Acutely increased ß-hydroxybutyrate plays a role in the prefrontal cortex to escape stressful conditions during the acute stress response.

Ketone bodies can be increased in the blood under certain physiological conditions, but their role under such conditions remains to be clarified. In the present study, we found the increment and usage of ß-hydroxybutyrate (BHB) in the prefrontal cortex (PFC) during acute stress. BHB levels increased in the blood and PFC after 30-min acute immobilization stress, and BHB dehydrogenase 1 increased in the PFC simultaneously, but not in the hippocampus. Moreover, increased levels of acetyl-CoA, pyruvate carboxylase, and glutamate dehydrogenase 1 were found in the PFC, implicating the metabolism of increased BHB in the brain. Thus, we checked the levels of glutamate, glutamine, and GABA and found increased levels of glutamate and glutamine in the stressed group compared with that in the control group in the PFC. Exogenous administration of BHB enhanced struggling behaviors under stressful conditions. Our results suggest that the metabolism of BHB from peripheral blood in the PFC may contribute to acute stress responses to escape stressful conditions.

Decreased levels of RGS4 in the paraventricular nucleus facilitate GABAergic inhibition during the acute stress response.

A healthy acute stress response requires both rapid increase and rapid clearance of blood corticosteroids. We previously showed that regulators of G-protein signaling 4 (RGS4), which decreases in the paraventricular nucleus (PVN) during acute stress, forms a complex with the GABAB receptor. In the present study, we show that this decrease in RGS4 levels in the PVN during an acute stress response facilitates the return of blood corticosteroids to basal levels. Moreover, the effect of RGS4 decrease is attenuated by a GABAB receptor antagonist. These results suggest that RGS4 in the PVN regulates blood corticosteroid-related GABAB receptor signaling during the acute stress response.

Decreased expression of extracellular matrix proteins and trophic factors in the amygdala complex of depressed mice after chronic immobilization stress.

BACKGROUND: The amygdala plays an essential role in controlling emotional behaviors and has numerous connections to other brain regions. The functional role of the amygdala has been highlighted by various studies of stress-induced behavioral changes. Here we investigated gene expression changes in the amygdala in the chronic immobilization stress (CIS)-induced depression model. RESULTS: Eight genes were decreased in the amygdala of CIS mice, including genes for neurotrophic factors and extracellular matrix proteins. Among these, osteoglycin, fibromodulin, insulin-like growth factor 2 (Igf2), and insulin-like growth factor binding protein 2 (Igfbp2) were further analyzed for histological expression changes. The expression of osteoglycin and fibromodulin simultaneously decreased in the medial, basolateral, and central amygdala regions. However, Igf2 and Igfbp2 decreased specifically in the central nucleus of the amygdala. Interestingly, this decrease was found only in the amygdala of mice showing higher immobility, but not in mice displaying lower immobility, although the CIS regimen was the same for both groups. CONCLUSIONS: These results suggest that the responsiveness of the amygdala may play a role in the sensitivity of CIS-induced behavioral changes in mice.

Isoflavone-Enriched Soybean Leaves (Glycine Max) Alleviate Cognitive Impairment Induced by Ovariectomy and Modulate PI3K/Akt Signaling in the Hippocampus of C57BL6 Mice.

(1) Background: The estrogen decline during perimenopause can induce various disorders, including cognitive impairment. Phytoestrogens, such as isoflavones, lignans, and coumestans, have been tried as a popular alternative to avoid the side effects of conventional hormone replacement therapy, but their exact mechanisms and risk are not fully elucidated. In this study, we investigated the effects of isoflavone-enriched soybean leaves (IESLs) on the cognitive impairment induced by ovariectomy in female mice. (2) Methods: Ovariectomy was performed at 9 weeks of age to mimic menopausal women, and the behavior tests for cognition were conducted 15 weeks after the first administration. IESLs were administered for 18 weeks. (3) Results: The present study showed the effects of IESLs on the cognitive function in the OVX (ovariectomized) mice. Ovariectomy markedly increased the body weight and fat accumulation in the liver and perirenal fat, but IESL treatment significantly inhibited them. In the behavioral tests, ovariectomy impaired cognitive functions, but administration of IESLs restored it. In addition, in the OVX mice, administration of IESLs restored decreased estrogen receptor (ER) ß and PI3K/Akt expression in the hippocampus. (4) Conclusions: The positive effects of IESLs on cognitive functions may be closely related to the ER-mediated PI3/Akt signaling pathway in the hippocampus.

Ingestion of Bis(2-ethylhexyl) phthalate (DEHP) during adolescence causes depressive-like behaviors through hypoactive glutamatergic signaling in the medial prefrontal cortex.

Over the past decades, the production and use of hazardous chemicals has increased worldwide, and the incidence of neurological diseases is increasing proportionately. Among these chemicals, Bis(2-ethylhexyl) phthalate (DEHP) is the most common member of the phthalate family used as a plasticizer. The present study assessed the consequences of daily DEHP ingestion and its effects on brain functions related to depressive-like behaviors. Adolescent C57BL/6 male mice ingested different concentrations of DEHP in their diet (2, 20, and 200 mg/kg of diet), and behavioral changes in anxiety, despair, anhedonia, and sociality were investigated. DEHP exposure evoked depressive-like behaviors in a dose-dependent manner for each symptom. The levels of corticosterone and reactive oxygen species/reactive nitrogen species increased in DEHP-exposed groups, suggesting chronic stress-like responses. In the medial prefrontal cortex (mPFC), glutamate and glutamine were decreased, and glutamine synthetase showed lower activity compared to the control group, suggesting imbalanced glutamatergic signaling. Measuring the spontaneous excitatory postsynaptic current of glutamatergic neurons, we found that DEHP ingestion resulted in hypoactive glutamatergic signaling in the mPFC.

Glutamine Supplementation Prevents Chronic Stress-Induced Mild Cognitive Impairment.

We recently reported that glutamine (Gln) supplementation protected glutamatergic neurotransmission from the harmful effects of chronic stress. Altered glutamatergic neurotransmission is one of the main causes of cognitive disorders. However, the cognitive enhancer function of Gln has not been clearly demonstrated thus far. Here, we evaluated whether and how Gln supplementation actually affects chronic stress-induced cognitive impairment. Using a chronic immobilization stress (CIS) mouse model, we confirmed that chronic stress induced mild cognitive impairment (MCI) and neuronal damage in the hippocampus. In contrast, Gln-supplemented mice did not show evidence of MCI. To investigate possible underlying mechanisms, we confirmed that CIS increased plasma corticosterone levels as well as brain and plasma levels of reactive oxygen/nitrogen species. CIS also increased levels of inducible nitric oxide synthase and NADPH oxidase subunits (p47phox and p67phox) in both the prefrontal cortex and CA1 region of the hippocampus. CIS decreased the number of synaptic puncta in the prefrontal cortex and hippocampus, but these effects were inhibited by Gln supplementation. Taken together, the present results suggest that Gln is an effective agent against chronic stress-induced MCI.

The Root of Polygonum multiflorum Thunb. Alleviates Non-Alcoholic Steatosis and Insulin Resistance in High Fat Diet-Fed Mice.

Non-alcoholic steatosis and insulin resistance are critical health problems and cause metabolic complications worldwide. In this study, we investigated the molecular mechanism of Polygonum multiflorum Thunb. (PM) against hepatic lipid accumulation and insulin resistance by using in vitro and in vivo models. PM extract significantly attenuated the accumulation of lipid droplets and hepatic triglyceride in free fatty acid (FFA)-exposed HepG2 cells. PM extract increased the AMPK and ACC phosphorylation and GLUT4 expression, whose levels were downregulated in FFA-exposed cells. PM extract also decreased precursor and mature forms of SREBP-1 in FFA-exposed cells. C57BL/6 mice fed with normal diet (ND) or high-fat diet (HFD) were administered PM extract (100 mg/kg) or vehicle orally for 16 weeks. PM extract attenuated the increases of the epididymal and perirenal fats on HFD feeding. PM extract markedly reduced hepatic lipid accumulation and fasting glucose levels, and improved glucose and insulin sensitivity in HFD-fed mice. HFD-fed mice decreased the AMPK and ACC phosphorylation and GLUT4 expression, and increased precursor and mature forms of SREBP-1; these changes were significantly restored by PM extract. In conclusion, PM extract alleviates non-alcoholic steatosis and insulin resistance through modulating the expression of proteins on lipid metabolism and glucose transport in the liver.

Anti-Stress and Anti-Depressive Effects of Spinach Extracts on a Chronic Stress-Induced Depression Mouse Model through Lowering Blood Corticosterone and Increasing Brain Glutamate and Glutamine Levels.

Spinach is one of the most widely consumed vegetables, and is known as for both physical and mental health maintenance. However, there is little information about how spinach protects one from stress. In the present study, we created three extracts from Spinach oleracea L., (frozen powder (FP), water extract (WE), and ethanol extract (EE)), and examined their anti-stress and anti-depressive effects on mouse using a chronic immobilization stress (CIS) regimen. FP, WE, and EE showed different free amino acid constituents. Calorie-balanced diets derived from each extract were tested for their ability to reduce blood corticosterone (CORT) levels in naïve mice. Diets supplemented with FP or EE induced lower blood CORT levels than a normal diet, but the WE diet did not. Mobility duration and sucrose preference were increased by FP and EE supplementation in the CIS-induced depression animal models. Moreover, FP and EE increased glutamate and glutamine levels in the medial prefrontal cortex (mPFC) compared with CIS-induced depressed group. These results suggest that spinach has anti-stress and anti-depressive properties by lowering CORT and increasing glutamate and glutamine levels in the mPFC.

Ganoderma lucidum Ameliorates Non-Alcoholic Steatosis by Upregulating Energy Metabolizing Enzymes in the Liver.

Non-alcoholic steatosis is a common health problem worldwide due to altered food habits and life styles, and it is intimately linked with various metabolic disorders. In the present study, we investigated the molecular mechanism of Ganoderma lucidum (GL) against the development of non-alcoholic steatosis using in vivo and in vitro settings. C57BL/6 mice fed with normal diet (ND) or high fat diet (HFD) were administered GL extract or vehicle for 16 weeks. HFD feeding increased serum alanine aminotransferase level and hepatic lipid droplet, but these increases were significantly attenuated by GL. GL inhibited the increases in epididymal and perirenal adipose tissue weights and serum cholesterol and LDL levels in HFD-fed mice. Fasting blood glucose levels were elevated in HFD-fed mice compared to ND-fed mice, and glucose and insulin sensitivities were deteriorated. These changes were markedly improved by GL. GL restored the reduction of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in the liver of HFD-fed mice, and increased AMPK and ACC phosphorylation in HepG2 and 3T3-L1 cells. GL induced GLUT4 protein expression in 3T3-L1 cells. Finally, GL attenuated lipid accumulation induced by free fatty acid in HepG2 cells. Taken together, our results indicate that GL has a potential to improve non-alcoholic steatosis and the associated complicated disorders via the induction of energy metabolizing enzymes.

Type 1 diabetes alters astrocytic properties related with neurotransmitter supply, causing abnormal neuronal activities.

Glutamine synthetase (GS), an astrocytic protein in the brain, mediates the process by which glutamate (Glu) is transformed into glutamine (Gln) during Glu and gamma-aminobutyric acid (GABA) de novo synthesis. There are many types of neural complications related with those neurotransmitters in type 1 diabetes (T1D) patients, but there is little information about the change GS. Therefore, we examined changes in GS activity and expression, as well as the amount of Glu, Gln, and GABA in the brain of a T1D animal model. Using primary culture we found that glucose fluctuation caused glial fibrillary acidic protein (GFAP) and GS changes but constant high glucose level didn׳t. In T1D mouse, GS expression increased in the prefrontal cortex (PFC) and hippocampus (HI), but decreased GS activity was only observed in the HI whereas GFAP expression decreased in both regions. Gln increased in both regions, but Glu and GABA were only increased in the HI of T1D animals where GS activity decreased with higher reactive oxygen/nitrogen species. Collectively, low GS activity may be closely related with high levels of Glu and GABA in the HI of T1D brain, and this would result in abnormal neurotransmissions.

The GABAB receptor associates with regulators of G-protein signaling 4 protein in the mouse prefrontal cortex and hypothalamus.

Regulators of G-protein signaling (RGS) proteins regulate certain G-protein-coupled receptor (GPCR)-mediated signaling pathways. The GABA(B) receptor (GABA(B)R) is a GPCR that plays a role in the stress response. Previous studies indicate that acute immobilization stress (AIS) decreases RGS4 in the prefrontal cortex (PFC) and hypothalamus (HY) and suggest the possibility of a signal complex composed of RGS4 and GABA(B)R. Therefore, in the present study, we tested whether RGS4 associates with GABA(B)R in these brain regions. We found the co-localization of RGS4 and GABA(B)R subtypes in the PFC and HY using double immunohistochemistry and confirmed a direct association between GABA(B2)R and RGS4 proteins using co-immunoprecipitation. Furthermore, we found that AIS decreased the amount of RGS4 bound to GABA(B2)R and the number of double-positive cells. These results indicate that GABA(B)R forms a signal complex with RGS4 and suggests that RGS4 is a regulator of GABA(B)R.

Enhancement of IGF-2-induced neurite outgrowth by IGF-binding protein-2 and osteoglycin in SH-SY5Y human neuroblastoma cells.

A previous study using a mouse model of depression showed that chronic immobilization stress (CIS) reduces levels of insulin-like growth factor (IGF)-2, IGF binding protein 2 (IGFBP2), osteoglycin, and fibromodulin in the amygdala. Here, using human neuroblastoma cells, we tested whether these four proteins cooperatively modulate neuronal plasticity. We found that IGF-2 and IGFBP2 synergistically increased neurite outgrowth via enhanced early signaling through the IGF type 1 receptor. Furthermore, we found that osteoglycin, a small leucine-rich proteoglycan, significantly increased IGF-2/IGFPB2-induced neurite outgrowth, but fibromodulin had no effect. We also found that central amygdala neurons of CIS-induced depressive mouse showed a decreased total dendritic length. These findings suggest that CIS-responsive proteins modulate neuronal morphology during chronic stress.

Acute stress responsive RGS proteins in the mouse brain.

Regulator of G-protein signaling (RGS) proteins play an important role in G-protein coupled receptor (GPCR) signaling and the activity of some GPCRs is modulated via RGS protein levels during stress response. The aim of this study was to investigate changes in RGS protein mRNA expressions in the mouse brain after 2h restraint stress. The mRNA level of 19 RGS proteins was analyzed using real-time PCR in six brain regions, which included the prefrontal cortex, amygdala, hippocampus, hypothalamus, striatum, and pituitary gland, from control and stressed mouse. We found that the level of mRNA of each RGS varied according to brain region and that two to eight RGS proteins exhibited changes in mRNA levels in each brain region by restraint stress. It was also revealed that RGS4 protein amount was consistent with mRNA level, indicating RGS4 protein may have regulatory roles in the acute stress response.