No description available.

Mitochondrial dysfunction is considered as a key mechanism of aging but little is known about the impact of mitochondrial biogenesis. Mitochondrial DNA (mtDNA) copy number control is an important aspect of mitochondrial biogenesis and is highly regulated in eukaryotic organisms. By studying mtDNA copy number, our aim is to gain a better understanding of the relationship between mitochondrial biogenesis and aging. We developed an optimized protocol for measuring mtDNA copy number in Caenorhabditis elegans using quantitative real-time PCR (qPCR). We investigated how mtDNA regulation is affected by a variety of aging-related pathways. We found the insulin/IGF-1 signaling (IIS) pathway regulates mtDNA content in a DAF-16- and UCP-4-dependent manner. By utilizing RNA interference (RNAi) against polg-1, we showed that mitochondrial stress likely modulates lifespan through the IIS pathway. Our work identifies IIS as a communications pathway between mitochondria and the nucleus in modulating mitochondrial biogenesis and lifespan in Caenorhabditis elegans.

Many obese individuals and type 2 diabetes mellitus (T2DM) patients have elevated levels of insulin. Hyperinsulinemia is a major cancer risk factor in T2DM individuals and activated insulin receptor (IR) has been linked to many types of cancer and poor survival. However, the mechanisms that account for the link between the hyper-active insulin signaling and cancer risk is not well understood. PTEN plays an antagonistic role in the canonical insulin signaling pathway, and is the second most commonly mutated tumour suppressor (after p53) found in human cancers. In many cancers the PTEN gene is not deleted, but instead the protein is lost. Therefore the regulation of PTEN protein in humans is of great importance. Here we hypothesized that the activated insulin signaling down-regulates PTEN. Considering that insulin signaling is highly conserved from C. elegans to human, I used C. elegans as a model and showed that DAF-2, the worm homolog of IR, is a negative regulator of DAF-18, the worm homolog of PTEN. In addition, I showed that DAF-28, the worm homolog of insulin, also negatively regulates DAF-18/PTEN. I used western blot and immunostaining to show that the protein level of DAF-18/PTEN is increased in the daf-2/IR and daf-28/insulin mutants. I further showed that daf-18/Pten is genetically epistatic to daf-2/IR in regulating neuronal development. I then employed human cell culture experiments and reported that this negative regulation is conserved in human cancer cell lines. I showed that knocking-down IR through siRNA up-regulates PTEN, and over-expressing a gain-of-function IR down-regulates PTEN. I also showed that insulin stimulation dramatically decreased PTEN and this decrease is dependent on IR. I further confirmed a physical association between IR and PTEN in both human and C. elegans, and reported that IR could phosphorylate PTEN. To provide mechanistic insight to DAF-18/PTEN regulation, I identified another protein, which is a ubiquitin ligase, that functions in insulin signaling to down-regulate DAF-18/PTEN. Additionally, I also provided evidence that insulin signaling cross talks with Eph receptor signaling. In summary, my findings will be informative for cancer biologists to study the roles of these genes in carcinogenesis. / Thesis (Ph.D, Biology) -- Queen's University, 2013-02-04 14:37:29.376

The canonical PI3K/Akt pathway is activated downstream of numerous receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors, and is a crucial regulator of growth and survival in metazoans. The deregulation of Akt is implicated in the pathogenesis of numerous diseases including cancer, making the identification of modifiers of its activity of high chemotherapeutic interest. In a transheterozygous genetic screen for modifiers of embryonic Akt function in Drosophila, in which the PI3K/Akt signaling pathway is conserved, we identified the A subunit of the vacuolar ATPase (Vha68-2) as a positive regulator of Dakt function. Our characterization of this genetic interaction in the larval stage of development revealed that Vha68-2 mutant phenotypes stereotypically mimicked the growth defects observed in mutants of the Drosophila insulin signaling pathway (ISP). The loss of Vha68-2 function, like Dakt-deficiency, was found to result in organismal and cell-autonomous growth defects, and consistent with its putative role as a positive regulator of Dakt function, both the mutational and pharmacological inhibition of its activity were found to downregulate Akt iv activation. Genetic epistasis experiments in somatic clones of Vha68-2/dPTEN double mutants demonstrated that the loss of Vha68-2 function suppressed the growth defects associated with dPTEN-deficiency, placing Vha68-2 activity downstream of dPTEN in the ISP, while the examination of PI3K activity and PH domain-dependent membrane recruitment in pharmacologically inhibited larval tissues further placed Vha68-2 function downstream of PI3K. These findings were recapitulated in cultured NIH-3T3 cells, whose treatment with bafilomycin A1, a potent and specific inhibitor of V-ATPase, resulted in the downregulation of Akt phosphorylation, particularly in non-cytoplasmic intracellular compartments. Furthermore, cellular subfractionation of bafilomycin-treated NIH-3T3 cells demonstrated a decrease in the localization of Akt to early endocytic structures, and a downregulation in the localization and activation of Akt in the nuclei of both Drosophila and mammalian cells. Finally, the pharmacotherapeutic relevance of V-ATPase inhibition was addressed in two tumor models – multiple myeloma and glioblastoma – and our preliminary findings in these cancers, which are often associated with ectopic PI3K/Akt signaling, showed significant cytotoxic efficacy in vitro, warranting its consideration as a tractable pharmacological option in the treatment of cancer.

The preadipocyte is crucial for healthy adipose tissue (AT) remodeling, and insulin resistance in these cells may contribute to AT dysfunction. Chronic exposure to insulin and high glucose induces insulin resistance in the 3T3-L1 mouse adipocyte cell line in vitro, however, whether this occurs in human preadipocytes is not known. To investigate this, human preadipocytes were isolated from subcutaneous AT obtained from 6 female patients undergoing elective surgery (Research Ethics Board-approved). Human preadipocytes were incubated in 5 mM glucose or 25 mM glucose in the presence or absence of 0.6 nM insulin for 48 hours, followed by acute 100 nM insulin stimulation. 25 mM glucose + 0.6 nM insulin inhibited insulin-stimulated tyrosine phosphorylation of IR-β (77%) and IRS-1 (81%) compared to NG (p<0.01), however, insulin-stimulated Ser473 Akt phosphorylation was not affected. 25 mM glucose and/or 0.6 nM insulin did not significantly change levels of pro-inflammatory adipokines. 25 mM glucose and/or 0.6 nM, prior to and/or during 14 days of adipogenic induction, did not affect levels of adipogenic markers or intracellular triglyceride accumulation.

The calorie restriction (CR) and insulin/IGF-I-like signalling (IIS) are two pathways regulating the lifespan of C. elegans. Recent studies showed that glucose restriction extends the lifespan of C. elegans while excessive glucose shortens the lifespan of the worms. The first step of the glucose metabolism is the transport of glucose across the plasma membrane by the glucose transporters. The work described in this thesis aims to identify glucose transporters in C. elegans and to provide a primary investigation of the in vitro and in vivo function of the identified glucose transporter. Nine putative transporters have been cloned and expressed. Out of the nice cloned putative transporters in the C. elegans genome, H17B01.1 (H17) only is identified as a fully functional glucose transporter using an oocyte expression system in which glucose transport activity is directly measured. The two transcripts of H17 are both capable of transporting glucose with high affinity, as well as transporting trehalose. Heterologous expression of H17 in mammalian CHO-T cells suggests that the protein is localised both on the plasma membrane and in the cytosol. In vitro studies of H17 show that the protein does not respond to insulin stimulation when expressed in mammalian CHO-T cell and rat primary adipocyte systems. In vivo functional studies using H17 RNAi indicate that the worm’s lifespan is not affected by the H17 knockdown. However, glucose metabolism of C. elegans (as measured by glucose oxidation to CO2 and incorporation into fat reserves) is influenced by the decreased expression of H17, especially in the daf-2 mutant strain, e1370. However, the increase of glucose metabolism caused by H17 knockdown observed in daf-2 mutant is inhibited in the age-1 and akt-1 mutant strains. The findings reported in this thesis suggest that the H17 glucose transporter may play an important role glucose metabolism in C. elegans and that this transport and metabolism is influenced by insulin receptor activity and serine kinase cascades.

Insulin signaling plays a prominent role in regulation of dauer formation and longevity in Caenorhabditis elegans. Here, I show that insulin signaling also is required in benzaldehyde-starvation associative plasticity, where worms pre-exposed to the odor attractant benzaldehyde in the absence of food subsequently demonstrate a conditioned aversion response towards the odorant. Animals with mutations in ins-1, daf-2, and age-1 which encode the homolog of human insulin, insulin/IGF-1 receptor, and PI-3 kinase, respectively, have significant deficits in benzaldehyde-starvation associative plasticity. Using a conditional allele I show that the behavioral roles of DAF-2 signaling in associative plasticity can be dissociated, with DAF-2 signaling playing a more significant role in the memory retrieval than in memory acquisition. I propose DAF-2 signaling acts as a learning specific starvation signal in the memory acquisition phase of benzaldehyde-starvation associative plasticity but functions to switch benzaldehyde-sensing AWC neurons into an avoidance signaling mode during memory retrieval.

Synaptogenesis entails the development and establishment of functional synapses, which form the fundamental unit of communication in the nervous system. Initially identified in Caenorhabditis elegans (C. elegans), the FSN-1, F-box protein family has emerged as evolutionarily conserved binding partners of PHR family proteins, which regulate synaptogenesis. Previously, we have shown that FSN-1 and RPM-1 form a SCF/FSN-1/RPM-1 ubiquitin ligase complex that negatively regulates synapse growth by downregulating presynaptic targets, like the MAP kinase pathway. For my master’s thesis, I used a combination of both candidate and forward genetic approaches to identify additional components of signaling pathways that are regulated by FSN-1 during synaptogenesis. Our studies are among the first to suggest diverging roles for these partners and provide the first evidence for a mechanism through which the F-box protein regulates synaptogenesis via retrograde insulin/IGF/FOXO signaling and glucosaminidase/O-GlcNAc modifications.

Synaptogenesis entails the development and establishment of functional synapses, which form the fundamental unit of communication in the nervous system. Initially identified in Caenorhabditis elegans (C. elegans), the FSN-1, F-box protein family has emerged as evolutionarily conserved binding partners of PHR family proteins, which regulate synaptogenesis. Previously, we have shown that FSN-1 and RPM-1 form a SCF/FSN-1/RPM-1 ubiquitin ligase complex that negatively regulates synapse growth by downregulating presynaptic targets, like the MAP kinase pathway. For my master’s thesis, I used a combination of both candidate and forward genetic approaches to identify additional components of signaling pathways that are regulated by FSN-1 during synaptogenesis. Our studies are among the first to suggest diverging roles for these partners and provide the first evidence for a mechanism through which the F-box protein regulates synaptogenesis via retrograde insulin/IGF/FOXO signaling and glucosaminidase/O-GlcNAc modifications.

Insulin signaling plays a prominent role in regulation of dauer formation and longevity in Caenorhabditis elegans. Here, I show that insulin signaling also is required in benzaldehyde-starvation associative plasticity, where worms pre-exposed to the odor attractant benzaldehyde in the absence of food subsequently demonstrate a conditioned aversion response towards the odorant. Animals with mutations in ins-1, daf-2, and age-1 which encode the homolog of human insulin, insulin/IGF-1 receptor, and PI-3 kinase, respectively, have significant deficits in benzaldehyde-starvation associative plasticity. Using a conditional allele I show that the behavioral roles of DAF-2 signaling in associative plasticity can be dissociated, with DAF-2 signaling playing a more significant role in the memory retrieval than in memory acquisition. I propose DAF-2 signaling acts as a learning specific starvation signal in the memory acquisition phase of benzaldehyde-starvation associative plasticity but functions to switch benzaldehyde-sensing AWC neurons into an avoidance signaling mode during memory retrieval.