Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/beta-catenin signaling in the hypothalamus

Background: Insulin plays diverse roles in the brain. Although insulin produced by pancreatic β-cells that crosses the blood-brain barrier is a major source of brain insulin, recent studies suggest that insulin is also produced locally within the brain. However, the mechanisms underlying the production of brain-derived insulin (BDI) are not yet known. Results: Here, we examined the effect of Wnt3a on BDI production in a hypothalamic cell line and hypothalamic tissue. In N39 hypothalamic cells, Wnt3a treatment significantly increased the expression of the Ins2 gene, which encodes the insulin isoform predominant in the mouse brain, by activating Wnt/β-catenin signaling. The concentration of insulin was higher in culture medium of Wnt3a-treated cells than in that of untreated cells. Interestingly, neurogenic differentiation 1 (NeuroD1), a target of Wnt/β-catenin signaling and one of transcription factors for insulin, was also induced by Wnt3a treatment in a time- and dose-dependent manner. In addition, the treatment of BIO, a GSK3 inhibitor, also increased the expression of Ins2 and NeuroD1. Knockdown of NeuroD1 by lentiviral shRNAs reduced the basal expression of Ins2 and suppressed Wnt3a-induced Ins2 expression. To confirm the Wnt3a-induced increase in Ins2 expression in vivo, Wnt3a was injected into the hypothalamus of mice. Wnt3a increased the expression of NeuroD1 and Ins2 in the hypothalamus in a manner similar to that observed in vitro. Conclusion: Taken together, these results suggest that BDI production is regulated by the Wnt/β-catenin/NeuroD1 pathway in the hypothalamus. Our findings will help to unravel the regulation of BDI production in the hypothalamus.1

The physiological role of insulin synthesized in paraventricular nucleus of the hypothalamus

prohibitionI. Introduction 1 1. Insulin in the brain 1 2. Hypothalamus 2 3. Wnt/β-catenin signaling in the hypothalamus 2 4. Stress and hypothalamus 3 II. Materials and Methods 6 1. Animals 6 2. Cell culture and treatments 7 3. Quantitative real-time polymerase chain reaction (qRT-PCR) 7 4. Enzyme-linked immunosorbent assay (ELISA) 7 5. Immunofluorescence analysis 8 6. Immunoblot analysis 9 7. Lentivirus preparation 9 8. Stereotaxic injection of Wnt3a and lentivirus 10 9. in situ hybridization 11 10. Microdissection of hypothalamic sub regions 11 11. Statistical analysis 11 III. Results 14 1. Induction mechanism of insulin in the hypothalamus 14 1.1. Insulin is expressed in the brain 14 1.2. Metabolic challenges do not change insulin expression in the hypothalamus 14 1.3. Wnt3a increases the production of insulin in the hypothalamic cells, N39 16 1.4. Wnt3a upregulates insulin production through the canonical Wnt/β-catenin signaling 19 1.5. Wnt3a increases insulin production through NeuroD1 21 1.6. Wnt3a administration increases the expression of Ins2 and NeuroD1 in the hypothalamus 28 2. The physiological role of insulin synthesized in paraventricular nucleus of the hypothalamus (PVN) 30 2.1. Insulin is synthesized in the PVN 30 2.2. PVN insulin is needed to maintain pituitary GH production 30 2.3. PVN insulin regulates body length of young mice via modulating GH production 34 2.4. Acute restraint stress (RS) reduces GH production via suppression of PVN insulin 34 2.5. Chronic RS-induced suppression of PVN insulin results in growth retardation of young mice via reduction of GH production 39 IV. Discussion 44 IIV. Reference 50DOCTORdCollectio

Optimizing a three-dimensional spheroid clearing method for the imaging-based evaluation of cardiotoxicity

Background Toxicity evaluation based on two-dimensional cell culture shows differences from clinical results and has the disadvantage of not accurately reflecting cell-to-cell cross-signaling. Since almost all cells in the human body are arranged in a three-dimensional structure and constitute a tissue, the in vitro reproduction of a three-dimensional tissue composed of human cells can be used as an effective model for drug development and toxicity evaluation. The clearing technique improves image resolution and can implement three-dimensional bio-images throughout the organization, enabling more efficient toxicity evaluation of disease model analysis using spheroids. Methods We generate an 100-200um spheroids and optimized clearing condition. Next application and imaging of optimized clearing condition in the cardiac spheroid model. Results Here we first reported that the optical spheroid clearing protocol for the image-based toxicity prediction model. In our results, spheroid clearing significantly increases fluorescence intensity and enables image-based toxicity prediction. Conclusion We propose that these clearing methods of spheroid can be utilized for the image-based cardiotoxicity evaluation. Furthermore, we also present the possibility that our protocol can also be utilized for patient-tailored toxicity prediction evaluation

Silencing of hypothalamic FGF11 prevents diet-induced obesity

Fibroblast growth factor 11 (FGF11) is a member of the intracellular fibroblast growth factor family. Here, we report the central role of FGF11 in the regulation of metabolism. Lentiviral injection of Fgf11 shRNA into the arcuate nucleus of the mouse hypothalamus decreased weight gain and fat mass, increased brown adipose tissue thermogenesis, and improved glucose and insulin intolerances under high-fat diet conditions. Fgf11 was expressed in the NPY-expressing neurons, and Fgf11 knockdown considerably decreased Npy expression and projection, leading to increased expression of tyrosine hydroxylase in the paraventricular nucleus. Mechanistically, FGF11 regulated Npy gene expression through the glycogen synthase kinase 3-cAMP response element-binding protein pathway. Our study defines the physiological significance of hypothalamic FGF11 in the regulation of metabolism in response to overnutrition such as high-fat diet.TRU

Inhibition of cathepsins B and L increases caspase-dependent apoptosis under glucotoxicity.

<p>(A) INS-1 cells were treated with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM), and cathepsin L inhibitor (CLi, 20 μM) in INS-1 medium containing 11 mM or 30 mM glucose. Cell viability was assessed using the CellTiter-BlueⓇ Cell Viability assay and indicated as a percentage of the values measured at each time point (n = 15). Statistical significance was represented as * <i>p</i> <0.01, and *** <i>p</i> <0.001 compared with 11 mM glucose and 30 mM glucose. (B) Protein levels of active caspase-9 (cleaved cas-9), caspase-3 (cleaved cas-3) and Bcl-2 were measured in INS-1 cells cultured in 11 or 30 mM glucose medium for 48 hr. Cells were treated daily with E, CBi, and CLi. (C) Immunoblot analysis of pancreatic islets of SD rats treated with CBi and CLi in 11 mM or 30 mM glucose medium for 48 hr. (D) JNK inhibitor (SP600125, 5 μM) was treated with CBi and CLi in INS-1 cells cultured in 30 mM glucose medium for 24 hr.</p

Inhibition of cathepsins B and L triggers apoptosis in INS-1 cells.

<p>(A) INS-1 cells were treated with E64d (E, 20 μg/mL) and pepstatin A (P, 20 μg/mL) in 11 mM glucose medium for 48 hr, and caspase activation was monitored by western blotting using antibodies detecting cleaved, active forms of caspases. (B) INS-1 cells were treated with cathepsin B inhibitor (CBi, 20 μM), cathepsin L inhibitor (CLi, 20 μM), and cathepsin K inhibitor (CKi, 20 μM) for 48 hr in 11 mM glucose medium, and caspase activation was monitored by western blotting. (C) INS-1 cells stained with Annexin-V and Hoechst were observed using a fluorescence microscope for quantification of apoptotic cells. Cells were treated with E, CBi, CLi and staurosporine (STS, 0.5 μM) in 11 mM glucose medium. Lysosomal protease inhibitors were added daily, and STS was added 6 hr prior to staining (three independent counts). STS is a known prototypic inducer of apoptosis. *<i>p</i> <0.01, ** <i>p</i> <0.05 compared with 11 mM glucose. The scale bar represents 20 μm.</p

Inhibition of cathepsins B and L results in incomplete processing of cathepsins in lysosome, not in ER.

<p>(A) Immunoblot analysis with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM), and cathepsin L inhibitor (CLi, 20 μM)-treated INS-1 cells in 30 mM glucose for 24 or 48 hr. (B) Immunoblot analysis of pancreatic islets of SD rats treated daily with CBi and CLi in 30 mM glucose for 48 hr. (C) Cytoplasmic and nuclear fractions were prepared from INS-1 cells cultured in 30 mM glucose medium with cathepsin B and L inhibitors for 48 hr then immunoblotted. (D) Localization of pro-cathepsins and cathepsins after treatment with cathepsin B and L inhibitors in 30 mM glucose for 48 hr by cytosol and mito- / lyso- (mitochondria / lysosomal) fractionation. (E) Lysosome-enrichment extraction was prepared and separated by 10% SDS-PAGE. Fraction purity and loading were controlled by immunoblotting for LAMP2 (a lysosomal marker). Red dashed arrows indicate immature cathepsins, while block solid arrows indicate mature cathepsins. (F) Co-localization of cathepsin B (CatB) and L (CatL) with lysotracker was observed in INS-1 cells treated daily with CBi and CLi in 30 mM glucose for 48 hr, as detected by immunofluorescence analysis. (G) Co-localization of CatB / CatL and protein disulfide isomerase (PDI) observed in INS-1 cells treated daily with CBi and CLi in 30 mM glucose for 48 hr. Nuclei were stained with Hoechst 33342 dye. The scale bar represents 5 μm.</p

Inhibition of cathepsins B and L leads to LC3 accumulation.

<p>(A) Activity of cathepsins B and L in 30 mM glucose with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM) or cathepsin L inhibitor (CLi, 20 μM) was measured at 24 hr, 48 hr and 72 hr (n = 9). Statistical significance is represented as *** <i>p</i> <0.001 compared with 30 mM glucose. (B) INS-1 cells cultured in 30 mM glucose for 24 or 48 hr were treated daily with E, P, CBi, and CLi. LC3-II accumulation was assessed by immunoblot analysis. (C) Islets from SD rat cultured in 30 mM glucose and treated with CBi, and CLi. Level of LC3 was measured by immunoblot assay. (D) GFP-LC3/INS-1 stable cells were treated daily with E, P, CBi, and CLiin in 30 mM glucose for 48 hr. The graph indicates the number of GFP-LC3 puncta counted among GFP positive cells (n = 5). *** <i>p</i> <0.001 compared with 30 mM glucose. The scale bar represents 5 μm.</p

Lysosomal dysfunction by inhibition of cathepsins B and L causes cell death.

<p>In the macroautophagy pathway, impaired organelles are enclosed by a phagophore or isolation-membrane (IM), expansion of which gives rise to the autophagosome, a double-membrane vacuole that engulfs cellular components. Subsequently, the autophagosome fuses with lysosomes, in which lysosomal cathepsins, i.e. cathepsin B, D, L, etc. play a key role to allow normal function of the lysosome. Fusion of autophagosomes with lysosomes forms autolysosomes, playing a role in the degradation of cytoplasmic organelles. Inhibition of cathepsins B and L resulted in the accumulation of abnormal unprocessed cathepsins (pro-cathepsins) in the lysosomes. Abnormal accumulation of pro-cathepsins in the lysosomes leads to impaired autophagic process, especially fusion with autophagosomes, resulting in enlarged lysosomes. This lysosomal dysfunction indicates the phenomenon manifesting in lysosomal storage, finally inducing cell death by the activation of caspases.</p