Il Yong Kim

Strain-specific differences in cell proliferation and differentiation in the dentate gyrus of C57BL/6N and C3H/HeN mice fed a high fat diet

The authors investigated strain-specific cell proliferation and differentiation differences in the dentate gyri of C57BL/6N (susceptible strain to obesity) and C3H/HeN (resistant strain to obesity) mice. In addition, the influences of a high fat diet (HD) on neuronal differentiation in C57BL/6N and C3H/HeN mice fed a low-fat diet (LD) or HD for 4 or 12 weeks were investigated. Body weight and body weight gains were significantly higher in HD-fed C57BL/6N and C3H/HeN mice than in LD-fed C57BL/6N and C3H/HeN mice. In particular, body weight gains were significantly higher in C57BL/6N mice than in C3H/HeN mice. In both of HD- and LD-fed C57BL/6N and C3H/HeN mice for 4 weeks, some Ki67 and many DCX immunoreactive cells were detected in the subgranular zone of the dentate gyrus. In HD-fed C57BL/6N and C3H/HeN mice, the number of Ki67 immunoreactive cells and DCX immunoreactivities in the dentate gyri were significantly lower than in LD-fed C57BL/6N and C3H/HeN mice. However, the number of Ki67 immunoreactive cells and DCX immunoreactivities in HD-fed C57BL/6N mice were significantly lower than in HD-fed C3H/HeN mice. These results suggest that C57BL/6N mice are more vulnerable to HD induced obesity than C3H/HeN mice. In addition, the feeding of HD was found to exacerbate reduced cell proliferation and differentiation in the dentate gyri of C57BL/6N mice as compared with that in C3H/HeN mice.

Stronger proteasomal inhibition and higher CHOP induction are responsible for more effective induction of paraptosis by dimethoxycurcumin than curcumin

Although curcumin suppresses the growth of a variety of cancer cells, its poor absorption and low systemic bioavailability have limited its translation into clinics as an anticancer agent. In this study, we show that dimethoxycurcumin (DMC), a methylated, more stable analog of curcumin, is significantly more potent than curcumin in inducing cell death and reducing the clonogenicity of malignant breast cancer cells. Furthermore, DMC reduces the tumor growth of xenografted MDA-MB 435S cells more strongly than curcumin. We found that DMC induces paraptosis accompanied by excessive dilation of mitochondria and the endoplasmic reticulum (ER); this is similar to curcumin, but a much lower concentration of DMC is required to induce this process. DMC inhibits the proteasomal activity more strongly than curcumin, possibly causing severe ER stress and contributing to the observed dilation. DMC treatment upregulates the protein levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and Noxa, and the small interfering RNA-mediated suppression of CHOP, but not Noxa, markedly attenuates DMC-induced ER dilation and cell death. Interestingly, DMC does not affect the viability, proteasomal activity or CHOP protein levels of human mammary epithelial cells, suggesting that DMC effectively induces paraptosis selectively in breast cancer cells, while sparing normal cells. Taken together, these results suggest that DMC triggers a stronger proteasome inhibition and higher induction of CHOP compared with curcumin, giving it more potent anticancer effects on malignant breast cancer cells.

1H NMR-based metabolomic study on resistance to diet-induced obesity in AHNAK knock-out mice.

AHNAK is a giant protein of approximately 700 kDa identified in human neuroblastomas and skin epithelial cells. Recently, we found that AHNAK knock-out (AHNAK(-/-)) mice have a strong resistance to high-fat diet-induced obesity. In this study, we applied (1)H NMR-based metabolomics with multivariate statistical analysis to compare the altered metabolic patterns detected in urine from high-fat diet (HFD) fed wild-type and AHNAK(-/-) mice and investigate the mechanisms underlying the resistance to high-fat diet-induced obesity in AHNAK(-/-) mice. In global profiling, principal components analysis showed a clear separation between the chow diet and HFD groups; wild-type and AHNAK(-/-) mice were more distinctly separated in the HFD group compared to the chow diet group. Based on target profiling, the urinary metabolites of HFD-fed AHNAK(-/-) mice gave higher levels of methionine, putrescine, tartrate, urocanate, sucrose, glucose, threonine, and 3-hydroxyisovalerate. Furthermore, two-way ANOVAs indicated that diet type, genetic type, and their interaction (gene × diet) affect the metabolite changes differently. Most metabolites were affected by diet type, and putrescine, threonine, urocanate, and tartrate were also affected by genetic type. In addition, cis-aconitate, succinate, glycine, histidine, methylamine (MA), phenylacetylglycine (PAG), methionine, putrescine, uroconate, and tartrate showed interaction effects. Through the pattern changes in urinary metabolites of HFD-fed AHNAK(-/-) mice, our data suggest that the strong resistance to HFD-induced obesity in AHNAK(-/-) mice comes from perturbations of amino acids, such as methionine, putrescine, threonine, and histidine, which are related to fat metabolism. The changes in metabolites affected by microflora such as PAG and MA were also observed. In addition, resistance to obesity in HFD-fed AHNAK(-/-) mice was not related to an activated tricarboxylic acid cycle. These findings demonstrate that (1)H NMR-based metabolic profiling of urine is suitable for elucidating possible biological pathways perturbed by functional loss of AHNAK on HFD feeding and could elucidate the mechanism underlying the resistance to high-fat diet-induced obesity in AHNAK(-/-) mice.

Expression of tissue-type transglutaminase (tTG) and the effect of tTG inhibitor on the hippocampal CA1 region after transient ischemia in gerbils

Chronological changes of tissue-type transglutaminase (tTG) were observed in the hippocampal CA1 region after transient forebrain ischemia in gerbils. In the sham-operated group, tTG immunoreactivity was weakly detected in blood vessels which were immunostained with platelet endothelial cell adhesion molecule-1 (PECAM-1), and tTG immunoreactivity in blood vessels was highest 5 days after ischemia/reperfusion. In addition, tTG immunoreaction was expressed in microglia which were immunostained with Iba-1 at 4 days post-ischemia, and tTG immunoreactivity in the microglia was also highest at 5 days post-ischemia. In Western blot analysis, tTG protein levels in the CA1 region after ischemia/reperfusion began to increase 3 days after ischemia/reperfusion and peaked 5 days after ischemia/reperfusion. The expression of tTG in PECAM-1-immunoreactive blood vessels may be associated with integrin regulation or transendothelial migration of leukocytes in the ischemic CA1 region. In this study, we also observed the effect of cystamine, a tTG inhibitor, against ischemic damage. Administration of cystamine protected in certain degree neuronal damage from ischemic damage in the CA1 region. These results suggest that tTG may be associated with neuronal death in the hippocampal CA1 region induced by ischemia/reperfusion.

Effects of treadmill exercise on cyclooxygenase-2 in the hippocampus in type 2 diabetic rats: Correlation with the neuroblasts

Cyclooxygenase (COX) is a rate-limiting enzyme in synthesis of prostaglandins from arachidonic acid. In this study, we observed the effects of a physical exercise on COX-2 immunoreactivity in the hippocampus using immunohistochemistry in rats. In addition, we examined effects of administration of a COX-2 inhibitor, celecoxib, on neuroblast differentiation. At 6weeks of age, Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats were put on a treadmill with or without running for 1h/session/day for 5weeks. The running speed was gradually increased from 16 to 22m/min with 2m/min per 2weeks. In the ZLC and ZDF rats, COX-2 immunoreaction was detected in the granule cell layer of the dentate gyrus and in the stratum pyramidale of the CA2/3 region; COX-2 immunoreaction in the CA1 region was hardly detected. In the exercised-ZLC and ZDF rats, COX-2 immunoreactivity was significantly increased compared to that in the ZLC and ZDF rats, showing that COX-2 immunoreactivity in the exercised-ZDF rats was slightly low than that in the exercised-ZDF rats. In addition, weak COX-2 immunoreactivity was shown in the CA1 region by exercise. On the other hand, the repeated oral administration of celecoxib to 4-week-old ZDF rats significantly decreased the neuroblasts in the subgranular zone of the dentate gyrus. These results suggest that COX-2 may be associated with the increase of synaptic plasticity or contacts in the hippocampus.

Regulatory mechanism of hypothalamo-pituitary-adrenal (HPA) axis and neuronal changes after adrenalectomy in type 2 diabetes.

Diabetes, especially type 2, is closely associated with hypothalamo-pituitary-adrenal (HPA) axis regulation. Short-term effects of adrenalectomy (ADX) in type 2 diabetes are well characterized; however, there have been few reports on the long-term effects of ADX in genetically engineered type 2 diabetes and the neuroendocrine system. We performed bilateral ADX in Zucker Lean Control rats (ZLC; ADX-ZLC), Zucker Diabetic Fatty rats (ZDF; ADX-ZDF), and sham control rats to evaluate how the HPA axis would be regulated in long-term corticosterone deficient type 2 diabetic animals. We evaluated arginine vasopressin (AVP), glucocorticoid receptor (GR), and corticotropin-releasing hormone (CRH) expression with immunohistochemistry (IHC), immunofluorescence, real-time PCR, and Western blot analysis in each treatment group 7 weeks post ADX to assess HPA axis regulatory patterns in connection with type 2 diabetes. Additionally, mRNA expression of AVP and CRH receptors (V1aR, V1bR, CRHR1, and CRHR2) was also measured and adrenocorticotropin hormone (ACTH) immunoreactivity was surveyed by IHC to add to data regarding the regulatory mechanism. AVP and CRH protein expression levels increased after ADX in the hypothalamus of diabetic rats based on IHC results; however, we found that the subtypes of each receptor may be regulated differently in ADX groups compared to sham groups. Immunoreactivity of ACTH in the pituitary gland was enhanced in ADX groups and GR expression levels in the hypothalamic paraventricular nuclei (PVN) remained high, as determined by IHC as well as Western blot analysis. Without the negative feedback system of corticosterone, CRH is highly enhanced and may primarily combine with CRHR1 to stimulate negative feedback through ACTH in the pituitary gland in type 2 diabetic rats with long-term ADX. Although the negative feedback signal was not transmitted appropriately following long-term ADX with type 2 diabetes, a high GR protein level was maintained as in type 2 diabetes. The long-termed lack of corticosterone in the blood stream is a very important factor for normal regulation of the HPA axis even in diabetic animals. From the data, we can conclude that the stimulated HPA axis regulation in the developing type 2 diabetic animals following long-term adrenalectomy has remained elevated rather than diminished. Therefore, the current study may provide useful information to better understand patients suffering from both type 2 diabetes and Addisons disease.

RORα controls hepatic lipid homeostasis via negative regulation of PPARγ transcriptional network

The retinoic acid receptor-related orphan receptor-α (RORα) is an important regulator of various biological processes, including cerebellum development, circadian rhythm and cancer. Here, we show that hepatic RORα controls lipid homeostasis by negatively regulating transcriptional activity of peroxisome proliferators-activated receptor-γ (PPARγ) that mediates hepatic lipid metabolism. Liver-specific Rorα-deficient mice develop hepatic steatosis, obesity and insulin resistance when challenged with a high-fat diet (HFD). Global transcriptome analysis reveals that liver-specific deletion of Rorα leads to the dysregulation of PPARγ signaling and increases hepatic glucose and lipid metabolism. RORα specifically binds and recruits histone deacetylase 3 (HDAC3) to PPARγ target promoters for the transcriptional repression of PPARγ. PPARγ antagonism restores metabolic homeostasis in HFD-fed liver-specific Rorα deficient mice. Our data indicate that RORα has a pivotal role in the regulation of hepatic lipid homeostasis. Therapeutic strategies designed to modulate RORα activity may be beneficial for the treatment of metabolic disorders.Hepatic steatosis development may result from dysregulation of lipid metabolism, which is finely tuned by several transcription factors including the PPAR family. Here Kim et al. show that the nuclear receptor RORα inhibits PPARγ-mediated transcriptional activity by interacting with HDAC3 and competing for the promoters of lipogenic genes.

Evaluation of treadmill exercise effect on muscular lipid profiles of diabetic fatty rats by nanoflow liquid chromatography–tandem mass spectrometry

We compare comprehensive quantitative profiling of lipids at the molecular level from skeletal muscle tissues (gastrocnemius and soleus) of Zucker diabetic fatty rats and Zucker lean control rats during treadmill exercise by nanoflow liquid chromatography–tandem mass spectrometry. Because type II diabetes is caused by decreased insulin sensitivity due to excess lipids accumulated in skeletal muscle tissue, lipidomic analysis of muscle tissues under treadmill exercise can help unveil the mechanism of lipid-associated insulin resistance. In total, 314 lipid species, including phospholipids, sphingolipids, ceramides, diacylglycerols (DAGs), and triacylglycerols (TAGs), were analyzed to examine diabetes-related lipid species and responses to treadmill exercise. Most lysophospholipid levels increased with diabetes. While DAG levels (10 from the gastrocnemius and 13 from the soleus) were >3-fold higher in diabetic rats, levels of most of these decreased after exercise in soleus but not in gastrocnemius. Levels of 5 highly abundant TAGs (52:1 and 54:3 in the gastrocnemius and 48:2, 50:2, and 52:4 in the soleus) displaying 2-fold increases in diabetic rats decreased after exercise in the soleus but not in the gastrocnemius in most cases. Thus, aerobic exercise has a stronger influence on lipid levels in the soleus than in the gastrocnemius in type 2 diabetic rats.

AHNAK deficiency promotes browning and lipolysis in mice via increased responsiveness to β-adrenergic signalling

In adipose tissue, agonists of the β3-adrenergic receptor (ADRB3) regulate lipolysis, lipid oxidation, and thermogenesis. The deficiency in the thermogenesis induced by neuroblast differentiation-associated protein AHNAK in white adipose tissue (WAT) of mice fed a high-fat diet suggests that AHNAK may stimulate energy expenditure via development of beige fat. Here, we report that AHNAK deficiency promoted browning and thermogenic gene expression in WAT but not in brown adipose tissue of mice stimulated with the ADRB3 agonist CL-316243. Consistent with the increased thermogenesis, Ahnak−/− mice exhibited an increase in energy expenditure, accompanied by elevated mitochondrial biogenesis in WAT depots in response to CL-316243. Additionally, AHNAK-deficient WAT contained more eosinophils and higher levels of type 2 cytokines (IL-4/IL-13) to promote browning of WAT in response to CL-316243. This was associated with enhanced sympathetic tone in the WAT via upregulation of adrb3 and tyrosine hydroxylase (TH) in response to β-adrenergic activation. CL-316243 activated PKA signalling and enhanced lipolysis, as evidenced by increased phosphorylation of hormone-sensitive lipase and release of free glycerol in Ahnak−/− mice compared to wild-type mice. Overall, these findings suggest an important role of AHNAK in the regulation of thermogenesis and lipolysis in WAT via β-adrenergic signalling.

Identification of the responsible proteins for increased selenium bioavailability in the brain of transgenic rats overexpressing selenoprotein M

The present study was conducted to investigate whether the high antioxidant activity induced by selenium (Sel) treatment and selenoprotein M (SelM) overexpression affected the protein profile of the brain cortex. To accomplish this, the changes in global protein expression were measured in transgenic (Tg) rats expressing human SelM (CMV/hSelM) and non‑Tg rats using two-dimensional electrophoresis (2-DE). The results revealed that: ⅰ) CMV/hSelM Tg rats showed a high level of enzyme activity for antioxidant protein in the brain cortex compared to non-Tg rats; ⅱ) the high activity of these enzymes induced a decrease in total antioxidant concentration and γ-secretase activity in CMV/hSelM Tg rats; ⅲ) five proteins were upregulated and three were downregulated by SelM overexpression; ⅳ) among the five upregulated proteins, two associated with creatine kinase B-type (B-CK) and E3 ubiquitin-protein ligase RING1 (RING finger protein 1) were further increased in the two groups following Sel treatment, whereas synaptotagmin-15 (SytXV), eukaryotic translation initiation factor 4H (eIF-4H) and lactate dehydrogenase B (LDH-B) were increased or decreased under the same conditions; ⅴ) the three downregulated proteins did not induce a significant change in expression following Sel treatment; and ⅵ) the protein expression level alterations of the two selected spots (B-CK and SytXV) identified by 2-DE were extremely similar to the results from western blot analysis. Overall, the results of the present study provide primary novel biological evidence that new functional protein groups and individual proteins in the brain cortex of CMV/hSelM Tg rats are associated with Sel biology, including the response to Sel treatment and SelM overexpression.