The Hulver laboratory has recently found that the neuropeptide Y Y1 receptor (NPY Y1R) mRNA expression is elevated in skeletal muscle of obese humans (Hulver, unpublished). The goal of this research is to study the role of the NPY Y1R in skeletal muscle lipid metabolism. Rat L6, mouse C2C12, and human primary myotubes were incubated in 14C palmitate labeled fatty acid oxidation medium containing 80ng/mL, 250ng/mL, and 500ng/mL of NPY and for a three hour period. Experiments were repeated with the addition of 17mg/mL diprotin A to each NPY treatment. Fatty acid oxidation (FAO) and the percentage of lipids stored within the myotubes as diacylglyceride (DAG) and triaclyglyceride (TAG) were measured. Analyses were repeated in rat L6 and mouse C2C12 following a three hour incubation in 14C palmitate labeled fatty acid oxidation medium containing 1µg/mL, 10µg/mL, and 50µg/mL of the NPY Y1R ligand, [Leu31, Pro34] neuropeptide Y (Bachem, Torrance, CA). Incubation of human primary myotubes in NPY treatments with the addition of diprotin A significantly increased TAG accumulation (p< 0.05). Mouse C2C12 mytoube incubation in 500ng/mL NPY with diprotin A increased FAO (p 0.05). All other NPY and NPY Y1R ligand treatments in had no significant effect on FAO or the accumulation of TAG and DAG. / Master of Science

While cigarette smoking is a common-knowledge way to stay lean, it has long been known as a risk factor for diabetes and obesity. Here we establish that smoking causes fat gain and metabolic disruption in mice, effects which are exacerbated by a high-fat, high-sugar diet. We found that smoke exposure increases levels of ceramide—the lipid responsible for diet-induced insulin resistance—and that blocking ceramide production with the pharmacological inhibitor myriocin restored insulin sensitivity, stopped weight gain, and rescued mitochondrial respiration in vivo and in vitro.We also sought to assess the impact of the RAGE ligand HMGB1 on skeletal muscle metabolism. We found that respiration between vehicle and HMGB1-injected red gastrocnemius was comparable. In myotubes, adding myriocin treatment to the HMGB1 cells increased respiration above HMGB1 treatment alone. HMGB1 increased oxidative stress in cultured myotubes and increased the transcript levels of Spt2, the enzyme responsible for the rate-limiting step in ceramide synthesis, although transcript levels of markers of mitochondrial fission and fusion leave us unsure of HMGB1's impact on mitochondrial dynamics. HMGB1, even at an exceptionally low dose over only 2 weeks, did cause significant impairment in glucose and insulin tolerance tests. Considering HMGB1's accessibility as a therapeutic target, its involvement in metabolic disruption is worth pursuing further.

Yes / Objective: Many patients with acromegaly, a hormonal disorder with excessive growth hormone (GH) production, report pain in joints. We undertook this study to characterize the joint pathology of mice with overexpression of bovine GH (bGH) or a GH receptor antagonist (GHa) and to investigate the effect of GH on regulation of chondrocyte cellular metabolism. Methods: Knee joints from mice overexpressing bGH or GHa and wild-type (WT) control mice were examined using histology and micro–computed tomography for osteoarthritic (OA) pathologies. Additionally, cartilage from bGH mice was used for metabolomics analysis. Mouse primary chondrocytes from bGH and WT mice, with or without pegvisomant treatment, were used for quantitative polymerase chain reaction and Seahorse respirometry analyses. Results: Both male and female bGH mice at ~13 months of age had increased knee joint degeneration, which was characterized by loss of cartilage structure, expansion of hypertrophic chondrocytes, synovitis, and subchondral plate thinning. The joint pathologies were also demonstrated by significantly higher Osteoarthritis Research Society International and Mankin scores in bGH mice compared to WT control mice. Metabolomics analysis revealed changes in a wide range of metabolic pathways in bGH mice, including beta-alanine metabolism, tryptophan metabolism, lysine degradation, and ascorbate and aldarate metabolism. Also, bGH chondrocytes up-regulated fatty acid oxidation and increased expression of Col10a. Joints of GHa mice were remarkably protected from developing age-associated joint degeneration, with smooth articular joint surface. Conclusion: This study showed that an excessive amount of GH promotes joint degeneration in mice, which was associated with chondrocyte metabolic dysfunction and hypertrophic changes, whereas antagonizing GH action through a GHa protects mice from OA development. / Dr. Zhu's work was supported by Ohio University, the Arthritis National Research Foundation (grant 833836), a FIRST award from the American Society for Bone and Mineral Research, the NIH (grant R15-AR-080813), and a Hevolution Foundation AGE grant (AGE-008). Dr. Davis’ work was supported by a medical student seed grant from Ohio University. Dr. Lotz's work was supported by the NIH (grant R37-AG-059418). Dr. Berryman was supported by the NIH (grant R01-AG-059779). Dr. Kopchick was supported by the State of Ohio's Eminent Scholar Program that includes a gift from Milton and Lawrence Goll and the AMVETS, and by the NIH (grant R01-AG-059779).

Sialylation, a posttranslational modification of both glycolipids and glycoproteins, is typically found on the terminal positions of glycan chains. Unique among most other sugars, sialic acids are nine carbon sugars that are negatively charged and undergo a variety of side group modifications, which contribute to its role in cell-cell interactions and receptor recognition. While the majority of Drosophila glycoproteins do not terminate in sialic acid compared to mammalian glycoproteins, a sialic acid synthetic pathway is present in Drosophila but it is developmentally regulated and appears to be restricted to the nervous system (Kim et al., 2002; Koles et al., 2004). In order to investigate the role of the sialic acid pathway in Drosophila, we generated a null mutation of the sialic acid synthase gene (SAS) by imprecise excision of a nearby transposable element. Homozygous null flies exhibit partial lethality, male sterility and undergo age dependent neurodegeneration as evidenced by loss of locomotion and increased vacuolization in the brain. Mutant flies also have a shortened life span and display increased sensitivity to heat as they age. To identify protein targets of sialylation that possibly contributed to these phenotypes, a very sensitive solid-phase extraction method was used to capture sialylated glycopeptides from head extracts of wild type and SAS null flies. In collaboration with M. Betenbaugh and H. Zhang at Johns Hopkins University, I identified three sialylated peptides; the major peptide target was derived from the Shaker voltage-dependent potassium channel. The other two peptides were encoded by genes of unknown function. Electrophysiological measurements performed on control and SAS mutant larvae at the pre- and post-synaptic larval neuromuscular junction (in collaboration with T. Dean and A. Seghal, University of Pennsylvania) showed that loss of sialylation induced a depolarizing shift in the gating parameters of the Shaker ion channels, similar to what was previously reported in mammalian cell culture (Johnson and Bennett, 2007). Pre-synaptic neurons from the mutants displayed a two to three fold increase in the number of miniature excitatory peaks suggesting that the neurons were hyperactive. Since Shaker is a major target of sialylation in brain neurons, I suggest that the loss of sialylation of Shaker plays a major role in the neurodegeneration phenotype observed in our SAS null mutant flies. SAS mutant flies are unusually sensitive to starvation, typical of flies that cannot maintain metabolic homeostasis. Upon 24 h of starvation, mutant flies differed from control flies in that they consumed most of their triglyceride stocks, they displayed poor locomotion, had smaller sized cells in their fat body, and reduced their glycogen stores significantly. Mutant flies expressed and likely secreted an excess of insulin like protein, hyperinsulinemia, for the first five days after eclosion. However, as the flies aged (14-21 days) they had high hemolymph sugar, low insulin like protein expression and fewer number of insulin producing cells. All these phenotypes are similar to diabetic patients, as diabetic patients also have metabolic inflexibility, high blood sugar, low insulin secretion, and fewer number of insulin producing cells. It is now known that the Shaker homolog KV1 is expressed in human pancreatic β cells which secrete insulin (Ma et al., 2011). I propose that in our mutants the failure to sialylate the Shaker channel, which is known to be present in insulin producing cells (IPC ), will result in those cells secreting an excess of insulin , which in turn causes the metabolic defects leading to a diabetic state. By studying our sialic acid null mutants we can obtain useful information about how diabetes develop. Using the powerful genetics of flies, we can perform screens to identify novel genes that either enhance or reduce the sensitivity to starvation of our SAS mutants and thus play a role in the development or potential treatment of diabetes / Biology

La sclérose latérale amyotrophique est une maladie neurodégénérative associée à un dysfonctionnement métabolique. Des altérations du métabolisme des lipides, décrites chez les patients SLA et les animaux modèles, pourraient participer à la mise en place des premières étapes de la maladie. L’objectif de cette thèse était d’étudier le rôle de la stéaroyl-coenzyme A désaturase 1 (SCD1), une enzyme clé du métabolisme des lipides, dans la SLA. En étudiant le profil d’acides gras périphériques dans un modèle de souris SLA, les souris SOD1m, nous avons vu une diminution de l’activité de la SCD1 dès les stades précoces (subcliniques) de la maladie. Cette diminution pourrait expliquer, à elle seule, les altérations du métabolisme des lipides caractéristiques de la SLA. La répercussion de la perte de l’activité de la SCD1 sur l’axe moteur a été étudiée. Une délétion du gène ou une inhibition pharmacologique de la SCD1 améliore la récupération fonctionnelle après lésion du nerf sciatique chez la souris sauvage. Nous avons cherché à voir si la perte d’activité de la SCD1 trouvée chez les souris SOD1m est un mécanisme de protection mis en place pour lutter contre l’évolution de la SLA. Nous avons traité des souris SOD1m avec un inhibiteur de l’activité de la SCD1. Le traitement a conduit à une augmentation du métabolisme oxydatif, une préservation de l’intégrité neuromusculaire ainsi qu’une amélioration de la survie des motoneurones. Nousconcluons que l’inhibition de la SCD1 représente une cible thérapeutique prometteuse dans la SLA. / Amyotrophic lateral sclerosis is a neurodegenerative disease, associated with metabolic dysfunction. Alteration of lipid metabolism has been documented in ALS patients and animal models, and could participate to the first pathological steps of the disease. The objective of this thesis was to study the role of stearoyl-CoA desaturase 1 (SCD1), a key enzyme of lipid metabolism, in ALS. By studying the profile of peripheral fatty acids in an animal model of ALS, the SOD1 mice, we found that SCD1 activity was strongly reduced at early (sub-clinical) disease stage, and that this reduction could explain in itself the alteration of lipid metabolism characteristic of ALS. The impact of loss of SCD1 activity for the motor axis was then studied. Genetic deletion or pharmacological inhibition of SCD1 enhanced functional recovery after sciatic nerve injury in mice. Wefurther explored if the loss of SCD1 activity found in SOD1 mice is a protective mechanism elicited in response to ALS. We treated SOD1 mice with an inhibitor of SCD1 activity. The treatment resulted in exacerbated muscular oxidative metabolism,preservation of neuromuscular integrity and enhanced motor neuron survival. We conclude that inhibition of SCD1 represents a promising therapeutic target for ALS.