BACKGROUND: Brown adipose tissue (BAT) is a thermoregulatory tissue that may have a positive influence on metabolic health by improving glucose homeostasis, reducing adiposity and increasing energy expenditure. It is enriched with uncoupling protein-1 (UCP1) and therefore produces heat by uncoupling oxidative phosphorylation from ATP production. It has long been known that infants are born with BAT, however, only recently has BAT been reported in children and adults. In humans, BAT is predominantly located in the supraclavicular (SCV) region, however there are smaller depots in the peri-renal and mediastinal areas. BAT has primarily been studied in humans using Positron Emission Tomography/Computed Tomography (PET/CT). Such studies have demonstrated that children appear to have a higher prevalence of BAT than adults, however this methodology is not suitable for widespread research in healthy children. Therefore non-invasive methods that accurately measure BAT are required. The factors influencing BAT activity are of interest as this tissue may act as a desirable therapeutic target for metabolic diseases such as obesity and diabetes. PURPOSE: The purpose of this thesis project was two fold; Part 1 involved the examination of SCV skin temperature as a measure of BAT activity in children and the objective of Part 2 was to determine if SCV skin temperature had a relationship to body composition and physical activity in children. METHODS: We recruited 33 pre-pubertal boys (ages 8-10) to this cross-sectional study. The study included 3 visits, in which we measured lean mass and fat mass with dual-energy X-ray absorptiometry, resting energy expenditure (REE) before and after a 30 minute 12ºC cold exposure with indirect calorimetry, objective physical activity with an accelerometer and SCV temperature (measured every 2.5 minutes during a 30 minute, 12ºC cold exposure) with an Infrared Thermal Camera. Lean mass and Fat mass were quantified as lean mass index (LMI) and fat mass index (FMI). Physical activity was quantified as total accelerometer counts per minute (CPM) and minutes of moderate to vigorous physical activity (MVPA) per day. For Part 1, we assessed the precision of IR-thermal imaging of SCV skin temperature by examining the reproducibility of eight skin temperature outcomes over two trials. Furthermore, we assessed the accuracy of these eight skin temperature outcomes by investigating their association with energy expenditure. For Part 2 we evaluated the relationship of FMI, LMI and physical activity (MVPA) with SCV skin temperature in a multivariate analysis. RESULTS: Following these analyses, post-cooling skin temperature had the highest reproducibility of the eight skin temperature outcomes (intraclass correlation coefficient of 0.95, p<0.001) and was also significantly correlated with energy expenditure (Pearson correlation=0.392 p=0.032). Therefore, we used this outcome measure when examining the relationship between SCV skin temperature, body composition and physical activity. Fat mass index (FMI) was inversely related to post-cooling SCV skin temperature (β= -0.125, p<0.001). Minutes of moderate to vigorous physical activity and lean mass index (LMI) were not related to post-cooling SCV skin temperature. CONCLUSION: This study determined that post-cooling SCV skin temperature may be useful for detecting BAT in children and it is inversely related to adiposity. / Thesis / Master of Science (MSc)

OBJECTIVE: Obesity is a chronic disease with high incidence worldwide, which promotes the risk of incidence of type 2 diabetes (T2D). Obesity-induced adipocyte expansion promotes local chronic inflammation in the adipose tissue which is considered a contributing factor to insulin resistance, hyperinsulinemia, and T2D. Many organs, including adipose tissue, involve in the dysregulation of glucose homeostasis in T2D. The crosstalk between adipose tissue/adipocytes and pancreatic ß-cells has provoked scientists' interest for years. Here in this thesis, we focused on the effect carried out by adipocyte-specific GPS2 depletion on insulin secretion from pancreatic ß-cells. METHODS: Conditioned media collected over 24 h from both primary adipocyte and adipose tissue explant cultures from high fat diet (HFD)-fed WT and adipocyte-specific GPS2 knock-out (GPS2-AKO) mice were used to treat INS-1 clonal pancreatic ß-cells or primary islets from chow-diet WT mice. Conditioned media was diluted 1:8 in culture media of clonal INS-1 cells (cultured in media with 4 mM or 11 mM glucose chronically) and primary islets (cultured in media with 11 mM glucose) and incubated for 18 h before measuring insulin secretion. The isolated islets from chow-diet WT mice were also be treated with the primary adipocytes conditioned media from eWAT (epididymal white adipose tissue) of HFD-fed WT and GPS2-AKO mice. In addition, the effect of exosomes extracted from primary adipocyte conditioned media of HFD-fed WT and GPS2-AKO mice on GSIS was investigated in clonal INS-1 cells. Glucose-stimulated insulin secretion (GSIS) was measured to assess differences in insulin secretion by INS-1 cells and islets from mice in response to signaling from WT or GPS2-AKO adipocytes. RESULTS: Adipocyte conditioned media from both WT and GPS2-AKO mice reduced GSIS from INS-1 cells by the same extent compared to a non-treated control. The same result was obtained using media conditioned by adipose tissue explant culture. Exosomes isolated from adipocyte conditioned media from both WT and GPS2-AKO mice also reduced GSIS from INS-1 cells with no significant difference between WT and GPS2-AKO. Islets isolated from chow-diet WT mice treated with adipocyte conditioned media from eWAT of WT and GPS2-AKO mice also showed no significant difference between WT and GPS2-AKO in GSIS compared to our non-treated control. CONCLUSIONS: Both conditioned media and exosomes from primary adipocytes of HFD-fed mice inhibits GSIS from INS-1 cells and isolated islets, but no difference was observed between WT and GPS2-AKO mice. We conclude that the deletion of GPS2 in adipocytes does not influence GSIS from pancreatic ß-cells under our experimental conditions. Conditioned media-induced inhibition of GSIS is mediated by factors that may contribute to adipocyte-ß-cell crosstalk in-vivo. / 2025-11-02T00:00:00Z

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Adipose tissue is a type of connective tissue whose purpose was once thought to be limited to fat storage but is now understood to be a key factor in the pathogenesis of different metabolic diseases, including obesity and type-II diabetes. Adipose tissue consists largely of adipocytes, cells responsible for fat and releasing energy in form of lipids. Different classes of fatty acids, such as saturated and unsaturated have different biological effects on adipocytes. Lipid droplets are the primary organelles in adipocytes that store these fatty acids in form of lipids, and the development of engineered adipose tissues would benefit from improved techniques for analysis of lipid droplet composition, distributions, and dynamics based as a function of fatty acid saturation. Conventional microscopic techniques, such as fluorescence microscopy, provides excellent selectivity of lipid-based structures inside adipose tissue cellular structures based on staining with compound dyes. However, fluorescence staining limits multiplex imaging, and requires time consuming steps in preparing the samples for imaging. Therefore, developing a label-free, high resolution imaging platform with sensitivity to lipid composition could enable analysis of structural and compositional differentiation of lipid droplets within adipocytes during differentiation could give valuable insights into the importance of lipid droplets role in metabolism. As an important step towards achieving this goal, a femtosecond based CARS microscopy imaging platform has been developed to perform in vitro, label-free, imaging of fatty acid composition within engineered adipose tissues. / Bioengineering

BACKGROUND: There has been renewed interest in the study of brown adipose tissue (BAT) as a potential therapeutic target for obesity, diabetes and non-alcoholic fatty liver disease (NAFLD). There is now much evidence to suggest that BAT is not only important in thermogenesis but also plays an important role in metabolism. In adults, cold-induced BAT activation has led to a significant increase in insulin sensitivity and energy expenditure as well as decreased blood sugar levels. Thus, it is important to identify factors associated with these metabolic disorders such as the presence and activity of BAT to better understand if and how BAT can be targeted to treat these disorders. However, as a potential therapeutic target, it is important to develop accurate, precise, robust and reproducible non-invasive modalities to measure BAT. PROJECT OBJECTIVES: 1) Develop and assess protocols for the use of MRI in measuring BAT characteristics and activity 2) Examine the relationship between BAT MR outcomes and known covariates such as age, sex, body fat percentage and outdoor temperature in adult humans 3) Determine if there is any association between BAT outcomes and liver fat in adult humans, before and after adjusting for potential covariates of liver fat such as age, sex and body fat percentage METHODS: In total, 36 healthy participants (i.e. no conditions or medications that could influence BAT metabolism and/or liver disease) aged 18 to 60 years were recruited to this cross-sectional study. There were two study visits. In visit 1, anthropometrics (i.e. height, weight and waist circumference), blood pressure and body composition (via dual x-ray energy absorptiometry) were measured. Additionally, fasting bloodwork was collected and a 75-g oral glucose tolerance test (OGTT) was administered. During visit 2, participants were exposed to a standardized cold exposure set at 18°C for 3 hours using a water-perfused suit. MRI scans were acquired to evaluate changes in fat-fraction (FF%) and T2* relaxation (T2*) (BAT MR outcomes), liver fat and abdominal fat after a cold exposure. During the cold exposure protocol, mean skin temperature (MST) was monitored using 12 wireless temperature loggers placed at different sites of the body while electromyography (EMG) was used to measure shivering intensity. RESULTS: In the current study, an MRI protocol capable of detecting BAT in the supraclavicular (SCV) region was developed. This protocol included the use of FF and T2* masks to more accurately characterize BAT in the SCV region. Additionally, the MR segmentation protocol was found to be very reliable, as demonstrated by excellent ICC values (i.e. ICCagreement and ICCconsistency ≥ 0.90) for all BAT MR outcomes irrespective of cold exposure. As expected, FF% (mean difference = -2.97; p < 0.0001*) and T2* (mean difference = -0.84; p < 0.0001*) values in the SCV significantly decreased after cold exposure, consistent with BAT activation. Furthermore, the decline in both FF% and T2* after cooling was specific to the SCV region, as these changes did not occur in the posterior neck fat. In examining the relationship between BAT MR outcomes and known covariates of BAT (i.e. age, sex, body fat percentage and outdoor temperature), it is important to note that lower FF% or T2* values are reflective of a browner phenotype while a greater reduction in FF% is indicative of higher BAT activity. BAT characteristics (A: pre-cold FF%; B: pre-cold T2*) and BAT activity (C: FF% reduction) were correlated with age (A: r = 0.54; p = 0.0007*; B: r = 0.42; p = 0.0112*; C: r = -0.39; p = 0.0213*) and body fat percentage (A: r = 0.83; p < 0.0001*; B: r = 0.58; p = 0.0002*; C: r = -0.64; p < 0.0001*). That is, higher age and body fat were associated with a less brown phenotype prior to cold exposure and with less BAT activity (i.e. lower FF% decline) in response to cold exposure. However, no associations were found between BAT MR outcomes and sex or outdoor temperature. Lastly, liver fat was associated with higher values of pre-cold FF% (r = 0.60; p < 0.0001*) and pre-cold T2* (r = 0.47; p = 0.0040*) while FF% reduction was inversely correlated with liver fat (r = -0.38; p = 0.0295*). Additionally, the relationship between BAT MR outcomes and liver fat still existed after adjusting for age and sex while its effects were mediated by adiposity. CONCLUSION: In this study, a highly reliable MR segmentation protocol was developed that is capable of measuring BAT characteristics and activity irrespective of cold exposure. Additionally, the cold exposure protocol used was sufficient to elicit changes in BAT MR outcomes, as demonstrated by significant changes in FF% and T2* after cooling. Consistent with previous studies, BAT outcomes (as measured by MRI) were associated with age and body fat percentage. Lastly, findings in this thesis provide strong supporting data that BAT may regulate liver lipid content, however, the extent and mechanisms remain to be determined. / Thesis / Master of Science (MSc)

Uncoupling protein, a mitochondrial inner membrane protein found in mammalian brown adipose tissue, functions as an uncoupler of oxidative phosphorylation by serving as a proton carrier when activated, resulting in heat production, the function of the tissue. Unlike most nuclear-encoded mitochondrial proteins, uncoupling protein is not made with a cleavable presequence. With the availability of an uncoupling protein cDNA clone, the region responsible for targeting uncoupling protein to mitochondria was examined using in vitro transcription and translation and import into isolated mitochondria. In order to localize the targeting sequence of uncoupling protein, fusion proteins containing portions of uncoupling protein and uncoupling protein modified by site-directed mutagenesis were constructed and analysed for their ability to be imported. Previously it has been shown that there was a targeting signal within uncoupling protein amino acids 13 to 105 (Liu et al., 1988). However, amino acids 13 to 51 did not target a passenger protein to mitochondria (Liu et al., 1988). Here the role of amino acids 53 to 105 of uncoupling protein in targeting was examined with two new constructs, uncoupling protein amino acids 53 to 105 joined to rat ornithine carbamoyltransferase amino acids 147 to 354 and to mouse dihydrofolate reductase. These two constructs along with uncoupling protein with amino acids 2 to 51 deleted were imported into mitochondria consistent with uncoupling protein amino acids 53 to 105 having a potential targeting role in uncoupling protein. Further, these three constructs were processed upon import. The major processed forms of all three constructs are approximately 20 amino acids smaller than the initial translation product. Both fusion constructs also have an intermediate-sized processed form approximately 14 amino acids smaller than the initial translation product. Processing suggests that at least the amino terminus of these proteins has reached the mitochondrial matrix. The location of the proteins was examined using Na2CO3 extraction. Uncoupling protein and U13-105-OCT (uncoupling protein amino acids 13 to 105 joined to ornithine carbamoyltransferase amino acids 147 to 354) were found in the membrane fraction while the processed forms of Ud2-51 (uncoupling protein with amino acids 2 to 52 deleted) and U53-105-DHFR (uncoupling protein amino acids 53 to 105 joined to dihydrofolate reductase) were found in the aqueous fraction suggesting that uncoupling protein amino acids 2 to 52/53 are involved in membrane localization. Analysis of Ud2-35 (uncoupling protein with amino acids 2 to 35 deleted) revealed that it was associated with both the membrane and aqueous fractions. Analysis of uncoupling protein amino acids 53 to 105 revealed the potential existence of two positively charged amphipathic a-helices. Based on the sizes of processed forms and on the helical wheel projection for the first possible sequence, arginine54 , lysine56 and lysine67 were changed to glutamines, individually and in various combinations using oligonucleotide site-directed mutagenesis. All mutant proteins were imported into mitochondria even when all three basic amino acids were replaced. The results suggest that this portion of uncoupling protein, amino acids 54 to 67, is not a targeting signal in the protein. / Thesis / Master of Science (MS)

Obesity is a disease characterized by chronic inflammation and the progressive loss in systemic insulin sensitivity. One of the more effective medications in the treatment of insulin resistance have been the thiazolidinediones (TZDs), which act through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma ). Due to the many side-effects of TZDs, our laboratory sought out a natural phytochemical, abscisic acid (ABA), with chemical similarities to TZDs. Our first study demonstrated that ABA activates PPARgamma in vitro and significantly ameliorates white adipose tissue (WAT) inflammation and glucose tolerance in db/db mice. We next further examined the effect of ABA on the phenotype of adipose tissue macrophages (ATMs). In doing so, we discovered two separate ATM populations which differed in their expression of the macrophage surface glycoprotein and maturation marker F4/80 (F4/80hi vs F4/80lo). Dietary ABA-supplementation significantly reduced F4/80hiCCR2+ ATMs and had no effect on the F4/80lo population. Utilizing a tissue-specific knockout generated through Cre-lox recombination, we were able to determine that this effect was dependent on PPARgamma in immune cells. To further characterize the differences between the ATM subsets that were affected by ABA, we performed a multi-organ assessment (i.e., WAT, skeletal muscle and liver) of the effect of diet-induced obesity on the phenotype of infiltrating macrophages and T cells into metabolic organs. Based on our new data, we formulated a model by which F4/80hiCCR2hi ATMs infiltrate WAT and ultimately induce a CD11c+ pro-inflammatory phenotype in the resident F4/80loCCR2lo subset. Ultimately, our findings provide evidence that ABA has potential as an alternative preventive intervention, expound the role of PPARgamma in immune cells and, in general, expand our knowledge concerning the immunopathogenesis of obesity-induced insulin resistance. / Ph. D.

The aims of the first study were to determine the effects of early weaning followed by a period of high-grain feeding on signaling protein phosphorylation in muscle and on acetate clearance in growing steers.  Twelve Angus X Simmental steers were used in the study.  Six steers were weaned early (EW) and fed a high-gain diet for 148 d and the remainder were weaned at the normal age (NW).  Longissimus muscle tissue biopsies were collected to determine total and phosphorylated forms of AMP kinase and downstream proteins of the mTOR signaling pathway.   Of the 12, 8 steers were used to assess acetate clearance at different age points.  During early grain feeding, phosphorylated:total ratios of rpS6 and S6K1 were greater in EW steers and they had lower acetate clearance rates and greater synthesis rates.   The objectives of second study were to assess the effects of early grain feeding on acetate and glucose turnover rates, palmitate synthesis, and on acetate and glucose preference by subcutaneous, intramuscular and visceral adipose tissues in finishing steers.  Sixteen Angus x Simmental steers were infused continuously with [2H3] acetate (n = 8) or [U-13C6] glucose (n = 8), over a 12 h period immediately prior to harvest.  Plasma acetate and glucose enrichment, and palmitate enrichment in different adipose tissue locations were determined.  There were no treatment effects on acetate or glucose turnover rates or palmitate fractional synthesis rates (FSR).  Acetate turnover and palmitate FSR from acetate were greater than the corresponding rates from glucose.  There were no differences in preference for acetate or glucose among the fat depots. In conclusion, phosphorylation ratios of signaling proteins were not affected treatment.  Acetate clearance increased when steers were heavier and older.  Thus older calves are able to clear more acetate per unit of time and body mass than younger calves reflective of an enhanced ability to utilize the substrate.  There were no differences in preference for acetate and glucose among the major fat depots.  Thus diets leading to high glucose supply will not preferentially direct energy storage to intramuscular stores. / Ph. D.

Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>