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Aims: Thiazolidinediones have been advocated as second or third line insulin sensitizing agents in the management of type 2 diabetes (T2DM). Their widespread use has been hampered by concerns about their cardiovascular safety, including fluid retention. Metformin is established as first-line glucose-lowering pharmacotherapy in T2DM. It has also been suggested that it may have benefits in alleviating insulin resistance in type 1 diabetes (T1DM). This thesis examined: (i) cardiovascular, renal and metabolic differences between individuals with T2DM ‘tolerant’ or ‘intolerant’ of TZDs; (ii) risk factors for TZD-associated oedema in T2DM; and (iii) the potential for metformin as adjunct therapy in T1DM. Methods: (i) A small clinical study characterising TZD tolerant and intolerant individuals with T2DM; (ii) A population-based epidemiological study of TZD induced oedema in individuals with T2DM in Tayside, Scotland (using incident loop diuretic prescription as a surrogate); (iii) A systematic review and meta-analysis of published studies of adjunct metformin in T1DM. Results (i) During a five-day high sodium diet, two known TZD-intolerant individuals with T2DM had reductions in haematocrit, aldosterone, and diastolic BP and increases in ANP and central and peripheral augmentation indices which were outwith reference ranges derived from nine TZD-tolerant individuals; (ii) Predictors of time to loop diuretic prescription included age, body mass index, systolic BP, haematocrit, ALT and macrovascular disease but rates of this outcome did not differ by therapy: 4.3% (TZDs) vs 4.7% (other agents) [unadjusted OR 0.909 (95% CI 0.690, 1.196); p = 0.493]; (iii) In meta-analysis of nine small studies in T1DM (192.8 patient-years of follow-up), metformin was associated with a reduction in total daily insulin dose (6.6 units/day; p < 0.001) but no studies examined cardiovascular surrogates or outcomes. Conclusions: Hypotheses were generated for several potential biomarkers predictive of TZD-induced oedema but the clinical importance of TZDs as a risk factor for oedema in individuals with T2DM was questioned. As there is some evidence for the safety of metformin as an adjunct therapy in T1DM but little evidence of efficacy, larger studies are warranted.

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzenesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases, as it will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family. Furthermore, we carried out ITC studies of the interactions between mitoNEET and thiazolidinediones (TZDs), a group of compounds/drugs that bind to peroxisome proliferator-activated receptor-gamma (PPAR-γ) to manage and treat type 2 diabetes mellitus. The binding affinities or KDs as well as the thermodynamic parameters such as enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) were determined for each thiazolidinedione compound employed in this work. Moreover, the reaction stoichiometry (n) and c values were also determined for each interaction between a thiazolidinedione and mitoNEET. These studies may offer insightful perspective into designing compounds that may have higher affinity for mitoNEET in drug discovery efforts.

This dissertation explores the relationship between mitochondrial physiology and development of therapeutics. Mitochondrial dysfunction is associated with both acute and chronic forms of pathophysiology. This work aims to address development efforts at the cell culture and drug-target levels with respect to mitochondria. At the cell culture level, I characterize an approach that has been shown to improve the physiological dependency on mitochondria in tumor-derived cells. I demonstrate that prolonged replacement of glucose with galactose in culture medium induces a global metabolic shift in hepatocellular carcinoma (HepG2) cells to closer reflect a primary hepatocyte phenotype (Chapter 2). I characterize this shift by performing metabolomics and transcriptomics on HepG2 cells adapted to either glucose or galactose-based media for several weeks. At the drug development level, I explore the relationship between the thiazolidinedione (TZD) class of drugs and mitochondrial physiology. I characterize the effects of a classical TZD (pioglitazone) and two experimental TZDs (NL-1 and NL-2) on mitochondrial bioenergetics in cells derived from mammalian liver, muscle, and adipose tissues (Chapter 3). At the target development level, I examine biochemical activities that may regulate the function of a mitochondrial [2Fe-2S] protein which has been shown to bind TZDs. I provide evidence that purified MitoNEET is controlled at multiple biochemical levels by small molecular weight biological thiols and lipid-derived electrophiles (Chapter 4). Finally, I examine the role of both TZDs and NEET proteins in the regulation of cell death. I specifically demonstrate a unique role for NL-1 in protecting HepG2 cells against ferroptotic cell death (Chapter 5). Taken together, these studies provide insights that can leverage mitochondrial physiology to develop novel and effective treatments for a range of pathophysiological conditions.

Type 2 diabetes mellitus is strongly associated with increased risk of cardiovascular disease. Despite availability of several antidiabetic medications, the cardiovascular outcomes remain unchanged. Activation of renin angiotensin system is one of the critical factors in development of diabetic complications. Hyperglycemia causes an increase in tissue and circulating Angiotensin II. Angiotensin converting enzyme (ACE2) and neprilysin (NEP) are angiotensin (1-7) forming enzymes. ACE2 was first cloned from heart failure patients and has cardioprotective and renoprotective properties. Combination of angiotensin receptor antagonism and NEP inhibition is a new therapeutic strategy for treatment of heart failure. The aim of this study was to investigate whether there is an alteration of cardiac NEP and ACE2 in db/db mice and to study the effect of glycemic control with rosiglitazone and pioglitazone on these enzymes. Lean control and db/db mice were fed 20 mg/Kg/day rosiglitazone and pioglitazone. Diabetic mice demonstrated hyperglycemia which was attenuated by rosiglitazone and pioglitazone treatment. Western blot revealed two bands for NEP and ACE2 in both lean and db/db mice in both the studies. Mature NEP was downregulated while fragment was upregulated in db/db mice. Expression of mature ACE2 was upregulated in db/db mice while the fragment was not affected. Both rosiglitazone and pioglitazone had no effect on cardiac NEP and ACE2 expression. There was a significant increase in cardiac ADAM 17 expression in db/db mice. Another aim was to optimize the fluorescence based enzyme activity assay for renal and cardiac ACE2 and NEP. Protease inhibitors for prolyl endopeptidase, prolyl carboxypeptidase, aminopeptidase A-M were introduced in the assay buffer. Addition of protease inhibitors improved the specificity of the renal ACE2 activity assay. Protease inhibitors however, did not affect renal NEP activity assay. This is the first study to report the presence of two immunoreactive bands for cardiac ACE2 and NEP expression. We also report the possibility of fully mature form of cardiac NEP being degraded to form fragmented form. To the best of our knowledge, this study is also the first to report an upregulation in cardiac ACE2 in db/db mice.

The overall objective of this study was to evaluate the impact of cardiovascular safety concerns on the utilization of the thiazolidinediones (TZDs), pioglitazone and rosiglitazone, and other oral antidiabetic drugs. In May 2007, a meta-analysis was published that found a potential increased risk of myocardial infarction (MI) associated with TZDs, particularly with rosiglitazone. A two-year retrospective study of patients diagnosed with type 2 diabetes (ICD9 250.xx) using prescription and medical databases from the Scott & White Health System (SWHS) was conducted. Patients aged 18 or older who were continuously enrolled with SWHS from 2006 to 2008 and had high adherence (Medication Adherence Ratio ≥80%) for either pioglitazone or rosiglitazone during the pre-safety warning period (May 2006- April 2007) were included. Patients were followed through the post-safety warning period (May 2007 - October 2008) or occurrence of event (discontinuation of index TZD drug). Patients who discontinued their index TZD drug by April 2008 were identified if they had a prescription filled for a new oral antidiabetic drugs (OADs), and followed until October 2008 or occurrence of event (discontinuation of new OAD). Cox proportional hazards models were used to assess the rate of and time to discontinuation of index TZD and new OAD with adjustment of age, gender and Charlson Comorbidity Index (CCI). A total of 531 patients (58 percent male; mean age [SD] = 61 [9.1] years) were included in the final analysis, 255 and 276 patients in the rosiglitazone and pioglitazone groups, respectively. The rate of discontinuation for the pioglitazone and rosiglitazone groups began to separate within 90 days of the index event (meta-analysis published in May 2007). In the pioglitazone group, the rate of discontinuation was significantly lower than in the rosiglitazone group ( HR = 0.56; 95% CI = 0.47, 0.67). A total of 21 patients did not experience discontinuation of their index medication. Among patients receiving a new OAD after discontinuing their index TZD (N = 95 rosiglitazone and N = 33 pioglitazone patients), there was no statistical significant in the rate of discontinuing their new OAD between the rosiglitazone and pioglitazone groups (HR = 0.98; 95% CI = 0.61, 1.59). However, patients who received metformin/sulfonylurea combinations had a lower rate of discontinuation compared to patients who received sulfonylureas (HR = 0.38; 94% CI = 0.21, 0.66). The analysis showed the cardiovascular safety concern of TZDs had a significant impact on the utilization of oral antidiabetic drug utilization.

The ligand-dependent transcription factor, peroxisome proliferator activated receptor, (PPAR-gamma), is a member of the nuclear hormone receptor superfamily and a target of antidiabetic thiazolidinediones (TZDs). Initially characterized for its effects in glucose and lipid metabolism, PPAR-gamma activation has been shown to play critical roles in the inhibition of inflammatory vascular diseases such as atherosclerosis. Since PPAR-gamma agonists have receptor-independent actions, knockout studies are required to determine whether PPAR-gamma is necessary for the inhibitory effects of the PPAR-gamma agonists. Due to the pleiotropic effects of the PPAR-gamma activation, establishing which cell types are responsible for the anti-inflammatory effects observed in whole organism studies is difficult without the use of the cell-specific knockouts. To unravel the complex role of PPAR-gamma in inflammation and to begin categorizing relative contributions of PPAR-gamma activity in different cell types, we created two PPAR-gamma knockout models: pancreatic epithelial cell knockout and vascular endothelial cell knockout. These knockout models not only provided insight into the important role PPAR-gamma plays in the control of cell inflammation but also showed that TZDs act directly through PPAR-gamma in suppressing inflammation in an in vivo model of acute pancreatitis and in an in vitro models of endothelial cell inflammation. We show that PPAR-gamma in acinar cells, and not in inflammatory cells, is the target of the TZD anti-inflammatory activity in the model of acute pancreatitis. In vascular endothelial cells, PPAR-gamma is required for TZD activity and PPAR-gamma activation inhibits the function of a pro-inflammatory transcription factor NF-kappaB by decreasing the formation of functional transcriptional complexes at the promoters of NF-kappaB target genes.

Thiazolidinediones, which are peroxisome-proliferator-activated receptor γ (PPARγ) agonists that are used as insulin sensitizers for treatment of type 2 diabetes, have been reported to induce apoptosis in cancer cells. The mechanisms by which thiazolidinediones induce apoptosis are undefined. Here, we show that the induction of apoptosis by thiazolidinediones in prostate cancer cell lines is independent of PPARγ activation. Two prostate cancer lines, PC-3 (PPARγ-expressing) and LNCaP (PPARγ-deficient), are both sensitive to apoptosis induction by troglitazone. Also, a troglitazone analogue that lacks binding affinity to PPARγ retained the same ability to induce apoptosis as troglitazone. Moreover, rosiglitazone and pioglitazone, PPARγ agonists with higher binding affinities than troglitazone, do not exhibit correlative apoptotic effects even at high concentration. Target identification studies showed that the apoptotic effect of troglitazone, ciglitazone, and their analogues is in part attributable to their ability to inhibit antiapoptotic protein Bcl-2 and Bcl-xL function. Fluorescence polarization assay indicated that thiazolidinediones disrupt protein-protein interactions between Bak peptide and Bcl-2/Bcl-xL protein at the BH3 domain binding pocket. In addition, co-immunoprecipitation studies showed that treatment of PC-3 cells with troglitazone and its analogue reduced association of Bcl-2 and Bcl-xL with Bak. Transfected LNCaP cells expressing different levels of Bcl-xL were protected from apoptosis after treatment with iii troglitazone and its analogue in a manner that paralleled Bcl-xL expression level. This troglitazone-inducing apoptotic effect involved cytochrome c release and caspase activation. Although troglitazone exhibits Bcl-2/Bcl-xL inhibitory activity, the potency is weak. To improve its potency, three series of analogues are designed and synthesized based on the structure of troglitazone. Among all the analogues synthesized, TG-88 was the best inhibitor of Bcl-2/Bcl-xL function in vitro, and also repressed PC-3 xenograft tumor growth in a nude mouse model.