Thesis (MSc (Biochemistry))--University of Limpopo (Medunsa Campus), 2009. / Background and Purpose The stem bark, roots and leaves of Sclerocarya birrea (S. birrea), {(A. Rich) Hochst}, subspecies caffra (Sond) Kokwaro are widely used in South Africa and some African countries as folk medicine in the treatment and management of a variety of human ailments, including diabetes mellitus. Although the blood glucose lowering effect of the stem bark extract of S. birrea have been confirmed using experimental animal models of diabetes, there is no clear understanding of the mechanism(s) whereby S. birrea stem bark extracts and/or their components exert their blood glucose lowering effects. The primary aim of the current study was to study the in vitro inhibitory effects of S. birrea stem bark extracts on the activities of selected diabetic related carbohydrate metabolizing enzymes (α-amylase, α-glucosidase and glucose 6-phosphatase). The current study also investigated the acute in vivo effect of S. birrea stem bark acetone extract on postprandial blood glucose levels after oral sucrose loading as well as the effect of S. birrea stem bark aqueous extract on hepatic glucose 6-phosphatase activity. In addition, the long term (21 days) effects of S. birrea stem bark acetone extract on fasting blood glucose levels, plasma insulin levels, plasma triglyceride and body weight in normal and alloxan induced diabetic rats were also investigated. Methods For in vitro studies: Crude hexane, acetone, methanolic and aqueous extracts of the stem bark extract of S. birrea were prepared by means of a sequential solvent extraction procedure and screened for inhibitory activities against human urinary α-amylase, rat pancreatic α-amylase, Bacillus stearothermophilus α-glucosidase and rabbit liver glucose 6-phosphatase using standard procedures for assaying the activities of these enzymes. IC50 values and mode of inhibition of extracts demonstrating appreciable inhibitory activity against α-amylase and α-glucosidase were determined and compared with those of acarbose, a known inhibitor of these two enzymes. The IC50 value and mode of inhibition of extracts demonstrating appreciable inhibitory activity against glucose 6-phosphatase were determined and compared with those of sodium orthovavadate and sodium tungstate, known inhibitors of glucose 6-phosphatase. In vivo studies: In vivo studies were conducted in normal and alloxan induced diabetic male WKY rats. Diabetes was induced in rats that had been fasted for 12 h by a single intraperitoneal injection of 140 mg/kg body weight of alloxan monohydrate freshly dissolved in sterile normal saline. The effect of S. birrea stem bark acetone extract on postprandial blood glucose level was determined in 18 h fasted diabetic and normal rats by administering orally, the plant extract (300 mg/kg) 30 minutes before an oral sucrose loading and measuring postprandial blood glucose levels after sucrose loading by means of a MediSense’s Optimum Xceed Glucometer (MOXG). In addition, rat intestinal dissacharidase (α-glucosidase/sucrase) activity was determined in homogenate of small intestine of rats sacrificed one hour after given orally either plant extract or acarbose. The in vivo effect of S. birrea stem bark extract on glucose 6-phosphatase was determined by measuring the activity of hepatic glucose 6-phosphatase at the end of the study. For the determination of the long term (chronic) effect of S. birrea stem bark crude acetone extract on blood glucose levels, body weight and water intake, alloxan induced diabetic and normal WKY rats were treated daily with S. birrea stem bark crude acetone extract (300 mg/kg) for 21 days. Fasting blood glucose levels and changes in body weight were determined on day 0, 7, 14 and 21 after initiation of treatment by means of a MOXG and gravimetrically respectively. Water intake was determined on the same days that blood glucose levels were determined by measuring the amount of water left overnight by each rat and subtracting this amount from the initial amount water given to each rat. Blood was also collected at the end of the study for the measurement of plasma glucose, triglyceride and insulin levels. Plasma glucose and plasma triglyceride levels were measured using commercially available kits based respectively on the glucose oxidase and the glycerol blanked methods (Beckman Coulter®’s UniCell DXC 800 Synchron® Clinical System). Plasma insulin levels were determined by means of an enzyme linked immunosorbant assay (ELISA) adapted to the Beckman Coulter® Ireland Inc’s UniCell DXI 800 Access® Immunoassay System. Results In vitro studies: The crude methanolic and acetone S. birrea stem bark extracts strongly inhibited both human urinary α-amylase and rat pancreatic α-amylase in a competitive manner. The inhibitory effect of the crude methanolic extract on both enzymes was significantly stronger than acarbose. Hexane and acetone crude extracts of the stem-bark of S. birrea demonstrated the highest percentage inhibition against B. stearothermophilus α-glucosidase. The mode of inhibition of the crude hexane extract on B. stearothermophilus α-glucosidase appeared to be a noncompetitive one. However, the this plant extract appeared to be a less potent inhibitor of α-glucosidase enzyme than acarbose. Rabbit liver glucose 6-phophatase was strongly inhibited by the crude aqueous S, birrea stem bark extract in a competitive manner. In vivo studies: Administration of S birrea stem bark acetone extract 30 min before oral sucrose loading significantly suppressed (P < 0.01) the rise in postprandial blood glucose levels in treated rats compared to control rats. The crude extract also decreased significantly the intestinal disaccharidase activity of experimental rats compared to control rats. These observations suggest that the in vitro inhibitory effects of the crude hexane extract on α-glucosidase enzymes are applicable in vivo Daily, continuous oral treatment of alloxan–induced diabetic and normal WKY rats with S. birrea stem bark extract for 3 weeks resulted in significant reductions in fasting blood glucose levels and water intake of treated diabetic rats compared with diabetic controls. The extract, however, failed to bring about any significant change in the body weight, plasma insulin levels, plasma triglyceride levels and hepatic glucose 6-phosphatase of treated diabetic rats compared to diabetic control rats Conclusions The results of the current study suggest that the observed in vitro inhibitory effect of S. birrea stem bark acetone extract on alpha glucosidase enzymes are applicable in vivo whereas the observed in vitro inhibitory effect of S. birrea stem bark aqueous extract on glucose 6-phosphatase are not applicable in vivo. Furthermore, in the current study S. birrea stem bark acetone extract appears to lower blood glucose levels of alloxan induced diabetic rats without increasing their plasma insulin levels. Thus, it can be concluded on the basis of the current study that S. birrea stem bark acetone and hexane extracts exert their blood glucose lowering effect in alloxan induced diabetic rats in part, through inhibition of intestinal brush border α-glucosidase enzymes.

No description available.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

No description available.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Damage to peripheral nerves following trauma or disease has a number of consequences including burning pain, muscle wasting, paralysis, or organ dysfunction. The most common form of neuropathy is that associated with metabolic abnormality, notably diabetes. Many diabetics, especially those with poor blood sugar control, ultimately develop a distal symmetrical and painful neuropathy that initially affects the longest peripheral axons, but with time spreads proximally. Deficiency in neurotrophic support has been proposed to contribute to the development of diabetic neuropathy. Recently, peripheral gene delivery of vascular endothelial growth factor (VEGF), neurotrophin-3 (NT-3), NGF, BDNF or hepatocyte growth factor (HGF) has been shown to facilitate the continuous production of neurotrophic factors and alleviate the diabetic neuropathy. The role of glial cell-derived neurotrophic factor (GDNF) in the pathogenesis and therapeutics of diabetic neuropathy is not well defined. The main objectives of this research sought to inspect the protective effect of GDNF peripheral gene delivery during hyperglycemia- or constriction- induced sciatic nerve injury in rats. In present proposal, we propose to investigate the change in organization and expressions of GDNF signaling complex in the sciatic nerve following injury in the initial stage. Subsequently, the recombinant adenovirus was used gene delivery system for GDNF to evaluate the potential of intramuscular administration of gene delivery for prevent nerve degeneration, and the molecular mechanism of GDNF to ameliorate neuropathy will be clarified. The above study would enable us to test the hypothesis that the topical gene delivery might be a suitable strategy for the treatment of diabetic neuropathy and other disorders in peripheral nerve. Furthermore, the results of animal studies might be extrapolated for future clinical application.

In this dissertation, I investigated computing algorithms for automated retinal blood vessel detection. Changes in blood vessel structures are important indicators of many diseases such as diabetes, hypertension, etc. Blood vessel is also very useful in tracking of disease progression, and for biometric authentication. In this dissertation, I proposed two algorithms to detect blood vessel maps in retina. The first algorithm is based on integration of a Gaussian tracing scheme and a Gabor-variance filter. This algorithm traces the large blood vessel in retinal images enhanced with adaptive histogram equalization. Small vessels are traced on further enhanced images by a Gabor-variance filter. The second algorithm is called a radial contrast transform (RCT) algorithm, which converts the intensity information in spatial domain to a high dimensional radial contrast domain. Different feature descriptors are designed to improve the speed, sensitivity, and expandability of the vessel detection system. Performances comparison of the two algorithms with those in the literature shows favorable and robust results. Furthermore, a new performance measure based on central line of blood vessels is proposed as an alternative to more reliable assessment of detection schemes for small vessels, because the significant variations at the edges of small vessels need not be considered. The proposed algorithms were successfully tested in the field for early diabetic retinopathy (DR) screening. A highly modular code library to take advantage of the parallel processing power of multi-core computer architecture was tested in a clinical trial. Performance results showed that our scheme can achieve similar or even better performance than human expert readers for detection of micro-aneurysms on difficult images.

Activin is a dimeric protein of inhibin beta subunit, which is abundantly stored in normal bone matrix, presumably produced by osteoblasts in the process of normal bone formation. The expression of activins was examined in the wound healing of diabetic rats. In this study,insulin-dependent diabetes mellitus was induced in a group of mature Sprague-Dawley rats by injecting streptozotocin. Control animals were injected with citrate buffer only. After 3 weeks,all of rats underwent extraction of the right maxillary molars teeth after anesthesia. Rats were killed at varying intervals and the maxilla and calvaria were recovered in continuity. Tissue sections were stained with hematoxylin-eosin as well as immunohistochemical gent. Hematoxylin-eosin analyses showed that at 7 days after tooth extraction in the control and insulin-streptozotocin-treated rats there were, thick collagen fibers which formed a pretrabecular the scaffold dictated the direction of the forming trabeculae. However,the collagen fibers in the diabetic socket were thin and scanty, and only formed a narrow layer in the apical part of the socket. These histologic observations suggest that in uncontrolled, insulin-dependent diabetes, the formation of the collagenous framework in the tooth extraction socket is inhibited, resulting in delayed healing.The immunohistochemical analyses showed that at 7 days after tooth extraction in both control and insulin-streptozotocin-treated rats, osteoblasts were increased in extra-alveolar bone formation.Our findings also suggested that activin was actively involve in bone modeling during osteogenesis. These findings suggest that activin may play important role in the regulation of bone formation and it may be useful in the future for the wound healing in diabetic patients.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Previous in vitro studies showed that exchange protein directly activated by cyclic AMP 1 (Epac1), which is a cAMP mediator, plays an important role in maintenance of endothelial barrier function. Diabetic retinopathy is characterized by impairment of retinal blood vessel integrity leading to breakdown of blood retinal barrier, retinal hypoxia, and neuronal damage. Here, we hypothesize that Epac1 regulates endothelial permeability and protects retina from the retinal damage associated with diabetes. To test such hypothesis, we first demonstrated that human retinal microvascular endothelial cells (HRMECs) exposed to high glucose concentration at 25 mM or 35 mM showed the decreased Epac1 expression level. Our preliminary data also showed that Epac1-downstream activator, Rap1, a member of Ras GTPase, was also altered by different glucose levels. In addition, retina from type 2 diabetic, db/db, mice also showed the decreased Epac1 expression compared to that of non-diabetic, db/m, mice. To further determine the role of Epac1 in diabetic retinopathy, we made use of Epac1-deficient mice. The pathogenesis of diabetic retinopathy share similar characteristics to that of ischemic retinopathy, such as neuronal cell death, glial reactivity, and glutamate toxicity. Therefore, we used our previous retinal ischemic model, i.e., transient middle cerebral artery occlusion (tMCAO). Firstly, we determined the retinal morphology of Epac1-/- mice under normal condition at 3wks. At 3 wks old, the Epac1-/- retinae showed a significantly decreased thickness of outer plexiform layer (OPL) with a trend of increase in inner nuclear layer (INL) thickness. Interestingly, there were obviously more glutamine synthetase (GS)-positive M?ller cells and protein kinase C (PKC)-α positive rod bipolar cells in INL. In addition, there were more IgG-positive blood vessels in OPL. To further determine whether these phenotypes will lead to more severe retinal damage, Epac1-/- mice were exposed to 2 hours of MCAO followed by 22 hours of reperfusion, which we have previously shown to induce retinal ischemia. There was no obvious difference in retinal thickness and expressions of glial fibrillary acidic protein (GFAP) and GS in the contralateral sides of Epac1+/+ and Epac1-/- retina after tMCAO suggesting that the Epac1-deficiency may be compensated by either protein kinase A (PKA) or Epac2. However, Epac2 level was not altered by Epac1-deficiency by Western blot analysis. The ipsilateral sides of the retina of Epac1+/+ and Epac1-/- after tMCAO also did not show obvious difference in swelling and cell death in inner retina, GFAP, glutamate, GS, nitrotyrosine (NT), and peroxiredoxin 6 (Prx6), suggesting that Epac1-deficiency may have been compensated by other cAMP mediators, such as Epac2. However, Epac2 expression in the ipsilateral side of Epac1+/+ and Epac1-/- retinae was not significantly different, although the activities of Epac and PKA were not determined. Taken together, the Epac1-deficient mice would serve as a useful model to determine the role of Epac1 in retinal development, and to determine the detail mechanisms of pathogenesis of diabetic and ischemic retinopathy. / published_or_final_version / Anatomy / Master / Master of Philosophy