M. Sc. Thesis Abstracts

Regulation of the nuclear factor activated T cell (NFAT) family of transcription factors in the freeze tolerant wood frog, Rana sylvatica

Abstract:

During winter, wood frogs (Rana sylvatica) can endure whole body freezing with 65-70% of total body water converted to extracellular ice. As a result, cells experience extensive dehydration when water exits as well as anoxia due to interruption of blood flow. Adapting to such challenges requires metabolic rearrangement, partially mediated by transcription factor control over gene expression. Here, involvement of the nuclear factor of activated T-cells (NFAT) transcription factors, isoforms c1-c4, was analyzed in liver and skeletal muscle over freeze/thaw and anoxia/re-oxygenation cycles. Freezing activated NFATc3 in liver, leading to increased osteopontin expression and glycogen synthase kinase 3β repression (the latter potentially linked with glucose production as a cryoprotectant). Anoxia activated NFATc4 in liver, leading to increased atrial natriuretic peptide levels. Neither freezing nor anoxia significantly affected NFATs in skeletal muscle. Overall, the study indicates that NFATs have a crucial role to play in the natural cryoprotection of liver.

Involvement of reversible protein methylation in 13-lined ground squirrel hibernation

Abstract:

During winter hibernation, body temperature falls to near ambient levels, metabolism is shifted to favor lipid oxidation and transcriptional and translational activity is minimized in the face of limited resources and increased heat generation costs. In order to regulate such profound changes, mammals require control at least partly brought about by protein post-translational modifications. Protein lysine methylation provides a mechanism by which enzymes may alter the activity, stability and modification states of proteins relevant to hibernator physiology. Protein abundance of SMYD2, SUV39H1, SET8, SET7/9, G9a, ASH2L and RBBP5 in 13-lined ground squirrel (Ictidomys tridecemlineatus) skeletal muscle and liver was characterized. Tissue-specific regulation was seen and enzymes changed during either torpor, arousal, or transitory periods. Methylation of H3, HSP90, and p53 proteins were also quantified and typically followed patterns of modifying enzymes. Overall, these experiments show protein lysine methylation is differentially regulated during 13-lined ground squirrel hibernation.

An investigation of the relationship between dietary fiber, fecal bacterial composition, and colon cancer

Abstract:

Colon cancer (CC) is the second leading cause of all cancer-related deaths in North America. Dietary fiber (DF) may be an important risk factor in the aetiology and pathogenesis of CC. The anticancer effects of dietary fiber were investigated with a focus on fecal bacterial diversity and toxicity of bacterial metabolites in the aqueous phase of feces (fecal water: FW) that contains bile acids, short chain fatty acid, lactate, succinate, etc. Briefly, male Fischer-344 rats were randomized to one of 3 diets: alphacel (control), fructooligosaccharides (FOS) or wheat bran (WB) with a total fermentability level of 3% (wt/wt). Rats were injected with saline or azoxymethane (AOM) to induce tumors. FW toxicity was tested on HCT-116 cells. Rats fed alphacel and FOS diets had significantly more colon tumors than those fed WB. FW from both FOS and alphacel significantly increased apoptosis and DNA damage, and induced cell cycle arrest in HCT-116 cells after a 48 hr treatment whereas FW of WB had no effect on those cell parameters. Lower pH of FW was associated with more tumors incidence and higher cell toxicity. FOS diet was significantly associated with more Allobaculum sp. whereas Lactobacillus sp. and Clostridium XI sp. were associated with WB diet. These results suggest that dietary fiber can be an influence in CC development. This seems to be related to changes in bacterial population and bacterial metabolic activities.

Effect of fermentation rate of dietary fibre on short-term satiety, long-term food intake and gut hormone response in male rats

Abstract:

As obesity rates increased worldwide, nutritional strategies to reduce food intake as a weight management tool have gained much attention. Studies showed that dietary fibre can be protective against weight gain through fermentation that influences gut hormone levels to increase satiety and reduce food intake. Macronutrient-induced satiety to reduce meal size has also received a great interest. This study aimed to investigate the effects of macronutrients (carbohydrate, protein, fat) and fibres (different fermentation rates) on satiety, corresponding hormone responses (insulin, ghrelin, glucagon-likepeptide-1, peptide YY), and their relationships with food intake and body weight in rats. I found that diet containing fructooligosaccharides led to reduced long term food intake, weight gain and fat mass whereas wheat bran promoted food intake with unaffected weight gain. Both fructooligosaccharides and oil were associated with significantly lower food consumption and higher circulating PYY. My data suggested that satiety regulation is complex and can be strain-dependent.

Targeting of calcium calmodulin-dependent protein kinase II to membranes

Abstract:

Communication between neurons is mediated by the controlled release of neurotransmitters from small synaptic vesicles. Several proteins are involved in the regulation of neurotransmitter release. One such family of proteins is the calcium/calmodulin-dependent protein kinases. Calcium/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant members of this protein family. and is found in many tissues with specific isoforms usually predominating in certain tissues. CaMKII is found CO- purified with synaptic vesicles. The question of how CaMKII is attached to the synaptic vesicle is addressed in this thesis. It was hypothesized that CaMKII may interact with the phospholipid component of the vesicle membrane, which could mediate binding.

C-terminal deletion mutants of murine α-CaMKII were produced by PCR directional cloning and liposome-binding assays were performed on the purified proteins. Binding of murine α-CaMKII to several types of liposomes was not observed. Recent studies into targeting of this enzyme suggest a role for anchoring proteins. Murine α-CaMKII like the β isoform from rat muscle may interact with membranes through a putative non-kinase translation product (a putative anchoring protein), which encompasses amino acids 230-270 at N-terminal of the regulatory domain rather than through direct binding with phospholipids.

SMAD transcription factor expression in anoxia-tolerant Trachemys scripta elegans and dehydration-tolerant Xenopus laevis: A comparative study 

 

Abstract:

The TGF-β signaling pathway with its associated transcription factors (SMADs) was studied in the red-eared slider (Trachemys scripta elegans; liver, heart, white skeletal muscle, red skeletal muscle, and kidney) as a model for anoxia tolerance and the African clawed frog (Xenopus laevis; liver and muscle) as a model for dehydration resistance. In addition, protein expression levels of precursor cytokines were measured, as well as known genes and proteins downstream of the SMAD signaling pathway, such as c-Myc, and AP-1 proteins. The data showed that selective control of different SMAD isoforms occurred in response to environmental stress, the mechanism being reversible protein phosphorylation. Cellular responses and signaling profiles were observed to be highly tissue-specific and stress-specific. SMADs were shown to play a role in the hypometabolic response of both animal models through the regulation of cell cycle arrest, angiogenic processes, and oxidative injury management strategies.

Regulation of lactate dehydrogenase and glycerol-3-phosphate dehydrogenase in mammalian hibernation

 

Abstract:

Hibernation is a winter survival strategy for many small mammals. Animals sink into deep torpor, body temperature falls to near 0°C and physiological functions are strongly suppressed. Enzymes are the catalysts of metabolic pathways in cells and their appropriate control is critical to hibernation success. This thesis explores the properties and regulation of two key enzymes of carbohydrate metabolism (lactate dehydrogenase, LDH) and lipid metabolism (glycerol-3-phosphate dehydrogenase, G3PDH) purified from liver and skeletal muscle of ground squirrels (Urocitellus richardsonii). The studies showed that changes in pH, temperature, and inhibitors play roles in differentially regulating these enzymes between euthermic and torpid states. Furthermore, reversible protein phosphorylation proved to be a significant regulatory mechanism, producing a reduced activity state of skeletal muscle LDH and increased activity state of G3PDH in both skeletal muscle and liver during torpor. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly protein phosphorylation, contribute to reorganizing fuel metabolism during hibernation.

Metabolic regulation of glutamate dehydrogenase

 

Abstract:

Glutamate dehydrogenase (GDH), a key control enzyme interrelating carbohydrate and amino acid metabolism, was investigated with respect to coenzyme utilization and regulatory control via allosteric effectors. GDH from gall fly larva, Eurosta solidaginis, was purified and kinetic studies suggested that it was preferentially NADP-linked. Temperature effects on the regulatory properties of this GDH, including ATP/GTP interactions, may be responsible for the increase in proline seen at low temperature in the overwintering larva. GDH from the intertidal sea anemone, Anthopleura xanthogrammica, was purified and found to be NADP(H)-specific. This GDH was not regulated by nucleotide effectors but was strongly affected by ions. The dual coenzyme specific GDH from bovine liver was investigated with respect to its coenzyme preference. The data suggested that under increasing glutamate concentrations the preferred coenzyme was NADP, a preference which was promoted by the action of metabolic modulators.

Less is mTOR: Regulation of protein synthesis via the insulin signalling pathway in the anoxia-tolerant red-painted slider Trachemys scripta elegans

 

Abstract:

The red-eared slider turtle, Trachemys scripta elegans, can survive 3-4 months of anoxic submergence in cold water during the winter. The effect of hypoxia/anoxia on protein synthesis in red-eared sliders was investigated with a focus on the insulin-signaling pathway and analysis of the mammalian target of rapamycin (mTOR) and its upstream and downstream effectors in liver and white muscle. Expression of mTORC1 did not change in muscle but increased significantly in liver after 5 and 20 hours of anoxic submergence. Upstream effectors, AKT and RAPTOR, were also elevated in liver but suppressed in muscle. PRAS40 and TSC2 inhibitors of mTOR were differentially regulated in both tissues but generally suppressed. Downstream targets of mTOR signaling (eIF4E, 4E-BP1, P70S6K, S6) as well as the poly(A) binding protein also showed differential responses to anoxia. Overall, the data indicate that the early response to anoxia by turtles is maintenance of protein synthesis in liver but suppression in white muscle.

Reversible protein acetylation in the regulation of mammalian hibernation

 

Abstract:

To survive the winter, many small mammals use hibernation. Employing a remarkable strategy of metabolic rate depression these animals accrue profound energy savings by remaining in a torpid state over most of the winter. Global metabolic suppression is mediated by intricate molecular mechanisms, including the post-translational modification of cellular proteins. One such modification – reversible protein acetylation – is an important regulator of metabolism, but little is known about its relevance to hibernation. This thesis provides an initial characterization of possible functions of reversible protein acetylation, and several enzymes that mediate the process (protein lysine acetyltransferases (KATs) and deacetylases (SIRTs)), in the context of a rodent model of mammalian hibernation, the thirteen-lined ground squirrel, Ictidomys tridecemlineatus. Notably, SIRT and KAT protein expression and activities increased in skeletal muscle and brown adipose tissue, respectively, during torpor, in correlation with fluctuations in downstream target acetylation. Such changes identify roles for protein acetylation during hibernation.