Plant-Derived Bioactive Lipids Impacts Glucose Homeostasis and Energy Metabolism in Mice

There is a crucial need to identify and test sustainable alternatives to fish oil as a means to supplement dietary omega (n-3) fatty acids which have demonstrated health benefits to humans with metabolic syndrome and its associated diseases. Echium oil has a high content of the n-3 fatty acid stearidonic acid (SDA), a precursor of the bioactive lipids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found in fatty cold-water fish, with known or possible functions to improve metabolism and delay the onset of or prevent diabetes. To characterize the effects of dietary Echium oil (EO) vs. fish oil (FO), the oils were formulated into either a low-fat (10% kcal; LF) or high fat (60% kcal; HF) diabetogenic diet and fed to male C57BL/6 Tac mice for 12 weeks. Compared to the low-fat or high-fat controls without the supplementation of EO or FO, EO and FO diets had no effect on blood glucose concentrations or plasma insulin levels throughout the study. The EO-enriched HF diet improved glucose tolerance by week 12 compared to the HF-CON (p\u3c0.05) and HF-FO (p\u3c0.1) groups. EO supplementation reduced visceral fat weight without affecting body mass, promoted a metabolically favorable high polyunsaturated fatty acid (PUFA) to saturated fatty acid (SFA) ratio in adipose and muscle tissues compared to the HF-CON and HF-FO diet groups, and led to higher tissue EPA and DHA concentrations compared to both LF and HF CON (p\u3c0.1). Tissue EPA and DHA in EO were not as high as the concentrations found in mice fed the FO diets for both HF and LF. In conclusion, EO-supplemented diets in mice appear to have distinct effects from FO diets that may be exploited in future strategies to curtail metabolic disorders

Hepatocyte Nuclear Factor 3beta is Involved in Pancreatic Beta-Cell-Specific Transcription of the PDX-1 Gene

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdx-1 is expressed principally within insulin-secreting pancreatic islet b cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb 24.5 to 18.2 was used to drive a b-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp 22560 and 21880 (site 1), bp 21330 and 2800 (site 2), and bp 2260 and 1180 (site 3), were identified within the 5*-flanking region of the endogenous pdx-1 gene. Pancreatic b-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdx-1–herpes simplex virus thymidine kinase promoter expression constructs in transfected b-cell and non-b-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven b-galactosidase reporter construct was directed to islet b-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in b-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides 22007 and 21996. Gel shift analysis indicated that HNF3b present in islet b cells binds to this element. Immunohistochemical studies revealed that HNF3b was present within the nuclei of almost all islet b cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3b, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas

Structure and regulation of the human muscle-specific enolase gene

The enzyme enolase (E.C.4.2.1.11), catalyses the interconversion of 2-phosphoglycerate to phosphoenolpyruvate and occurs midway through the glycolytic pathway. Mammals and birds exhibit three specific isoenzymes; muscle-specific enolase [MSE] expressed in adult skeletal muscle, neuron-specific enolase (NSE) expressed primarily in neurons and non-neuronal enolase (NNE) expressed in foetal and other cell types. This thesis describes the characterisation of the human MSE gene and the studies of the transcriptional regulation of this gene. Chapter 1 reviews the current literature on MSE with respect to the other two isoenzymes, NSE and NNE and includes a brief summary of the aims of the project. Chapter 2 outlines the general methodology used throughout the project. Chapter 3 describes the characterisation of the human MSE gene. Its exon-intron structure is similar to that of the other isoforms. The 5' end displays features characteristic of a CpG-rich island. In keeping with its role as a muscle-specific gene, three muscle-specific regulatory elements, M-CAT, MEF-1 and CArG were identified. This study also demonstrates that the MSE transcript is expressed specifically in adult skeletal muscle. Chapter 4 describes methylation studies carried out to determine the role of DNA methylation in the regulation of MSE gene expression. The studies show that the 4kb CpG-rich island is methylation-free. One site however, in intron 6 is partially demethylated in skeletal muscle but fully methylated in sperm and brain. The implications of the methylation-free island and the site-specific demethylation are discussed. Chapter 5 describes the characterisation of the MSE promoter, by transient transfection studies and CAT assays in a mouse muscle cell line. Although the promoter region per se is not able to drive CAT gene expression, proximal and distal domains of the promoter region display 20-30 fold higher CAT activity equally, at both the myoblast and myotube stage. The role of the MSE gene as an early marker of gene expression, and the potential function of the muscle-specific regulatory elements are discussed. Chapter 6 presents a general discussion of the work carried out for this project and offers possible future directions for further investigations.</p

Vagal control of pancreatic β-cell proliferation

The physiological mechanisms that preserve pancreatic β-cell mass (BCM) are not fully understood. Although the regulation of islet function by the autonomic nervous system (ANS) is well established, its potential roles in BCM homeostasis and compensatory growth have not been adequately explored. The parasympathetic vagal branch of the ANS serves to facilitate gastrointestinal function, metabolism, and pancreatic islet regulation of glucose homeostasis, including insulin secretion. Given the functional importance of the vagus nerve and its branches to the liver, gut, and pancreas in control of digestion, motility, feeding behavior, and glucose metabolism, it may also play a role in BCM regulation. We have begun to examine the potential roles of the parasympathetic nervous system in short-term BCM maintenance by performing a selective bilateral celiac branch-vagus nerve transection (CVX) in normal Sprague-Dawley rats. CVX resulted in no detectable effects on basic metabolic parameters or food intake through 1 wk postsurgery. Although there were no differences in BCM or apoptosis in this 1-wk time frame, β-cell proliferation was reduced 50% in the CVX rats, correlating with a marked reduction in activated protein kinase B/Akt. Unexpectedly, acinar proliferation was increased 50% in these rats. These data suggest that the ANS, via the vagus nerve, contributes to the regulation of BCM maintenance at the level of cell proliferation and may also mediate the drive for enhanced growth under physiological conditions when insulin requirements have increased. Furthermore, the disparate effects of CVX on β-cell and acinar cells suggest that the endocrine and exocrine pancreas respond to different neural signals in regard to mass homeostasis