Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut non- endocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and co-localized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.

The Vo sector of the vacuolar H+-ATPase is a multi-subunit complex that forms a proteolipid pore. The largest subunit in this complex is the a subunit which has four isoforms (a1-a4). The isoform(s) critical for secretory vesicle acidification has yet to be identified. Using a cell line derived from rat pheochromocytoma in which Voa1 and/or Voa2 had been down-regulated this study revealed that Voa1, and to a lesser extent, Voa2 are critical for acidifying dense-core vesicles (DCVs). The acidification defects resulting from down-regulation of Voa1 and Voa1/ Voa2 were suppressed by the expression of knockdown-resistant Voa1. Defects in DCV acidification resulted in reductions in their transmitter uptake and storage. Lastly, Ca2+-dependent peptide secretion appeared normal in Voa1 and Voa1/ Voa2 knockdown cells. . This study demonstrated that Voa1 and Voa2 cooperatively regulate dense-core vesicle acidification as well as transmitter uptake/storage, while they may not be critical for dense-core vesicle exocytosis.

The Vo sector of the vacuolar H+-ATPase is a multi-subunit complex that forms a proteolipid pore. The largest subunit in this complex is the a subunit which has four isoforms (a1-a4). The isoform(s) critical for secretory vesicle acidification has yet to be identified. Using a cell line derived from rat pheochromocytoma in which Voa1 and/or Voa2 had been down-regulated this study revealed that Voa1, and to a lesser extent, Voa2 are critical for acidifying dense-core vesicles (DCVs). The acidification defects resulting from down-regulation of Voa1 and Voa1/ Voa2 were suppressed by the expression of knockdown-resistant Voa1. Defects in DCV acidification resulted in reductions in their transmitter uptake and storage. Lastly, Ca2+-dependent peptide secretion appeared normal in Voa1 and Voa1/ Voa2 knockdown cells. . This study demonstrated that Voa1 and Voa2 cooperatively regulate dense-core vesicle acidification as well as transmitter uptake/storage, while they may not be critical for dense-core vesicle exocytosis.

Eccentric exercise causes skeletal muscle damage, yet the acute cellular responses post-exercise have yet to be fully elucidated. To better understand the post-exercise response, heat shock protein (HSP) 72 content and nuclear factor-κB (NF-κB) activation where examined in Adult (A; 6 month) and Late middle-aged (LMA; 24 month) Fischer 344xBrown Norway rats. Animals were randomly divided into five groups (n=6): non-exercising controls (C), level (L) or eccentric (ECC) (-16°) running at 16m.m-1 and killed immediately post-exercise (0), 48 hours post-exercise (48). Following ECC, vastus intermedius (VI) from A and LMA showed more damage compared to L exercise. Neither age-group had significantly increased VI HSP72 content compared to C. Pooled results founded increased HSP72 content in ECC-48 compared to C (p<0.02). NF-κB activation in the VI was lower in LMA (p<0.001) and unchanged in WG when compared to AC. These findings suggest HSP72 is increased following eccentric exercise in the VI.

Left ventricular (LV) filling increases during exercise, but left atrial (LA) phasic function and its contribution to LV filling is poorly understood. Sixteen endurance-trained middle-aged males were studied at rest and during light (LE) and moderate (ME) intensity cycle-ergometry. Atrioventricular-plane displacement (AVPD) increased from rest to LE (from 14±2 mm to 18±2 mm, p<0.01), but did not increase further at ME. LA reservoir volume increased from rest to LE (from 32±8 mL to 40±10 mL, p<0.01). LA passive contribution increased at LE (from 21±5 mL to 27±8 mL, p<0.01), while LA active contribution increased from rest only at ME (from 12±5 mL to 23±9 mL, p<0.01). AVPD, and thus the longitudinal shortening of LV systole, contributes to LA filling primarily during LE, but is a limited mechanism beyond LE. These data suggest that LV filling appears to shift to a reliance on conduit function to increase LV filling at ME.

The hypothalamus is a central regulator of energy homeostasis. Recently, proglucagon-derived peptides have emerged as potential appetite regulators. The proglucagon gene is expressed in the periphery and also in selective hypothalamic neurons. The regulation of hypothalamic proglucagon by two key regulators of energy balance, insulin and leptin, remains unstudied. Central glucagon-like peptide (GLP)-1 receptor (GLP-1R) activation by exendin-4, a long-acting GLP-1R agonist, induces anorexia; however, the specific hypothalamic neuronal populations activated by exendin-4 remain largely unknown. The role of GLP-2 as a central appetite regulator is poorly understood. In this thesis, using murine hypothalamic cell lines and mice as experimental models, mechanisms involved in the direct regulation of proglucagon gene by insulin and leptin were studied, and the actions of exendin-4 and GLP-2 on hypothalamic neuropeptides were determined. It was found that insulin and leptin regulate hypothalamic proglucagon mRNA by activating Akt and signal transducer and activator of transcription 3, respectively. Insulin and leptin did not regulate human proglucagon promoter regions, but affected proglucagon mRNA stability. In mice, intracerebroventricular exendin-4 and GLP-2 induced anorexia, activated proopiomelanocortin- and neuropeptide Y-expressing neurons in the arcuate nucleus and neurotensin- and ghrelin-expressing neurons in major hypothalamic appetite-regulating regions. In the hypothalamic neuronal models, exendin-4 and GLP-2 activated cAMP-response element-binding protein/activating transcription factor-1, and regulated neurotensin and ghrelin mRNA levels via a protein kinase A-dependent mechanism. Overall, the in vivo and in vitro findings suggest that these neuropeptides may serve as potential downstream mediators of exendin-4 and GLP-2 action. This research demonstrates direct regulation of hypothalamic proglucagon by insulin and leptin in vitro, and reports a previously unrecognized link between central GLP-1R and GLP-2R activation and regulation of hypothalamic neuropeptides. A better understanding of the regulation of hypothalamic proglucagon and central GLP-1R and GLP-2R activation is important to further expand our knowledge of feeding circuits.

An acute increase in hypothalamic glucose and its downstream metabolite lactate lower glucose production (GP) and plasma glucose (PG) levels in normal rodents. However, the effectiveness of this nutrient-sensing mechanism in metabolic disease is unknown. We assessed the effects of intracerebroventricular (i.c.v.) or intra-hypothalamic glucose and lactate on in vivo glucose kinetics in conscious rats. Study I revealed that i.c.v. lactate lowered PG via a suppression of GP in rodents with uncontrolled diabetes and diet-induced insulin resistance. Study II demonstrated that i.c.v. glucose was ineffective at suppressing GP in uncontrolled diabetic rodents or rodents with a prior 24 h whole-body or hypothalamic hyperglycemic insult. When PG levels per se were normalized in diabetic rodents hypothalamic glucose sensing to lower GP was rescued. As such, sustained hyperglycemia per se impairs hypothalamic glucose effectiveness in diabetes. Further studies are necessary to determine defective mechanisms upstream of lactate metabolism hindering CNS glucose sensing.

Little is known of the temporal cerebral blood flow response to a chemical stimulus consisting of increased PCO2 measured over time. Currently, there is only one study suggesting multiple phases in the CBF-CO2 response. Time constants of middle cerebral artery blood velocity (MCAV) response to a change in PETCO2 have been reported to be between 3 and 99.4 s. We studied the MCAV response in 28 subjects (10 females) to a sustained +10 mmHg above baseline (10 min) acute increase of PETCO2. We found that there were three distinct MCAV response patterns among subjects. Additionally, the responses of males and females differed. These studies suggest that there are multiple overlapping mechanisms controlling the chemoresponse of cerebral blood vessels and that these mechanisms may differ between men and women.

One contentious issue regarding pancreatic acinar exocytosis concerns which SNAP25 isoform (SNAP23/29/47) mediates the various fusion events in this cell type. Based on dominant-negative over-expression studies, SNAP23 was hypothesized to be the putative isoform mediating apical exocytosis, basolateral exocytosis, and ZG-ZG fusion. Unfortunately, using a SNAP23 KD mouse model, 80% SNAP23 KD was insufficient to manifest any secretion phenotype. A novel syncollin-pHluorin exocytosis imaging technique initially meant to assess which fusion events are perturbed by SNAP23 KD was successfully developed and displayed improvements over previous imaging techniques. The syncollin-pHluorin imaging enabled visualization of apical and basolateral exocytosis as well as sequential ZG-ZG fusions. Combined with spinning disk microscopy, this assay allows 3D live exocytosis imaging with high temporal and spatial resolution. This novel imaging assay will be useful in visualizing apical, basolateral, sequential, and lateral fusion events for future acinar studies.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.