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.

Studies show that insulin induced activation and assembly of insulin receptor substrate-1 (IRS1) and phosphatidylinositol-3-kinase (PI3K), within remodelled actin structures is critical for GLUT4 translocation to the cell surface in muscle cells. This study identifies the F-actin binding protein, nexilin, as a novel IRS1 binding partner. Insulin stimulates nexilin to dissociate from IRS1 and interact with actin. Nexilin knockdown has no effect on insulin-stimulated IRS1 tyrosine phosphorylation, but does enhance insulin-stimulated IRS1-PI3K interaction, increasing PIP3 formation, PKB activation and glucose uptake. This study also shows that nexilin overexpression may have an inhibitory effect on PKB phosphorylation and glucose uptake in adipocytes. These findings suggest nexilin is a negative regulator of IRS1 action on PI3K and insulin-stimulated dissociation of IRS1-nexilin allows the formation of IRS1-PI3K complexes in cytoskeletal-membrane compartments. Nexilin also specifically associates with the PH domain of IRS1, and not IRS2, suggesting a mechanism for signaling specificity of these isoforms.

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.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

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.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

Postprandial overproduction of apolipoprotein B48 (apoB48)-containing lipoproteins has been observed in states of insulin resistance and is important to the sequelae of cardiovascular disease, but little is understood about factors that regulate their secretion. The glucagon-like peptides (GLPs) are released from ileal enteroendocrine L-cells following lipid ingestion. I hypothesized that the GLPs could acutely affect the production of apoB48-containing triglyceride (TG)-rich lipoproteins (TRL) in the small intestine. Using the Syrian golden hamster, I first characterized the gross effects of the GLPs on TRL secretion in response to an oral fat load and then continued to dissect the mechanisms of these changes using primary intestinal cell cultures and a variety of knockout mouse models. An exogenous GLP-1 receptor (GLP-1R) agonist was found to acutely inhibit chylomicron secretion in both hamsters and mouse models, and extending the bioactivity of endogenously-secreted GLP-1 with a dipeptidyl peptidase-4 inhibitor had suppressive effects in insulin-resistant fructose-fed hamsters. The insulinotropic and delayed gastric emptying functions do not completely account for the hypolipidemic effect of GLP-1R agonism, and the effect of the GLP-1R agonist exendin-4 could be seen directly in the apoB48 secretion of primary enterocytes. In contrast, the sister peptide GLP-2 was a potent acute stimulator of chylomicron secretion in hamsters and mice. The hyperlipidemic effect of GLP-2 could be attributed to an increased rate of luminal FA uptake mediated by the posttranslational modification of the FA transporter CD36, and CD36-deficient mice were found to be refractory to the stimulatory effects of GLP-2. The activity of nitric oxide synthase was also found to be essential to the hyperlipidemic action of GLP-2. I identified a set of intercellular communications that could contribute in mediating the action of GLP-2, in which GLP-2 secreted from the enteroendocrine L-cell stimulates intestinal subepithelial myofibroblasts to release vascular endothelial growth factor, which directly activated the enterocyte to secrete apoB48. In summary, this thesis demonstrates that two co-secreted postprandial hormones have considerable but completely opposite influences on chylomicron production. Changing the balance of the GLPs’ actions in vivo could provide a therapeutic strategy to combat postprandial dyslipidemia.

The metabolic syndrome is a collection of pathologies including dyslipidemia, obesity and insulin resistance. A thorough understanding of the mechanisms behind metabolic syndrome development would help in the development of treatment and prevention strategies. Our lab has previously shown that cholesterol feeding exacerbates features of the metabolic syndrome in high fat-, high fructose-fed mice. The nuclear receptor Liver X Receptor (LXR), a master transcriptional regulator of cholesterol, fat and carbohydrate metabolism in the liver, is endogenously activated by oxysterols, metabolic derivatives of cholesterol. In order to determine whether cholesterol exerts its metabolic effects via LXR activation, parallel studies were conducted comparing chronic cholesterol supplementation with LXR activation in the hamster. Results showed that cholesterol feeding and LXR activation caused similar dyslipidemia, glucose intolerance and upregulation of target mRNA and proteins. These data support the hypothesis that the dyslipidemic effects of dietary cholesterol are mediated at least in part by LXR.

ChREBP has emerged as one of the key controllers of hepatic lipogenesis. While the function of ChREBP has been extensively investigated, mechanisms underlying its transcriptional regulation remain largely unknown. We located a conserved POU-binding site within mammalian ChREBP promoters, and demonstrated that the POU homeodomain protein Oct-1 binds to this site in the human HepG2 cell line. Oct-1 transfection significantly repressed ChREBP promoter activity 50-75%. Conversely, knockdown of Oct-1 expression with shRNA significantly increased ChREBP expression levels. Furthermore, insulin treatment resulted in a two-fold activation of ChREBP promoter activity, and stimulated endogenous ChREBP expression. We found that the stimulatory effect of insulin on the ChREBP promoter is at least partially dependent on the presence of the POU-binding site, and that insulin treatment reduced Oct-1 expression. Our observations identify Oct-1 as a transcriptional repressor of ChREBP, and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.

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.