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The global number of obese people has reached pandemic proportions. High caloric diets and reduced physical exercise are to blame for this growing epidemic. Obesity has a very complex association with several other metabolic disorders, such as insulin resistance (IR), diabetes mellitus type 2 (DMT2) and cardiovascular disease. This puts a huge burden on health care systems world wide and claims many lives. Sutherlandia frutescens is a traditionally used herb, which is known to have anti-diabetic properties. However, the direct mode of action of S. frutescens still remains to be elucidated. The aim of this study was to investigate the developmental stages of high fat diet (HFD)-induced IR, to illuminate the pathogenesis of IR with a focal point on modifications in the lipid metabolism. Furthermore, the effects of S. frutescens as a treatment or prevention drug for IR and associated metabolic changes were examined. Two sets of experiments were conducted on male Wistar rats. In the first experiment rats, one week post weaning received a low fat diet (LFD), high fat diet (HFD) or HFD supplemented with S. frutescens (50mg/kg BW/d). Rats were sacrificed at week 0, 1, 2, 4, 8 and 12 in the feeding regime. In a second experiment rats were fed with a LFD or a HFD for 12 weeks and treated thereafter with S. frutescens (50mg/kg BW/d), metformin (13mg/kg BW/d) or water (control) for 28 days. Rats in the second experiment were sacrificed at week 12 to confirm IR while concurrently run rats were sacrificed after 28 days of treatment. For all the experiments rats were anaesthetized, blood was removed and rats were dissected. Plasma samples were analyzed for insulin, glucose, blood lipid parameters and cytokines. Liver, muscle and adipose tissue were analyzed for glucose uptake, total lipid content, lipid profile and fatty acid profile. It was shown that the intake of HFD caused IR and hyperinsulinaemia. The developmental stages in experiment one confirmed that an increase in plasma free fatty acids preceeded the onset of IR. Plasma and tissue lipid parameters (free fatty acid-, triglyceride- and cholesterol concentrations) showed pathological modifications in the HFD group. An ectopic accumulation of fat was observed in muscle and liver, as well as a change in membrane fatty acid profile. The results for circulating cytokines were somewhat inconclusive. Rats supplemented with S. frutescens did not develop HFD-induced IR (study one) or IR was reversed (study two). S. frutescens treatment also resulted in positive changes in plasma and tissue lipid parameters. In summary, an animal model for HFD-induced IR was established and the detrimental effect of elevated plasma FFA on glucose and lipid metabolism was observed. A novel discovery suggests that the anti-diabetic mode of action of S. frutescens is through modulation of lipid metabolism. It was also established that S. frutescens has the potential to prevent IR in vivo.

Obesity can lead to Type 2 Diabetes, both conditions increase in association with physical inactivity and high-energy diets, resulting in elevated blood glucose, decreased insulin sensitivity and increased insulin resistance. Sutherlandia frutescens (S.frutescens), an anti-diabetic plant, reverses and prevents insulin resistance in a rat model and human cell culture model. Gene expression analysis in hepatocyte cultures, identified genes down regulated in insulin resistance and up regulated by S.frutescens. These included genes encoding vesicle transporter proteins, hypothesised to be linked to hepatic lipid accumulation and lipid droplet formation during insulin resistance. The aim of this study was to investigate critical genes involved in lipid droplet formation, vesicle assembly and transport in high fat diet (HFD)-induced insulin resistant rat liver tissue during the development of insulin resistance and the reversal of these changes by S.frutescens. Rats were fed a low fat diet (LFD) or HFD supplemented with S.frutescens for 2, 4 and 8 weeks. Rats fed a HFD for 12 weeks developed insulin resistance, confirmed by plasma glucose and insulin levels (compared to normal controls). Groups of these rats were gavaged with S. frutescens (50mg/kg BW), Metformin (13mg/kg BW) or water for a further 4 weeks and starved for 12 hours, anaesthetized and blood removed by heart puncture. Liver was stored in RNA-Later™ for qRT-PCR and snap-frozen in liquid nitrogen for western blotting and confocal microscopy analysis. Changes in expression of vesicle transporter genes VAMP3 and NSF were analysed by qRT-PCR and changes in the protein expression by western blotting analysis. Proteins were localised within the liver by confocal immunohistochemistry using ZEN lite™ software. Statistical analysis was performed using One-Way ANOVA and unpaired t-test. mRNA gene expression of vesicle transport components VAMP3, NSF and SNAP25 showed relatively moderate changes with considerable individual variation within control or experimental groups. Uncorrelated changes in mRNA and protein products were found and may be due to differential regulation by siRNA. Proteins also showed altered staining patterns in high fat diet rats that reverted towards normal on S. frutescens treatment, potentially reflecting functional changes associated with transport of lipid-filled vesicles.

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While women are diagnosed with depression at twice the rate of men, pre-clinical research on this topic has relied heavily on the responses of male animals. This thesis examined the behavioural and physiological effects of environmental manipulation in the female Wistar-Kyoto rat, a putative animal model of depression. At postnatal day 52, baseline behavioural measures were collected in 36 Wistar and 36 Wistar-Kyoto female rats using the following tests: the elevated plus maze to assess anxiety, the forced swim test for depression-like behaviour, and sucrose preference test to assess hedonic status. At postnatal day 62, the rats were randomly assigned to one of three environments for 30 days: 1) standard housing - 3 rats in one large cage 2) isolated housing - 1 rat per small cage, or 3) environmental enrichment - 6 animals in a multistory cage filled with novel objects and a running wheel. Following 30 days in their housing condition, the same behavioural measures were again collected. Large differences between strains were found with the Wistar-Kyoto females showing significantly less mobility and activity in both the forced swim test and elevated plus maze. Sucrose preference was significantly higher after enrichment in both strains. Post-environment immune cytokine and corticosterone levels were also assessed in these animals at baseline and after the forced swim test. No difference in corticosterone between strains was found at baseline. However, Wistar-Kyoto females had significantly higher corticosterone levels than their Wistar counterparts after the forced swim test. In contrast, Wistar-Kyoto females showed significantly lower serum levels of the pro-inflammatory cytokine IL-1β than Wistar females. In the hippocampus, astroglial staining intensity was significantly increased in the CA1 of Wistar females after environmental enrichment. Glucocorticoid receptor staining in the CA3 was also increased after environmental enrichment in both the Wistar-Kyoto and Wistar animals. Finally there was a trend towards higher levels of glucocorticoid receptors in the amygdala in Wistar-Kyoto animals who experienced environmental enrichment. Taken together, this thesis provides evidence for the effect of environment, specifically enrichment, on behaviour and physiological systems. These results suggest that incorporating social and physical enrichment as part of clinical intervention may benefit individuals with depression.

The objective of these studies was to investigate mechanisms whereby cardiac sarcoplasmic reticulum (SR) calcium transport activity may be influenced by changes in the lipid environment of the SR membrane in the experimental endocrine disease states hypothyroidism and insulin-dependent diabetes mellitus. These endocrine disease states were studied to determine, respectively, if SR function is influenced by endogenous acylcarnitine associated with the SR membrane and if SR phospholipid acyl composition plays a role in diabetes-induced cardiomyopathy. The effects of endogenous acylcarnitines on SR calcium transport in hypothyroidism were of interest since it has previously been implicated that acyl carnitines play a regulatory role in SR function. SR calcium transport was not affected at two weeks, but was significantly reduced at four, six and eight weeks following thyroidectomy. Endogenous acyl carnitines were detectable in the SR membrane fraction isolated from both euthyroid control and thyroidectomized animals. The level of acyl carnitine associated with the SR did not correlate with calcium transport activity. Since acylcarnitine did not appear to play a role in the reduced SR calcium transport, SR calcium pump protein was quantified. The reduced SR calcium transport of thyroidectomized animals, manifest at four weeks, was shown to correlate with a reduction in SR acylphosphoprotein level. Therefore the reduced SR calcium transport activity of hypothyroidism is not related to the level of SR acyl carnitine, but rather a hypothyroid-induced reduction in SR calcium pump sites. Since omega-3 fatty acids affect parameters relevant to diabetes-induced cardiomyopathy, it was of interest to determine the cardiac effects of omega-3 fatty acid treatment of streptozocin (STZ)-induced diabetic animals. Omega-3 fatty acid treatment significantly reduced the development of diabetic cardiomyopathy and improved isolated cardiac SR calcium transport activity of STZ-induced diabetic animals. To determine if the cardiac and SR changes were related to membrane changes induced by omega-3 fatty acids, the fatty acyl composition of phospholipid was determined. Phospholipid analysis of cardiac phosphatidylcholine and phosphatidylethanolamine, and total SR phospholipid indicated modest changes in the omega-3 fatty acid component. Omega-3 fatty acid treatment produced slight (statistically insignificant) changes in SR cholesterol levels. Therefore a change in membrane phospholipid acyl composition may not account for the observed cardiac and SR functional changes. / Pharmaceutical Sciences, Faculty of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Posttraumatic stress disorder involves a constellation of neural and behavioral alterations in response to trauma exposure. Aside from symptoms involved in posttraumatic stress disorder diagnosis, patients frequently present with working memory impairments. Working memory training has been established as an effective intervention to reduce posttraumatic stress symptoms. Working memory is associated with posttraumatic stress disorder in that it is commonly impaired in patients and that training can reduce the severity of posttraumatic stress symptoms. Taken together, these points suggest the possibility of a shared mechanism between working memory and posttraumatic stress disorder but working memory has not been studied thoroughly in rodent models of posttraumatic stress disorder. The present study utilizes footshock trauma to induce a posttraumatic stress state in rats and evaluates the effect of trauma and trauma-paired cues on working memory performance. Results demonstrate the emergence of chronic deficits in working memory among traumatized animals three weeks post-trauma. Presentation of trauma-paired cues caused further decrement in working memory performance. Regression analysis indicates that the degree of working memory impairment in response to a trauma-paired cue can be significantly predicted by behavioral phenotypes typic of diagnostic symptoms for posttraumatic stress disorder. This study enhances existing animal models by replicating the clinical observations of working memory deficits associated with posttraumatic stress disorder. This will pave the way for future work to probe underlying mechanistic dysregulation of working memory following trauma exposure and for future development of novel treatment strategies.

Numerous species of the genus Staphylococcus other than Staphylococcus aureus are important pathogens in human clinical practice and veterinary medicine. With improved methods of identification and more precise classification, we have speciated over 500 strains of staphylococci representing 17 species and subspecies of non-S. aureus Staphylococcus. We have examined these strains for possible virulence factors which may play a role in their pathogenesis. Using transmission electron microscopy (TEM), we have demonstrated that small to large amounts of glycocalyx are found on staphylococcal cells. Animal models have shown that staphylococci cause abscess formation in the presence or absence of a foreign body implant. Molecular characterization of cell extracts of Staphylococcus intermedius show that this species elaborates a protein which is serologically similar to the enterotoxins of Staphylococcus aureus in ELISA tests, but differs markedly in other characteristics.

Indiana University-Purdue University Indianapolis (IUPUI) / Anxiety disorders are the most common class of mental disorders in the United States, and they both promote and exacerbate disorders of substance abuse. Mounting evidence of sex differences in the relationship between anxiety disorders and alcoholism supports the potential existence of an anxiety-dependent vulnerability to alcohol abuse in women compared with men. One potential point of overlap in the physiological systems involved in anxiety response and reward processing is the amygdala. Here, a model of chronic stress in rodents was employed to probe changes in the electrophysiological and biochemical properties of the amygdala at a post-stress baseline and during a post-stress first exposure to alcohol. Electrophysiological data revealed that neurons in the centromedial amygdala were more responsive to stimulation in the basolateral amygdala in females compared with males, but a history of chronic stress altered the female response to match that of males with or without a history of chronic stress. Protein analysis of postsynaptic glutamatergic receptor expression and phosphorylation in the amygdala did not indicate any differences based on sex or exposure to stress or alcohol. These data demonstrate a sex difference in stress-induced alterations in amygdala circuitry and indicate a potential role for this circuitry in the comorbidity of anxiety disorders and alcoholism.