| Part I expression of axon guiding molecules from WT and Aldh1a1-/- miceObjective to identify the axon guiding molecules in intraabdominal fat between WT and Aldh1a1-/- miceMethods 1. WT group (n=10,5 male and 5 female); Aldh1a1-/- group (n=10,5 male and 5 female). All mice were fed on high fat diet for 180 days and then sacrificed. Proteins and mRNA were extracted from iAb for western blot, nanostring and immunohistochemistry.2.3T3-L1, WT and Aldh1a1-/- cells were co-cultured with Forskolin (10uM). Two days later, culture medium was changed for neuronal differentiation medium. Morphology was examined under microscope on Day 5.3. WT and Aldh1a1-/- cells were cultured and started differentiation. Harvest cells and extract mRNA on Day 5. Affymetrix GeneChip and NanoString techniques were used to analyze the genetic pathways related to thermogenesis and axon guidance of WT and Aldh1a1-/- cells.Results 1.Higher expression of Peripherin and TH was detected in Aldh1a1-/- mice. No difference was found between WT and Aldh1a1-/- mice on Rbfox3. Aldh1a1-/- mice expressed lower Nestin and Synopsin than WT mice.2.Neuron-like morphology can be induced in WT and 3T3-L1 cells. Aldh1a1-/- cells had no potential to induce neurogenic morphology.3. Aldh1a1-/- cells express higher levels of Ppary, Papara, Ucp2, EPHA4, EFNA5, Itga3, Plce1, Sema3d, Sema3e, Adamts9. However, Aldh1a1-/- cells express lower levels of Gnao1, Gng11, Hhip and Slit2.Conclusions Aldh1a1-/- cells express molecules integrating stimulation of axon growth with matrix remodeling and neurotransmitter pathways in adipocytes.Part Ⅱ regulations of LTA axon guidance molecules to innervations in vitro and vivo studiesObjective To determine whether the expressions of LTA axon guidance molecules were impaired in obese mice (developed by high fat diet or genetic obese) and whether LTA axon guidance molecules could induce neurite growth.Methods 1. Set up three groups:A. WT mice,4 months (n=14), fed on regular diet. B. Three months old obese mice were bought from Jackson Laboratory and kept on high fat diet for 3 weeks (n=11). C. Three months old genetic obese mice (Ob/Ob) were bought from Jackson Laboratory and kept on high fat diet for 3 weeks (n=11). mRNA was extracted from iAb upon sacrifice for NanoString test to compare the difference of expressions of LTA axon guidance molecules among groups.2. DRG were got from 12-16 weeks female C57BL/6 and started primary culture. Five groups were setup:A:DRG+neuron culture medium. B:DRG+NT-3(1ng/ml)+ neuron culture medium. C. DRG+NGF (10ng/ml)+neuron culture medium. D. DRG+ neuron culture medium+differentiated medium from WT (E:Aldh1a1-/- ) (v/v:1/1). After co-culture for 24 hours, growth of neurons among groups we’re compared.3. Eighteen 3 months female WT mice, fed on high fat diet for 90 days. Nano particles procedure was described below. Then, mice were divided into 4 groups:control group (1ml PBS injected into iAb fat, n=5); empty capsules group (lml empty capsules injected into iAb fat, n=3); encapsulated WT cells group (0.5* 106 cells in lml PBS injected into iAb fat, n=5); encapsulated Aldh1a1-/- cells group (0.5* 106 cells in lml PBS injected into iAb fat, n=5). After injection, all mice were kept on high fat diet for 80 days. iAb fat with injected capsules was got and prepared for further protein study and immunohistochemistry study.Results 1. Expressions of LTA axon guidance molecules in obese mice were lower than normal mice.2. Neurons co-cultured in Aldh1a1-/- medium grew better than neurons co-cultured with NGF or NT3.3. Encapsulated Aldh1a1-/- cells not WT cells activated the expression of peripherin in surrounding adipocytes. The expression of peripherin in iAb injected encapsulated Aldh1a1-/- cells was higher than mice injected empty capsules. iAb injected Aldh1a1-/- cells expressed higher HT. However, no expression of HT was detected in iAb injected WT cells.Conclusions Expressions of LTA axon guidance molecules were impaired in obese mice. LTA axon guidance molecules could promote the growth of neurons in vivo and in vitro.Part Ⅲ effects of RA to LTA axon guidance molecules and relationships between obesity and impaired LTA axon guidance moleculesObjective To discuss effects of RA to LTA axon guidance molecules and relationships between obesity and impaired LTA axon guidance moleculesMethods 1. DRG cells were divided into two groups:A. neurons culture medium + Aldh1a1-/- culture medium (v/v:1/1). B. neurons culture medium+Aldh1a1-/- culture medium (v/v:1/1)+RA(100nM). RA was added on differentiation Day 0,2,5. Observe the morphological changes on Day 6.2. Differentiate 3T3-L1 cells with or without Raid (30nM). NanoString techniques were used to test expressions of Sema3d, Sema3e, EFNA5, AdamtS9.3. Add TTNPB (50nM) or BMS429 (100nM) to differentiated 3T3-L1 cells on Day 4 and Day 5. NanoString techniques were used to test expressions of Sema3d, Sema3e, EFNA5, AdamtS9.4. WT and Aldh1a1-/- mice,5 months old, fed on regular diet were sacrificed and plasma was extracted for volume of EFNA5. Differentiate WT and Aldh1a1-/- cells, compare expressions of EFNA5 between two culture medium.5. DRG cells were divide into two groups:A. culture medium without EFNA5. B. culture medium with EFNA5 (30ng/ml). Co-culture for 24 hours and observe the growth of neurons with XTI microscope.6. Brainbow mice (n=14), fed on high fat diet for 140 days were divided into two groups, PBS injected group (n=6, 100ul) and EFNA5 injected group (n=8,45ng/ml, 100ul). Injection to iAb fat happened every other day for one month. Sacrifice half of the mice randomly and compare the difference of expression of TH in iAb fat between two groups. The other half mice were put in metabolic cage and exposure to cold circumstances (4°for 6 hours) and compare the metabolic rates.7. Subcutaneous fat was extracted from 10 white female (5 BMI<30.5 BMI≥40). Expressions of Aldh1a1 , EFNA5 and EPHA4 were compared between groups.Results 1. RA has significant inhibitory effects on LTA axon guidance molecules.2. Compared to 3T3L1 cells cultured without Rald,3T3L1 cells cultured with Rald expressed higher genes related to LTA axon guidance molecules (Sema3d, Sema3e, EFNA5,AdamtS9).3. Expressions of EFNA5 and EPHA4 were regulated by RAR.4. Higher concentration of EFNA5 was found in Aldh1a1-/- mice than WT mice. Differentiated Aldh1a1-/- cells express even higher EFNA5.5. EFNA5 can promote the growth of DRG cells.6. Compared to mice injected PBS, mice injected EFNA5 into iAb fat express higher TH. After exposure to cold, mice injected EFNA5 show higher metabolic rates.7. Obese people express higher Aldhlal and lower EFNA5 and EPHA4 than lean people.Conclusions EFNA5 is under RAR control and contributes to axon guiding in vitro and possibly in vivo. Impaired expressions of LTA axon guidance molecules in adipocytes may explain obesity in mice even in human. |
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