Very long chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD deficient mice and patients’ clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD deficient mice were treated systemically with 1x10 12 vector genomes of rAAV9-VLCAD. Expression was detected in the liver, heart and muscle. Also substantial expression of VLCAD was noted in the brain, where it was expressed across different sections of the brain and in different cell types with different morphologies. Biochemical correction was observed in vector-treated mice beginning two weeks post-injection, as characterized by a significant drop in long chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks post injection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD-/- mice dropped below 20°C and the mice became lethargic, requiring euthanasia. In contrast all rAAV9-treated VLCAD-/- mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD-/- mice maintained euglycemia, whereas untreated VLCAD-/- mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

5-Aminolevulinic acid synthase-1 (ALAS-1) is the first and normally rate-controlling enzyme for hepatic heme biosynthesis. ALAS-1 is highly inducible, especially in liver, in response to changes in nutritional status, and to drugs that induce cytochrome P-450. The critical biochemical abnormality of the acute porphyrias, a group of disorders of heme synthesis, is an uncontrolled up-regulation of ALAS-1. High intakes of glucose or other metabolizable sugars and intravenous heme are the cornerstones of therapy for acute attacks of porphyrias and both repress the over-expression ALAS-1, although their mechanisms of action have not been fully characterized. In this work, the chick hepatoma cell line, LMH, was characterized with respect to its usefulness in studies of heme biosynthesis and compared with chick embryo liver cells (CELCs), a widely used model for studies of heme metabolism. The inducibility of ALAS-1 mRNA and enzyme activity and accumulation of porphyrins by chemicals were used to evaluate heme biosynthesis in LMH cells. Repression of ALAS-1 mRNA and induced activity by exogenous heme (20 μM) was shown to occur in LMH cells as in CELCs. In addition, a synergistic induction of ALAS-1 enzyme activity was observed in LMH cells, as shown previously in CELCs, by treatment with a barbiturate-like chemical, Glutethimide (Glut), in combination with an inhibitor of heme synthesis, 4,6-dioxoheptanoic acid (DHA). This induction of ALAS-1 enzyme activity is analogous to what occurs in patients with acute hepatic porphyrias and LMH cells were used to further characterize effects of Glut, DHA, glucose, and heme on ALAS-1. A "glucose effect" to decrease Glut and DHA-induced ALAS-1 enzyme activity was obtained in LMH cells and CELCs in the absence of serum or hormones. This "glucose effect" was further characterized in LMH cells using a construct containing approximately 9.1 kb of chick ALAS-1 5'- flanking and 5' -UTR region attached to a luciferase/reporter gene (pGcALAS9.1-Luc). Glut (50 μM) and DHA (250 μM) synergistically induced luciferase activity (5-fold) in LMH cells transiently transfected with pGcALAS9.l-Luc. Addition of glucose (11 or 33 mM), in a dose-dependent manner, decreased the Glut+DHA up-regulation of pGcALAS9.1-Luc activity. Gluconeogenic or glycolytic substrates such as fructose, galactose, glycerol and lactate, but not the non-metabolizable sugar sorbitol, also down-regulated pGcALAS9.1-Luc in LMH cells. The cAMP analog 8-CPT-cAMP, augmented Glut induction of ALAS-1, indicating that the glucose effect may be partly mediated by changes in cAMP levels. The remaining studies focused on delineating the synergistic effect of Glut and DHA, and heme-dependent repression of ALAS-1. The 9.1 kb construct was compared with a construct containing the first 3.5 kb (pGcALAS3.5-Luc). The drug and heme effects were shown to be separate as drug induction was present in -3.4 to +0.082 kb region while the heme responsiveness was present in the -9.1 to -3.4 kb region. Using computer sequence analysis, several consensus activator protein-1 (AP-1) sites were found in the 9.1 kb ALAS-1 sequence but no consensus direct repeat (DR)-4 or DR-5 type recognition sequences for nuclear receptors were identified in the drug-responsive 3.5 kb region. Deletion constructs containing +0.082 to -7.6 kb (pGcALAS7.6-Luc) and +0.082 to -6.2 kb (pGcALAS6.3-Luc) cALAS 5'- flanking and 5' - UTR region were generated and tested and pGcALAS6.3-Luc was shown to have heme-dependent repression of basal and Glut and DHA-induced activity. A recently identified 167 bp chick ALAS-1 drug responsive enhancer (DRE) was PCR amplified and inserted upstream of the 9.1 kb (pGcALAS9.1+DRE), a 0.399 kb (+0.082 to -0.317) (pGcALAS0.3+DRE), and pGL3SV40 construct (pGL3SV40+DRE). DRE mediated the up-regulation of pGL3SV40+DRE construct by Glut was ~ 15-30 fold but interestingly only 3.2 and 3.7-fold for pGcALAS9.l +DRE and pGcALAS0.3+DRE constructs, respectively. In summary, in LMH cells drugs up-regulate ALAS-1 through non-DRE element(s) in the first 3.5 kb of ALAS-1 5'-flanking and 5'-UTR region and heme down-regulates ALAS-1 and determines the extent of the drug response through element(s) in the -6.3 to -3.5 kb region of ALAS-1 5'- flanking region.

The intestine is the largest lymphoid organ in the body, challenged constantly by an enonnous quantity and diversity of antigens. Distinct from peripheral lymphocytes, intestinal lymphocytes have evolved unique mechanisms of tolerance and appear to govern mucosal processes such as "chronic physiologic inflammation" and oral tolerance. Failure of mucosal tolerance has been implicated in the pathogenesis of several diseases, including inflammatory bowel disease, celiac disease, and even autoimmune diabetes. One population of intestinal lymphocytes, intraepithelial lymphocytes (IELs), exists within the intestinal epithelium itself and remains poorly characterized. IELs respond to unique activation signals and appear to be in part responsible for the maintenance of epithelial integrity and mucosal tolerance. Type 1 diabetes is one of the most common chronic childhood illnesses and causes significant morbidity and mortality. Type 1 diabetes mellitus is an autoimmune disease that results from immune-mediated destruction of insulin-producing pancreatic beta cells and is characterized by an absolute insulin deficiency. Several animal models are used to study the immunopathogenesis of type 1 diabetes, including the BB rat and NOD mouse. BBDP rats spontaneously develop autoimmune diabetes mellitus and are severely deficient in peripheral T cells. BBDR rats do not spontaneously develop autoimmune diabetes, have nonnal numbers of peripheral T cells, and can be induced to become diabetic by injections of a cytotoxic anti-ART2a mAb and low doses of poly I:C. The cause of autoimmune diabetes in BB rats and humans is still unknown, but both genetic and environmental factors appear to participate. I hypothesize that one important class of environmental factors--diet and enteromicrobial agents--participates in this pathogenic process through the mediation of the gut immune system. In this dissertation, I report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The yield of rat IELs using this method is 5-10 fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrate that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis reveals 5 major subsets of IELs, including populations of γδ T and natural killer (NK) cells present at levels not previously detected. I also report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, IENK cells differ from splenic NK cells phenotypically, and a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. I conclude that rat IELs harbor a large population of NKR-P1A+ CD3-cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive intraepithelial NK cells may participate in the regulation of mucosal immunity. I next demonstrate that, prior to diabetes, both BBDP and ART2a-depleted BBDR rats have a reduced total number of IELs and exhibit a selective deficiency of IENK cell number and function as compared to control BBDR rats. The deficiency of BBDP rat IELs can be corrected by engraftment of bone marrow from histocompatible WF donors. These results suggest 1) that the peripheral lymphopenia in BBDP rats extends to the IEL compartment, particularly to IENK cells, 2) that in BBDR rats the diabetes-inducing treatment depletes IELs, particularly IENK cells, and 3) that the defect in BBDP rat IELs is intrinsic to hematopoietic cells, not intestinal stromal cells. I also establish that, unlike BBDR and WF rats, BBDP rats are also deficient in γδTCR+IELs, a population of T cells that may play a role in normal mucosal tolerance. In addition, I report preliminary data supporting the hypothesis that systemic autoreactivity may be initiated in the intestine; peripheral autoreactive lymphocyte populations appear to emanate first from mesenteric lymph nodes that drain the intestine, and such cells may initiate a type 2 autoimmune phenomenon driven by IL-4. Collectively, my findings support the hypothesis that a failure of mucosal tolerance in BBDP rats, perhaps secondary to deficiencies in one or more IEL subpopulations, participates in the pathogenesis of autoimmune diabetes in these animals by activating peripheral autoreactive T cells. The nature of the autoimmune response in BB rats (driven by IL-4) appears to be distinct from that of NOD mice. Despite the differences between these two well-accepted animal models of autoimmune diabetes, until more is known about the pathogenesis of type 1 DM in humans, lessons learned from both the BB rat and NOD mouse continue to be of tremendous benefit to our understanding of human disease.

In humans, Bloom’s Syndrome is caused by a mutation of the RecQ helicase BLM. Patients with Bloom’s Syndrome exhibit a high amount of genomic instability which results in a high incidence of cancer. Though Bloom’s Syndrome has been intensively studied, there are still many questions about the function of BLM which need to be answered. While it is clear that loss of BLM increases genomic instability, the other effects of genomic instability on the organism aside from cancer such as a potential effect on aging, have yet to be elucidated. In Chapter II, I identify new phenotypes in the C. elegans ortholog of BLM, him-6. him-6 mutants have an increased rate of cell death, a mortal germ line phenotype, and an increased rate of mutations. Upon further examination of the mutator phenotype, it was determined that the increased rate of mutations was caused by small insertions and deletions. The mutator phenotype identified in him-6 mutants closely mimics the cellular phenotype seen in Bloom’s Syndrome cells. This indicates that HIM-6 may behave in a similar fashion to BLM. In addition to the mutator phenotype, it was found that loss of him-6causes a shortened life span. This may provide evidence that there is a link between genomic stability and aging. In Chapter III, I identify a new role for the transcription factor DAF-16. DAF-16 in C. elegans has been intensively studied and regulates a wide variety of pathways. In this chapter, I demonstrate via the well established unc-93 assay that loss of daf-16 causes a subtle mutator phenotype in C. elegans. This indicates that DAF-16 may play a role in suppression of spontaneous mutation. When I examined other classic genomic instability phenotypes, I found at 25°C, the number of progeny in the DAF-16 mutants was significantly reduced compared to wild type worms. Additionally, I demonstrate daf-16(mu86)has a cell death defect. This study identifies several new phenotypes caused by a loss of him-6. These phenotypes provide further evidence that loss of him-6 causes genomic instability. In addition, this study also demonstrates that him-6 has a shortened life span which may be due to genomic instability. Secondly, this study identifies a new role for DAF-16 in preventing the occurrence of spontaneous mutations. This may indicate a novel function for DAF-16 in maintaining genomic stability.

NOD mice model human type 1 diabetes and have been used to investigate tolerance induction protocols for islet transplantation in a setting of autoimmunity. Costimulation blockade-based tolerance protocols that induce prolonged skin and permanent islet allograft survival in non-autoimmune mice have failed in NOD mice. To investigate the underlying mechanisms, we generated NOD hematopoietic chimeras. We were able to show that dendritic cell maturation defects seen in NOD mice are partially corrected in mixed hematopoietic chimeras. Furthermore, skin allograft survival was dependent upon the phenotype of the bone marrow donor, demonstrating that in the NOD the resistance to tolerance induction resides in the hematopoietic compartment. In addition, we studied congenic NOD mice bearing insulin dependent diabetes (Idd) loci that reduce diabetes incidence. The incidence of diabetes is reduced in NOD.B6 Idd3 mice, and virtually absent in NOD.B6 Idd3Idd5 mice. Islet allograft survival in NOD.B6 Idd3 mice is prolonged as compared to NOD mice, and in NOD.B6 Idd3Idd5 mice islet allograft survival is similar to that achieved in C57BL/6 mice. Alloreactive CD8 T cell depletion in NOD mice treated with costimulation blockade is impaired, but is partially restored in NOD.B6 Idd3 mice, and completely restored in NOD.B6 Idd3Idd5 mice. Idd3 results from variations in Il2 gene transcription. We hypothesized insufficient levels of IL-2 in NOD mice contributes to impaired deletion of alloreactive CD8 T cells and shortened islet allograft survival. We observed using synchimeric mice that co-administration of exogenous IL-2 to NOD mice treated with costimulation blockade led to deletion of alloreactive CD8 T cells comparable to that in C57BL/6 mice and prolonged islet allograft survival. However, some Idd loci impaired the induction of transplantation tolerance. These data suggest that Idd loci can facilitate or impair induction of transplantation tolerance by costimulation blockade, and that Idd3 (IL-2) is critical component in this process.

Serum vitamin D deficiency/insufficiency, Chronic Kidney Disease (CKD) and elevated blood pressure are important health concerns especially among minorities with type 2 diabetes. The effect of vitamin D3 supplementation (cholecalciferol) at 6,000 IU/day (d) vs. 4,000 IU/d on kidney function and cardiovascular disease markers among Hispanics and African Americans with type 2 diabetes and hypovitaminosis D (/ml) was evaluated. Subjects (n=63) were recruited from two clinics in Miami-Dade County, FL. Fasting venous blood and fresh, single-voided first morning urine samples were collected from each participant by a certified phlebotomist and analyzed by Solstas Lab Partners, Davie, FL. Linear mixed models were used to compare the interaction between time and intervention. Least Significant Difference (LSD) comparisons were used to detect significant differences within and between 4,000 IU/d and 6,000 IU/d groups from baseline, 3 and 6 months. In the 4,000 IU/d and 6,000 IU/d groups, a significant increase in serum 25-hydroxy vitamin D [25(OH)D] levels were observed from baseline [(19.9±1.1 ng/mL) and (21.4±1.3 ng/mL)] to 3 months [(36.1±2.2 ng/mL, p3 longer than 6 months may be needed to determine sustained long term effects in kidney and cardiovascular disease markers. Further research could provide more information for translation of these findings into recommendations for individuals with CKD, hypertension and type 2 diabetes. The efficacy of vitamin D3 supplementation as complementary therapy for CKD and blood pressure in minority and other ethnic groups needs further investigation in larger and longer duration randomized controlled trials.

Aging is associated with a loss of proliferation of the insulin-secreting beta cell, a possible contributing factor to the greatly increased rate of type-2 diabetes in the elderly. A landmark study from our lab previously illustrated that mild endoplasmic reticulum (ER) stress drives beta cell proliferation specifically through ATF6α, one arm of the tripartite Unfolded Protein Response (UPR). It is unknown if old beta cells differ from young beta cells in UPR signaling or proliferative response to ER stress or ATF6α activation. To investigate, young and old mouse islets were cultured ex vivo in high glucose, and beta cell proliferation was quantified by BrdU incorporation after treatment with low dose thapsigargin or activation of overexpressed ATF6α. In addition, levels of UPR signaling were compared by semi-quantitative Xbp1 splicing assay. Interestingly, although old beta cells displayed reduced proliferation in glucose compared to young beta cells, their proliferative response to low-dose thapsigargin and ATF6α activation were nearly identical, and no difference was found in Xbp1 splicing under high glucose or high ER stress conditions. These results suggest that the aged mouse beta cell does not have impaired UPR-responsive proliferation or aberrant UPR signaling when cultured ex vivo

Developing a tissue-engineered Blood Vessel Mimic (BVM) to represent diabetic macrovascular disease could expedite design of new vascular devices specifically tailored to diabetic patients. In contribution toward this model, this thesis assessed Human Umbilical Vein Endothelial Cell (HUVEC) responses to high glucose conditions. Interleukin 6 (IL-6) and Cluster of Differentiation 36 (CD36) were selected to signify oxidative stress activity, a hallmark of diabetic macrovascular disease. Next, activity of potential reference genes B2M, HPRT1, and ACTB was assessed. All genes were found to exceed acceptable variability, so the E-ΔC T method of data analysis was selected. Next, cellular responses to high glucose treatment at 10.5 mM glucose and 25.5 mM glucose for 7 and 14 days were measured by qPCR. IL-6 mRNA expression increased significantly (p<0.001) following treatment with 25.5 mM glucose at both timepoints. Finally, fluorescent staining for Reactive Oxygen Species (ROS) production and cell viability was performed on HUVECs treated with 10.5- and 25.5-mM glucose for 24 and 48 hours. No differences in ROS production or cell viability were detected due to uncontrolled cell damage during the two-hour staining and imaging procedure. This thesis was limited by low reaction efficiency in qPCR reactions due to mistaken purchasing of primers with included probe-quencher reporters. Measurement of reaction efficiency facilitated valid analysis of data collected using these primers. Imaging experiments were unsuccessful due to a lack of incubation equipment designated for cells undergoing live staining and imaging. Alternative imaging assessments of oxidative stress activity were proposed to circumvent this problem.

Werner syndrome (WS) is an autosomal recessive disorder associated with premature aging and cancer predisposition. WS cells show increased genomic instability and are hypersensitive to DNA-damaging agents. WS is caused by mutations of the WRN gene. WRN protein is a member of RecQ DNA helicase family. In addition to a conserved 3’–5’ helicase activity, the WRN protein contains unique 3’–5’ exonuclease activity. WRN recognizes specific DNA structures as substrates that are intermediates of DNA metabolism. WRN physically and functionally interacts with many other proteins that function in telomere maintenance, DNA replication, and DNA repair. The function of WRN is regulated by post–translational modifications that include phosphorylation, acetylation, and sumoylation. SIRT1 is a NAD-dependent histone deacetylase (HDAC) that deacetylates histones and a numbers of cellular proteins. SIRT1 regulates the functions of many proteins, which are important for apoptosis, cell proliferation, cellular metabolism, and DNA repair. SIRT1 is also regulated by other proteins or molecules from different levels to activate or inhibit its deacetylase activity. In this study, we found that SIRT1 interacts with and deacetylates WRN. We further identified the major acetylation sites at six lysine residues of the WRN protein and made a WRN acetylation mutant for functional analysis. We found that WRN acetylation increases its protein stability. Deacetylation of WRN by SIRT1 reverses this effect. CREB-binding protein (CBP) dramatically increased the half-life of wild-type WRN, while this increase was abrogated with the WRN acetylation mutant. We further found that WRN stability is regulated by the ubiquitination pathway, and that WRN acetylation by CBP dramatically reduces its ubiquitination level. We also found that acetylation of WRN decreases its helicase and exonuclease activities, and that SIRT1 reverses this effect. Acetylation of WRN alters its nuclear distribution. Down-regulation of SIRT1 increases WRN acetylation level and prevents WRN protein translocating back to nucleolus after DNA damage. Importantly, we found that WRN protein is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells that were stably expressing WRN acetylation mutant display significantly higher sensitivity to MMC than the cells expressing wild-type WRN. Taken together, these data demonstrated that acetylation pathway plays an important role in regulating WRN function in response to DNA damage. A model has been proposed based on our discoveries.

Background Obesity is an important risk factor for resistance to insulin-mediated glucose disposal, and is a precursor of type 2 diabetes and other disorders. Objectives To identify molecular pathways in adipose tissue and inflammatory cells that may result in obesity-associated insulin resistance, we exploited the fact that not all obese individuals are prone to insulin resistance. Thus the degree of obesity as a variable was removed by studying obese subjects of similar body mass index (BMI) who are insulin-sensitive (IS) versus insulin-resistant (IR). Methods Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from 10 obese-IR and 10 obese-IS patients undergoing gastric bypass surgery. In a secondary study, we isolated monocytes from 4 obese-IR, 3 obese-IS, and 4 nonobese-IS adolescent and young adult subjects for purposes of assessing differences in expression of inflammatory genes in monocytes using RT-PCR. Results Gene sets related to chemokine activity and chemokine receptor-binding were identified as most highly enriched in the omental tissue from obese-IR compared to obese-IS subjects, independent of BMI. Strikingly, insulin resistance, but not BMI, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size. In the adolescent and young adult cohort, expression of two cytokine signaling molecules (IL8, SOCS3) and two downstream products of the JNK pathway (JunB, c-Fos) showed increased expression in the obese-IR subjects compared to the obese-IS and nonobese-IS subjects, suggesting the presence of a proinflammatory phenotype in monocytes in obesity, which is exacerbated in the insulin resistant state. Conclusions Our findings demonstrate that inflammation of omental adipose tissue and activation of proinflammatory monocytes is strongly associated with insulin resistance in human obesity. Manipulation of these pathways may result in the prevention of or delay in the onset of obesity-related co-morbidities.