Deling Zhang

Astragalus polysaccharides decreased the expression of PTP1B through relieving ER stress induced activation of ATF6 in a rat model of type 2 diabetes

Protein tyrosine phosphatase 1B (PTP1B) was considered as a potential therapeutic target of type 2 diabetes (T2DM) because of its negative regulation of insulin signaling. It located on the cytosolic surface of endoplasmic reticulum (ER) and played an essential role in the ER stress signaling. Activating transcription factor 6 (ATF6) was an ER stress regulated transmembrane transcription factor that activated the transcription of ER molecular chaperones. We hypothesized that the expression of PTP1B may be regulated by ATF6 when ER stress happened. Our previous studies showed that Astragalus polysaccharide (APS) increased the insulin sensitivity through decreasing the overexpression of PTP1B in T2DM animal models. In this study, we intended to investigate the possible mechanisms involved in this effect. A rat model of T2DM was established using high fat diet associated with intraperitoneal injection of 25 mg/kg streptozocin; 25 mmol/l D-glucose and 5x10(-7) mol/l insulin were used as in vitro investigations to mimic T2DM-like environment. 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and pCI-Flag-ATF6(N)(2-366) plasmid were treated separately on human hepatocyte line HL-7702 to observe the effect of ATF6 on the expression of PTP1B. The results suggested that APS not only restored the glucose homeostasis but also reduced the ER stress in this rat model of T2DM; ATF6 was involved in mediating the expression of PTP1B when ER stress happened; APS decreased the expression of PTP1B at least partly through inhibiting the activation of ATF6.

Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation.

Aim:To establish the mechanism responsible for the stimulation of glucose uptake by Astragalus polysaccharide (APS), extracted from Astragalus membranaceus Bunge, in L6 myotubes in vitro.Methods:APS-stimulated glucose uptake in L6 myotubes was measured using the 2-deoxy-[3H]-D-glucose method. The adenine nucleotide contents in the cells were measured by HPLC. The phosphorylation of AMP-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) was examined using Western blot analysis. The cells transfected with 4P mutant AS160 (AS160-4P) were constructed using gene transfer approach.Results:Treatment of L6 myotubes with APS (100−1600 μg/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 μg/mL) for 36 h. The APS-stimulated glucose uptake was significantly attenuated by pretreatment with Compound C, a selective AMPK inhibitor or in the cells overexpressing AS160-4P. Treatment of L6 myotubes with APS strongly promoted the activation of AMPK. We further demonstrated that either Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) or liver kinase B1 (LKB1) mediated APS-induced activation of AMPK in L6 myotubes, and the increased cellular AMP: ATP ratio was also involved. Treatment of L6 myotubes with APS robustly enhanced the phosphorylation of AS160, which was significantly attenuated by pretreatment with Compound C.Conclusion:Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.

Promotion of PDGF-induced endothelial cell migration by phosphorylated VASP depends on PKA anchoring via AKAP

Vasodilator-stimulated phosphoprotein (VASP), an important substrate of PKA, plays a critical role in remodeling of actin cytoskeleton and actin-based cell motility. However, how PKA accurately transfers extracellular signals to VASP and then how phosphorylation of VASP regulates endothelial cell migration have not been clearly defined. Protein kinase A anchoring proteins (AKAPs) are considered to regulate intracellular-specific signal targeting of PKA via AKAP-mediated PKA anchoring. Thus, our study investigated the relationship among AKAP anchoring of PKA, PKA activity, and VASP phosphorylation, which is to clarify the exact role of VASP and its upstream regulatory mechanism in PKA-dependent migration. Our results show that chemotactic factor PDGF activated PKA, increased phosphorylation of VASP at Ser157, and enhanced ECV304 endothelial cell migration. However, phosphorylation site-directed mutation of VASP at Ser157 attenuated the chemotactic effect of PDGF on endothelial cells, suggesting phosphorylation of VASP at Ser157 promotes PKA-mediated endothelial cell migration. Furthermore, disrupting PKA anchoring to AKAP or PKA activity significantly attenuated the PKA activity, VASP phosphorylation, and subsequent cell migration. Meanwhile, disrupting PKA anchoring to AKAP abolished PDGF-induced lamellipodia formation and special VASP accumulation at leading edge of lamellipodia. These results indicate that PKA activation and PKA-mediated substrate responses in VASP phosphorylation and localization depend on PKA anchoring via AKAP in PDGF-induced endothelial cell migration. In conclusion, AKAP anchoring of PKA is an essential upstream event in regulation of PKA-mediated VASP phosphorylation and subsequent endothelial cell migration, which contributes to explore new methods for controlling endothelial cell migration related diseases and angiogenesis.

APPL1-Mediating Leptin Signaling Contributes to Proliferation and Migration of Cancer Cells.

Leptin has been implicated in tumorigenesis and tumor progression, particularly in obese patients. As a multifunctional adaptor protein, APPL1 (containing pleckstrin homology domain, phosphotyrosine binding domain, and a leucine zipper motif 1) plays a critical role in regulating adiponectin and insulin signaling pathways. Currently, high APPL1 level has been suggested to be related to metastases and progression of some types of cancer. However, the intercourse between leptin signaling pathway and APPL1 remains poorly understood. Here, we show that the protein levels and phosphorylation statues of APPL1were highly expressed in tissues from human hepatocellular carcinoma and triple-positive breast cancer. Leptin stimulated APPL1 phosphorylation in a time-dependent manner in both human hepatocellular carcinoma HepG2 cell and breast cancer MCF-7 cell. Overexpression or suppression of APPL1 promoted or attenuated, respectively, leptin-induced phosphorylation of STAT3, ERK1/2, and Akt in the cancer cells, accompanied with enhanced or mitigated cell proliferation and migration. In addition, we identified that APPL1 directly bound to both leptin receptor and STAT3. This interaction was significantly enhanced by leptin stimulation. Our results suggested that APPL1 positively mediated leptin signaling and promoted leptin-induced proliferation and migration of cancer cells. This finding reveals a novel mechanism by which leptin promotes the motility and growth of cancer cells.

Wogonin attenuates diabetic cardiomyopathy through its anti-inflammatory and anti-oxidative properties

Among diabetic cardiovascular complications cardiomyopathy is major event which if not well controlled culminates in cardiac failure. Wogonin from the root of Scutellaria baicalensis Georgi has shown specific anti-diabetes bioactivity. However, its effect on diabetic complications remains unclear. The main purpose of this study is to investigate the potential effects of wogonin on diabetic cardiomyopathy and to figure out its underlying mechanism. We found that wogonin administration suppressed hyperglycemia, improved cardiac function, and mitigated cardiac fibrosis in STZ-induced diabetic mice. Wogonin supplementation also attenuated diabetic-induced cardiomyocyte apoptosis and necrosis. In addition, wogonin treatment exhibited the properties of anti-oxidative stress and anti-inflammation in STZ diabetic mice, evidenced by improved activities of anti-oxidases including SOD1/2 and CAT, decreased ROS and MDA production, suppressed expression of inflammation factors such as IL-1β, IL-6, TNFα, and PAI-1, and inhibited NF-κB signaling. These results suggested that wogonin potentially mitigate hyperglycemia-related cardiomyocyte impairment through inhibiting inflammation and oxidative stress.

APPL1-mediated activation of STAT3 contributes to inhibitory effect of adiponectin on hepatic gluconeogenesis

Adiponectin has been shown to suppress hepatic gluconeogenesis. However, the signaling pathways underlying its action remain ill-defined. The purpose of this study was to examine the potential role of APPL1 in mediating anti-gluconeogenic ability of adiponectin. Primary hepatocytes were isolated from male C57BL/6 mice. Western blot and RT-PCR were performed to detect protein expression and mRNA level, respectively. The protein-protein association was determined by immunoprecipitation and GST pull-down assay. We found that APPL1 protein levels were negatively associated with expressions of proteins and mRNAs of gluconeogenesis enzymes under stimulation with adiponectin. In addition, adiponectin-stimulated STAT3 phosphorylation and acetylation were positively regulated by APPL1 and negative regulated by SirT1. Pharmacological and genetic inhibition of STAT3 mitigated impact of adiponectin on hepatic gluconeogenesis. Furthermore, adiponectin administration facilitated the binding of APPL1 to SirT1 and suppressed the association of SirT1 with STAT3. Taken together, our study showed that APPL1-SirT1-STAT3 pathway mediated adiponectin signaling in primary hepatocytes. This new finding provides a novel mechanism by which adiponectin suppresses hepatic gluconeogenesis.

Wogonin enhances intracellular adiponectin levels and suppresses adiponectin secretion in 3T3-L1 adipocytes

As an insulin sensitizer and modulator of inflammatory responses, adiponectin has become a therapeutic target for insulin resistance, diabetes, and diabetes-related complications. Wogonin possesses anti-oxidative, anti-inflammatory, and anti-diabetic abilities. However, its effect on generation and secretion of adiponectin is ill-defined in adipocytes. Here, we demonstrated that wogonin administration augmented intracellular adiponectin levels and attenuated adiponectin release in a dose- and time-dependent manner in mature 3T3-L1 adipocytes, along with a suppression of PKCδ phosphorylation. Wogonin treatment also prevented PKCδ overexpression-induced reduction of intracellular adiponectin levels and enhancement of adiponectin release. In addition, wogonin supplementation dramatically increased AMPK phosphorylation and SirT1 expression. Inhibition of either AMPK or SirT1 mitigated wogonin action on adiponectin production and release. Furthermore, inhibition of AMPK by its specific inhibitor markedly reduced wogonin-enhanced mRNA and protein expressions of SirT1. These results suggested that wogonin regulated expression and secretion of adiponectin via PKCδ/AMPK/SirT1 signaling pathway in mature 3T3-L1 adipocytes.

Tiron ameliorates high glucose-induced cardiac myocyte apoptosis by PKCδ-dependent inhibition of osteopontin

Tiron functions as an effective antioxidant alleviating the intracellular reactive oxygen species (ROS) or the acute toxic metal overload. Previous studies have shown that cardiac myocyte apoptosis can be effectively inhibited by tiron administration in streptozotocin (STZ)‐induced diabetic rats, primary neonatal rat cardiomyocytes (NRVMs), and H9c2 embryonic rat cardiomyocytes. However, the underlying signalling mechanism is ill‐defined. In the present study, we found that tiron supplementation significantly inhibited apoptosis of high glucose (HG)‐treated NRVMs and the left ventricular cardiomyocytes from STZ‐diabetic rat, accompanied with a reduction of osteopontin (OPN) levels as well as an inhibition of PKCδ phosphorylation. OPN knockdown protected NRVMs against HG‐induced cell apoptosis. In addition, genetic inhibition of PKCδ mitigated HG‐stimulated enhancement of intracellular OPN levels in NRVMs. These findings indicate that ROS‐mediated activation of PKCδ upregulated OPN expression, leading to cardiac myocyte apoptosis. Interfering with ROS/PKCδ pathway by antioxidants such as tiron provides an optional therapeutic strategy for treatment and prevention of apoptosis‐related cardiovascular diseases including diabetic cardiomyopathy.

Icariin ameliorates palmitate-induced insulin resistance through reducing thioredoxin-interacting protein (TXNIP) and suppressing ER stress in C2C12 myotubes

Both thioredoxin-interacting protein (TXNIP) and endoplasmic reticulum (ER) stress are implicated in skeletal muscle insulin resistance. Icariin has been found to mimic insulin action in normal skeletal muscle C2C12 cells and display anti-diabetic properties in diet-induced obese mice. However, the underlying molecular mechanism remains to be well-established. Herein, we tested the hypothesis that the protective effects of icariin on free fatty acid-induced insulin resistance were attributed to its regulation on TXNIP protein levels and ER stress in skeletal muscle cells. We found that TXNIP mediated the saturated fatty acid palmitate (PA)-induced insulin resistance in C2C12 myotubes. Icariin treatment significantly restored PA-reduced proteasome activity resulting in reduction of TXNIP protein and suppression of ER stress, as well as improvement of insulin sensitivity. Proteasome inhibition by its specific inhibitor MG132 obviously abolished the inhibitory effect of icariin on PA-induced insulin resistance. In addition, MG132 supplementation markedly abrogated the impacts of icariin on ER stress and TXNIP-mediated downstream events such as inflammation and STAT3 phosphorylation. These results clearly indicate that icariin improves PA-induced skeletal muscle insulin resistance through a proteasome-dependent mechanism, by which icariin downregulats TXNIP levels and inhibits ER stress.