moleküle in Krankheitsprozessen und bei der experimentellen Erprobung neuartiger Therapieverfahren, in denen solche Moleküle die Zielstrukturen darstellen. In der vorliegenden Arbeit wurde die CD4/DR17-Maus, welche das humane CD4- und das DR17-Molekül exprimiert, im MLD-STZ-induzierten Diabetes, einem Tiermodell für den Typ 1 Diabetes, eingesetzt. Die funktionelle Beteiligung der Transgene wurde durch einen Vergleich mit Segreganten untersucht, denen die Transgene teilweise fehlen. Als klinische Parameter sind Blutglukose und Glukosetoleranz erfaßt worden, histopathologisch wurden Insulitis und Insulingehalt der Inselzellen bestimmt. Ferner wurde getestet, ob sich durch Verabreichung von monoklonalen Antikörpern, die gegen das transgene hCD4- oder gegen das CD8-Molekül gerichtet sind, dieser STZ-induzierte Diabetes beeinflussen läßt. Mit Hilfe der durchflußzytometrischen Immunfluoreszenzanalyse von Blutzellen wurde zusätzlich überprüft, ob Veränderungen auf T-Zellen hinsichtlich der Expression der Aktivierungsmarker CD25, CD69 und CD71 während des STZ-induzierten Diabetes auftreten. Es wurde gezeigt, dass die CD4/DR17-transgene Maus nach der Behandlung mit mehrfachen subdiabetogenen Dosen von Streptozotozin eine transiente Hyperglykämie entwickelt, die mit einer verringerten Glukosetoleranz sowie Insulitiden und einem Rückgang des Insulingehaltes in den Langerhans’schen Inseln einhergeht. Vergleiche mit Segreganten zeigen, dass die Expression beider transgener Merkmale zur maximalen Ausprägung einer schwergradigen Insulitis beiträgt. Die Anwendung von monoklonalen Antikörpern gegen das transgene hCD4-Molekül nach Beginn der STZ-Behandlung hat den Diabetes nicht wirkungsvoll verzögert. Dagegen milderte eine Behandlung mit Antikörpern, die gegen das CD8-Molekül gerichtet sind, den Diabetesverlauf. Während des STZ-Diabetes veränderte sich die Expression von Aktivierungsmarkern auf Lymphozyten des peripheren Blutes nicht signifikant. Die Arbeit belegt, dass die CD4/DR17-Maus suszeptibel gegenüber der Induktion eines experimentellen Diabetes mit mehrfachen subdiabetogenen Dosen von Streptozotozin ist. Die transgenen Moleküle hCD4 und DR17 sind dabei am Krankheitsprozeß beteiligt. / Summary Thure Adler The streptozotocin-induced diabetes in the transgenic CD4/DR17 mouse From the Institute of Immunology, Faculty of Veterinary Medicine and the Institute of Clinical Immunology and Transfusion Medicine, Faculty of Medicine, University of Leipzig 80 pages, 24 figures, 20 tables, 217 references Today, transgenic animals that express human molecules are getting important tools in functional studies and experimental, therapeutical attempts, that target these molecules. In this study, the CD4/DR17 mouse expressing the human CD4 and the human DR17 molecules and with a defective murine CD4 gene, was used in the multiple low-dose streptozotocin-induced (MLD-STZ) diabetes model, a model for type 1 diabetes. The functional involvement of the transgenic molecules in the development of the MLD-STZ-diabetes was analysed by comparing CD4/DR17 mice and segregants that lack one or more of the transgenes. The described parameters included the measurement of blood glucose levels and oral glucose tolerance tests, histopathologically grading of insulitis and determination of the content of insulin in pancreatic islets by immunohistological methods. In addition, the model was used to test the potential therapeutic effect of the administration of monoclonal antibodies against hCD4 or CD8. Furthermore, alterations of the expression of the activation markers CD25, CD69 and CD71 during the experimentally induced diabetes has been measured by FACS analysis. The study shows, that CD4/DR17 mice develop a transient hyperglycemia after MLD-STZ treatment, accompanied by a reduced tolerance to oral glucose, insulitis and the reduction of the content of insulin in the pancreatic islets. The full incidence of insulitis requires the expression of both transgenes. The treatment performed with monoclonal antibodies against the transgenic hCD4 after STZ-treatment could not meliorate the diabetic course, while the treatment with anti CD8 antibodies moderated the diabetic process. After STZ-treatment the expression of activation marker of peripheral T-cells did not alter significantly. Thus, the CD4/DR17 mouse is shown to be susceptible to the induction of experimental diabetes with MLD-STZ. The transgenic molecules CD4 and DR17 are involved in the pathogenesis of the disease.

Background: Diabetic kidney disease is the leading cause of end-stage renal disease. Administration of ACE inhibitors or/and SGLT2 inhibitors show renoprotective effects in diabetic and other kidney diseases. The underlying renoprotective mechanisms of SGLT2 inhibition, especially in combination with ACE inhibition, are incompletely understood. We used longitudinal intravital microscopy to directly elucidate glomerular hemodynamics on a single nephron level in response to the ACE inhibitor enalapril or/and the SGLT2 inhibitor empagliflozin. Methods: Five weeks after the induction of diabetes by streptozotocin, male C57BL/6 mice were treated with enalapril, empagliflozin, enalapril/empagliflozin or placebo for 3 days. To identify hemodynamic regulation mechanisms, longitudinal intravital multiphoton microscopy was employed to measure single nephron glomerular filtration rate (snGFR) and afferent/efferent arteriole width. Results: Diabetic mice presented a significant hyperfiltration. Compared to placebo treatment, snGFR was reduced in response to enalapril, empagliflozin, or enalapril/empagliflozin administration under diabetic conditions. While enalapril treatment caused significant dilation of the efferent arteriole (12.55 ± 1.46 µm vs. control 11.92 ± 1.04 µm, p < 0.05), empagliflozin led to a decreased afferent arteriole diameter (11.19 ± 2.55 µm vs. control 12.35 ± 1.32 µm, p < 0.05) in diabetic mice. Unexpectedly under diabetic conditions, the combined treatment with enalapril/empagliflozin had no effects on both afferent and efferent arteriole diameter change. Conclusion: SGLT2 inhibition, besides ACE inhibition, is an essential hemodynamic regulator of glomerular filtration during diabetes mellitus. Nevertheless, additional mechanisms—independent from hemodynamic regulation—are involved in the nephroprotective effects especially of the combination therapy and should be further explored in future studies.

Zusammenfassung Thure Adler Der Streptozotozin-induzierte Diabetes in der transgenen CD4/DR17-Maus Aus dem Institut für Immunologie der Veterinärmedizinischen Fakultät und dem Institut für Klinische Immunologie und Transfusionsmedizin der Medizinischen Fakultät der Universität Leipzig, 80 Seiten, 24 Abbildungen, 20 Tabellen, 217 Literaturangaben Die Verwendung transgener Tiere, die humane Moleküle exprimieren, gewinnt zunehmend an Bedeutung bei der Erforschung der Funktionen solcher Moleküle in Krankheitsprozessen und bei der experimentellen Erprobung neuartiger Therapieverfahren, in denen solche Moleküle die Zielstrukturen darstellen. In der vorliegenden Arbeit wurde die CD4/DR17-Maus, welche das humane CD4- und das DR17-Molekül exprimiert, im MLD-STZ-induzierten Diabetes, einem Tiermodell für den Typ 1 Diabetes, eingesetzt. Die funktionelle Beteiligung der Transgene wurde durch einen Vergleich mit Segreganten untersucht, denen die Transgene teilweise fehlen. Als klinische Parameter sind Blutglukose und Glukosetoleranz erfaßt worden, histopathologisch wurden Insulitis und Insulingehalt der Inselzellen bestimmt. Ferner wurde getestet, ob sich durch Verabreichung von monoklonalen Antikörpern, die gegen das transgene hCD4- oder gegen das CD8-Molekül gerichtet sind, dieser STZ-induzierte Diabetes beeinflussen läßt. Mit Hilfe der durchflußzytometrischen Immunfluoreszenzanalyse von Blutzellen wurde zusätzlich überprüft, ob Veränderungen auf T-Zellen hinsichtlich der Expression der Aktivierungsmarker CD25, CD69 und CD71 während des STZ-induzierten Diabetes auftreten. Es wurde gezeigt, dass die CD4/DR17-transgene Maus nach der Behandlung mit mehrfachen subdiabetogenen Dosen von Streptozotozin eine transiente Hyperglykämie entwickelt, die mit einer verringerten Glukosetoleranz sowie Insulitiden und einem Rückgang des Insulingehaltes in den Langerhans’schen Inseln einhergeht. Vergleiche mit Segreganten zeigen, dass die Expression beider transgener Merkmale zur maximalen Ausprägung einer schwergradigen Insulitis beiträgt. Die Anwendung von monoklonalen Antikörpern gegen das transgene hCD4-Molekül nach Beginn der STZ-Behandlung hat den Diabetes nicht wirkungsvoll verzögert. Dagegen milderte eine Behandlung mit Antikörpern, die gegen das CD8-Molekül gerichtet sind, den Diabetesverlauf. Während des STZ-Diabetes veränderte sich die Expression von Aktivierungsmarkern auf Lymphozyten des peripheren Blutes nicht signifikant. Die Arbeit belegt, dass die CD4/DR17-Maus suszeptibel gegenüber der Induktion eines experimentellen Diabetes mit mehrfachen subdiabetogenen Dosen von Streptozotozin ist. Die transgenen Moleküle hCD4 und DR17 sind dabei am Krankheitsprozeß beteiligt. / Summary Thure Adler The streptozotocin-induced diabetes in the transgenic CD4/DR17 mouse From the Institute of Immunology, Faculty of Veterinary Medicine and the Institute of Clinical Immunology and Transfusion Medicine, Faculty of Medicine, University of Leipzig 80 pages, 24 figures, 20 tables, 217 references Today, transgenic animals that express human molecules are getting important tools in functional studies and experimental, therapeutical attempts, that target these molecules. In this study, the CD4/DR17 mouse expressing the human CD4 and the human DR17 molecules and with a defective murine CD4 gene, was used in the multiple low-dose streptozotocin-induced (MLD-STZ) diabetes model, a model for type 1 diabetes. The functional involvement of the transgenic molecules in the development of the MLD-STZ-diabetes was analysed by comparing CD4/DR17 mice and segregants that lack one or more of the transgenes. The described parameters included the measurement of blood glucose levels and oral glucose tolerance tests, histopathologically grading of insulitis and determination of the content of insulin in pancreatic islets by immunohistological methods. In addition, the model was used to test the potential therapeutic effect of the administration of monoclonal antibodies against hCD4 or CD8. Furthermore, alterations of the expression of the activation markers CD25, CD69 and CD71 during the experimentally induced diabetes has been measured by FACS analysis. The study shows, that CD4/DR17 mice develop a transient hyperglycemia after MLD-STZ treatment, accompanied by a reduced tolerance to oral glucose, insulitis and the reduction of the content of insulin in the pancreatic islets. The full incidence of insulitis requires the expression of both transgenes. The treatment performed with monoclonal antibodies against the transgenic hCD4 after STZ-treatment could not meliorate the diabetic course, while the treatment with anti CD8 antibodies moderated the diabetic process. After STZ-treatment the expression of activation marker of peripheral T-cells did not alter significantly. Thus, the CD4/DR17 mouse is shown to be susceptible to the induction of experimental diabetes with MLD-STZ. The transgenic molecules CD4 and DR17 are involved in the pathogenesis of the disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Intestinal smooth muscle development involves complex transcriptional regulation leading to cell differentiation of the circular, longitudinal and muscularis mucosae layers. Differentiated intestinal smooth muscle cells express high levels of smooth muscle-specific contractile and regulatory proteins, including telokin. Telokin is regulatory protein that is highly expressed in visceral smooth muscle. Analysis of cis-elements required for transcriptional regulation of the telokin promoter by using hypoxanthine-guanine phosphoribosyltransferase (Hprt)-targeted reporter transgenes revealed that a 10 base pair large CC(AT)₆GG ciselement, called CArG box is required for promoter activity in all tissues. We also determined that an additional 100 base pair region is necessary for transgene activity in intestinal smooth muscle cells. To examine how transcriptional regulation of intestinal smooth muscle may be altered under pathological conditions we examined the effects of diabetes on colonic smooth muscle. Approximately 76% of diabetic patients develop gastrointestinal (GI) symptoms such as constipation due to intestinal dysmotility. Mice were treated with low-dose streptozotocin to induce a type 1 diabetes-like hyperglycemia. CT scans revealed decreased overall GI tract motility after 7 weeks of hyperglycemia. Acute (1 week) and chronic (7 weeks) diabetic mice also had decreased potassium chloride (KCl)-induced colon smooth muscle contractility. We hypothesized that decreased smooth muscle contractility at least in part, was due to alteration of contractile protein gene expression. However, diabetic mice showed no changes in mRNA or protein levels of smooth muscle contractile proteins. We determined that the decreased colonic contractility was associated with an attenuated intracellular calcium increase, as measured by ratio-metric imaging of Fura-2 fluorescence in isolated colonic smooth muscle strips. This attenuated calcium increase resulted in decreased myosin light chain phosphorylation, thus explaining the decreased contractility of the colon. Chronic diabetes was also associated with increased basal calcium levels. Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) analysis revealed significant changes in calcium handling proteins in chronic diabetes that were not seen in the acute state.These changes most likely reflect compensatory mechanisms activated by the initial impaired calcium response. Overall my results suggest that type 1 diabetes in mice leads to decreased colon motility in part due to altered calcium handling without altering contractile protein expression.