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Obesity is recognized as a risk factor for adverse pregnancy outcomes. Maternal obesity prevalence has increased in parallel with that in the general population and is associated with an increase in morbidity and mortality for both mother and baby. Obese mothers are more likely to develop gestational diabetes, hypertensive disorders including preeclampsia, thromboembolic complications, miscarriage, and have an increased need for induction of labour. Babies born from obese mothers can be abnormally large (macrosomia) or small for gestational age, and have a higher risk of perinatal death and congenital malformation. Pregnancy induces marked and dynamic changes in energy metabolism, however, the direct effects of pregnancy adipose tissue biology in both normal lean and obese women is still largely unknown. The aim of this thesis was to delineate novel mechanisms by which pregnancy affects adipose tissue biology, and thus infer how obesity might adversely affect pregnancy outcomes. We used an animal model of obesity during pregnancy in which mice were given a high fat diet (HF) to make them obese. We identified that pregnancy was associated with an unexpected curtailment of visceral (mesenteric) adipose tissue mass in HF mice and with an attenuation, rather than worsening of the metabolic impairment expected from the combination of excess dietary fat and insulin resistance/glucose intolerance of pregnancy. To determine the underlying molecular mechanism contributing to this phenotype global gene expression microarray with subsequent pathway analysis and qRT-PCR validation was employed within the visceral adipose tissue. In visceral fat of HF pregnant mice, gene pathways for de novo lipogenesis and lipid storage, inflammation, retinol metabolism, insulin like growth factor and estrogenic signaling showed altered regulation. Given the known role of estrogen on adipose tissue and inflammatory cell function, a hypothesis was generated that altered estrogen receptor (ER)α expression/activation/increased estradiol presence within mesenteric fat formed a unifying molecular mechanism underlying the altered adipose biology and relative amelioration of the metabolic phenotype in HF pregnant mice. To test the ER α hypothesis, a female clonal adipocyte cell line, Chub-S7, and primary visceral and subcutaneous adipocytes from pregnant obese and lean patients were treated with the ERα selective agonist, PPT. PPT downregulated mRNA levels of key genes involved in de novo lipogenesis (ME1, FANS and SCD1 Dgat2), consistent with a direct role for ERα activation in curtailment of fat expansion. Although the primary human study lacked sufficient power to adequately address the hypothesis, PPT significantly suppressed SCD1 mRNA levels in visceral adipocytes of lean women. In parallel with the curtailment of mesenteric fat expansion, HF pregnant mice were found to have increased liver weight and liver triglyceride content. However, this “fatty liver” phenotype was not associated with increased mRNA levels of genes involved in hepatic triglyceride uptake or de novo lipogenesis. This increase in liver triglycerides may be due to an excessive influx of fatty acids from mesenteric fat through the portal vein. In conclusion, pregnancy in obese animals is associated with a beneficial curtailment in mesenteric fat expansion, normalization of metabolic disturbances and reduced adipose inflammation. Increased ERα activation within adipocytes may play a critical role in this phenotype.

Peripheral artery disease (PAD) and the consecutive build-up of an atherosclerotic plaque restricting blood flow to the lower limbs lead to critical limb ischaemia, one of the most common circulation problems in the world. Although a small number of interventions (such as surgery or revascularization treatments) are available, patients with this condition are often too ill for these procedures, giving a poor prognosis for the disease. Several strategies to promote neovascularization using different stem cell populations with angiogenic potential have been proposed as plausible therapies. Perivascular cells (PCs), key structural components of the wall of small and large blood vessels have numerous advantages over other cell types since they are highly abundant, easy to obtain from the stromal vascular fraction (SVF) of human adipose tissue (an ethically approved source) and have mesenchymal and angiogenic properties. The work described in this thesis addressed the hypothesis that PCs isolated from human white adipose tissue would promote the recovery of blood flow in an ischaemic hindlimb by increasing blood vessel number and blood perfusion to the foot. To investigate whether PCs from human white adipose tissue could rapidly increase neovascularization and, therefore, be used as a possible therapeutic treatment for PAD and critical limb ischaemia, the initial aim was to validate, characterise and demonstrate the properties of the murine equivalent of these cells, in order to establish a direct link between the injected cells and the ones natively found in the mouse. This was then followed by the use of murine models of angiogenesis to determine whether transplanted human PCs stimulate angiogenesis in vivo. Initial studies using immunohistochemistry, fluorescence-activated cell sorting (FACS) and in vitro mesodermal differentiation demonstrated that perivascular cells (namely pericytes and adventitial cells) are present in multiple mouse organs, can be sorted to purity, and have mesenchymal stem cell (MSC) properties. These cells had similar characteristics to their human counterparts, thus validating the mouse as a suitable model for determining whether transplanted human PCs could stimulate angiogenesis. Using in vitro and two in vivo (sponge implantation and hindlimb ischaemia) models, it was shown that human PCs have angiogenic properties being capable of tube formation and interaction with endothelial cells, as well as promoting angiogenesis within sponges. Contrary to expectations, PCs did not increase blood perfusion to the mouse ischaemic hindlimb, despite increasing microcirculation within the skeletal muscle and myofibre regeneration. This work showed that PCs obtained from human adipose tissue have important therapeutic implications in promoting angiogenesis and skeletal muscle regeneration but failed to increase arteriogenesis which is the key mechanism allowing the restoration of blood perfusion.

archives@tulane.edu / Background: Individuals with human immunodeficiency virus (HIV) and undergoing antiretroviral therapy (ART) exhibit high levels of circulating inflammatory cytokines and proteins, which are strongly correlated with shortened time to death and disease. To target damaging inflammation at the source, the drivers of inflammation must be identified. Adipose tissue is a massive organ that contains adipocytes and immune cells capable of producing pro-inflammatory mediators. Dysregulated adipose tissue is implicated in the pathogenesis of obesity and related diseases, such as type 2 diabetes, that are likewise reported in persons with chronic HIV infection. Adipose tissue was therefore explored as a contributor to circulating inflammation in patients with HIV using the rhesus macaque model. Simian immunodeficiency virus (SIV) closely models HIV regarding pathogenesis, including CD4+ T cell depletion, induction of a viral reservoir, and development of opportunistic infections before succumbing to Acquired Immunodeficiency Syndrome (AIDS) and death. Methods: Subcutaneous adipose tissue (SQAT) from SIV-infected rhesus macaques was characterized using confocal microscopy to describe the major immune cell subsets. Adipose tissue homogenates and plasma were analyzed for expression of genes and proteins related to inflammatory processes using antibody and RNA-based fluorescent multiplex bead technology for protein and gene quantitation, respectively. The functions of adipose tissue immune cells during SIV infection were measured with stimulation and phagocytosis assays. / 1 / Marissa Fahlberg

Fibroblast Growth Factor 21 (FGF21) is an endocrine hormone derived from the liver that exerts pleiotropic effects on the body to maintain overall metabolic homeostasis. During the past decade, there has been an enormous effort to understand the physiological roles of FGF21 in regulating metabolism and to identify the mechanism for its potent pharmacological effects to reverse diabetes and obesity. Through both human and rodent studies, it is now evident that FGF21 is dynamically regulated by nutrient sensing and consequently functions as a critical regulator of nutrient homeostasis. In addition, recent studies with new genetic and molecular tools have provided critical insight into the actions of this exciting endocrine factor. Dissection of these FGF21-regulated pathways has tremendous potential for new targeted therapies to treat metabolic disease. The goals of this thesis are 1) to identify FGF21’s physiological role as a carbohydrate-regulated signal of macronutrient-specific satiety and 2) to determine the mechanism and tissues responsible for mediating the pharmacological effects of FGF21. To address the first goal, we used different FGF21 genetic knockout mouse models to determine if loss of FGF21 would affect macronutrient preference. We found that loss of FGF21 led to an increase in simple sugar intake whereas this had no effect on other macronutrients such as lipid or protein. To further characterize the relationship between carbohydrates and FGF21, in vitro and in vivo techniques revealed that FGF21 transcription in the liver increased in response to carbohydrate intake and this was dependent on the presence of a transcription factor activated by carbohydrates, ChREBP. We next addressed whether or not increased FGF21 levels would affect preference for simple sugars. We found that in response to increased circulating levels of FGF21, either through genetic overexpression or pharmacological administration, FGF21 would lead to a significant decrease in caloric and non-caloric sweeteners. Finally, we were able to determine that FGF21 was signaling to the hypothalamus to mediate this suppression of simple sugar intake through region specific knockout of the co-receptor beta-klotho. To address the pharmacological actions of FGF21, we generated an adipose-specific KLB KO mouse using mice that express Cre-recombinase under the adiponectin promoter. These mice lack the co-receptor for FGF21 in adipose tissue and are a more reliable adipose knockout model than previous studies that have used aP2-Cre mice. We were able to determine that the acute glucose lowering effects of FGF21 are mediated through direct signaling to adipose tissue and that FGF21 enhances insulin sensitivity by increasing glucose uptake in brown adipose tissue. However, FGF21 mediates its chronic effects, including lowering body weight and triglycerides, by signaling to some other non-adipose tissue. Overall our work has shown that FGF21 can significantly regulate glucose metabolism through multiple mechanisms.

Graduation date: 2004

Graduation date: 1996