Impaired Akt Phosphorylation Research Articles

Eicosapentaenoic Acid-Induced Inhibition of Metabolic Inflammation Is Associated with Preserved Mitochondrial Function and Insulin Sensitivity in Human Primary Myotubes

Abstract Objectives The aim of this study was to investigate whether metabolic inflammation in skeletal muscle may be prevented by eicosapentaenoic acid (EPA) and if this is associated with an improvement in markers of mitochondrial function and insulin sensitivity. Methods Human primary myotubes were treated for 24 hours with palmitic acid (PA, 500 µM) in hyperglycaemic conditions (13 mM glucose), referred to as nutrient overload, in the presence or absence of EPA (100 µM). After the treatments, the expression of peroxisome proliferator-activated receptor-γ coactivator 1-alpha (PGC1α) and interleukin-6 (IL-6) was assessed by q-PCR. Western blot was used to asses the abundance of the inhibitor of nuclear factor kappa-B (IKBα), mitochondrial electron transport chain complex proteins, the phosphorylation of AKT (Ser473) and AKT substrate 160 (AS 160) (Thr642) in response to insulin, the activation of 5'-AMP-activated protein kinase (AMPK) and the inhibition of acetyl-CoA carboxylase (ACC). Mitochondrial dynamics was assessed by immunocytochemistry. Results Nutrient excess activated the proinflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) signalling as indicated by the upregulation of IL-6 mRNA (P < 0.001) and a tendency to decrease in IKBα (P = 0.0654), tended to downregulate PGC1α (P = 0.0589) and promoted mitochondrial fragmentation (P < 0.001), all of which were counteracted by EPA. Furthermore, EPA induced complex III-core protein 2 (P < 0.05) relative to control cells, an effect that was absent in the myotubes exposed only to PA and hyperglycaemia. EPA, when administrated in combination with PA and hyperglycaemia, induced the phosphorylation of AMPK (P < 0.05) and its downstream target ACC (P < 0.05) relative to cells exposed to nutrient overload alone. Finally, while fuel surplus impaired insulin-induced phosphorylation of AKT (P < 0.01) and AS160 (P < 0.05), these effects were prevented by EPA. Conclusions EPA inhibited NFkB signalling which was associated with an attenuation of the deleterious effects of PA and hyperglycaemia on markers of mitochondrial function and insulin sensitivity. Thus, EPA may represent a valuable nutritional tool to preserve skeletal muscle mitochondrial function and metabolic health during periods of nutrient overload. Funding Sources CSIRO's Precision Health Future Science Platform (FSP).

NLRP3 inflammasome mediate palmitate-induced endothelial dysfunction

AimsFree fatty acids (FFA) is a key contributor to insulin resistance and endothelial dysfunction. However, the precise mechanism underlying the role of FFA remains elusive. This study aimed to investigate the role of NLRP3 (NOD-like receptor pyrin domain containing-3) inflammasome in FFA induced endothelial dysfunction. Main methodsHUVECs were transfected with NLRP3 siRNA and then stimulated with LPS and palmitate. C57 BL/6 J mice transfected with NLRP3 Lenti-Virus were fed with a high-fat diet (HFD). The levels of NLRP3 inflammasome, AMPKα (AMP-activated protein kinase), endothelial nitric oxide synthase (eNOS) and the activity of the insulin signal pathway, in endothelial cells were determined via Western blotting. Endothelial function was determined by measuring the level of endothelium-dependent vasodilatation. Key findingsFFA could activate NLRP3 inflammasome and induce IL-1β release both in vitro. and in vivo. Using siRNA and Lenti-Virus to inhibit NLRP3 abolished palmitate-induced IL-1β release and restored impaired phosphorylation of IRS-1 (Tyr), Akt (Ser473) and eNOS (Ser1177) and ACh-mediated endothelium-dependent vasorelaxation induced by palmitate. AMPKα activator AICAR(5-aminoimidazole-4-carbox-amide-1-β-d-ribofuranoside) inhibited NLRP3 inflammasome activation and decreased IL-1β release and restored impaired insulin signal pathway induced by palmitate. SignificanceNLRP3 inflammasome activation via AMPKα inactivation mediated palmitate-induced endothelial dysfunction through involves IL-1β-induced insulin signal pathway.

Everolimus enhances TRAIL-mediated anti-tumor activity of liver resident natural killer cells in mice.

In transplantation, innate immunity plays a pivotal role in immunosurveillance and host defence against microbes and neoplastic cells. Liver-resident NK cells express TNF-related apoptosis-inducing ligand (TRAIL), which distinguishes them from conventional NK cells. In this study, we investigated the impact of mTOR inhibition on liver-resident NK cells in comparison with that on splenic NK cells in a mouse model. In mice that received everolimus (EVR) for 7days (range: 0.0125-0.25mg/kg/day), the proportion of splenic NK cells was unchanged, whereas the number of liver NK cells including TRAIL+ NK subpopulation increased for all doses of EVR. Consistently, liver-resident NK cells from the EVR-treated mice displayed enhanced cytotoxicity against TRAIL-sensitive neoplastic cells. EVR treatment inhibited the transition of the immature subset of liver NK cells to a mature state. The negative regulator of NK cells FoxO1 was activated as a consequence of impaired mTORC2-dependent AKT phosphorylation. Activated FoxO1 both reduced T-bet expression and induced TRAIL expression, thereby inhibiting NK cell maturation and promoting the antitumour activity of the immature subset of liver NK cells in response to EVR treatment. These findings indicate that EVR treatment enhances the antitumour activity of immature liver-resident NK cells through TRAIL upregulation.

Oncostatin M, a muscle-secreted myokine, recovers high-glucose-induced impairment of Akt phosphorylation by Fos induction in hippocampal neuron cells.

Oncostatin M is a muscle-secreted myokine that has various effects on neuronal function, however, the underlying molecular mechanism has been poorly defined. In this study, we showed that Oncostatin M increased the phosphorylation of Akt and ERK, proteins crucial for neuron cell survival and proliferation. Furthermore, Oncostatin M increased the expression of c-Fos, a protein with significant involvement in neuronal cell proliferation and survival, through both Akt and ERK. Oncostatin M also increased intracellular calcium concentrations that act upstream of Akt and ERK. Treatment with Oncostatin M led to the recovery of high-glucose-induced impairment of Akt phosphorylation. Thus, Oncostatin M can protect neuronal cell damage related to high-glucose conditions, showing potential as a therapeutic agent.

248-LB: Deficiency of APPL1 in Macrophages Triggers Adipose Tissue Inflammation and Insulin Resistance by Potentiating NLRP3 Inflammasome Activation

Adipose tissue inflammation participates in the pathogenesis of type 2 diabetes in obesity. While the activation of the multiprotein complex known as NLRP3 inflammasome and subsequent release of IL-1β contribute to the development of adipose tissue inflammation and insulin resistance, the mechanisms of its regulation remain unclear. Here we revealed that APPL1, an adaptor protein essential for adiponectin and insulin signaling, is upregulated in the stromal vascular fraction of visceral fat of db/db diabetic mice. Also, lack of APPL1 in bone marrow-derived macrophages specifically enhanced inflammatory response mediated by NLRP3 inflammasome. Mice with hematopoietic cells-specific APPL1 deficiency (APPL1-BMT-KO) were then generated using bone marrow transplantation approach and subjected to high-fat diet (HFD) feeding. After 36 weeks of HFD feeding, APPL1-BMT-KO mice exhibited increased insulin resistance and glucose intolerance, accompanied with elevated IL-1β in serum, liver and epididymal adipose tissue and impaired Akt phosphorylation in the subcutaneous adipose tissue and liver. In vitro experiments suggest that ablation of APPL1 in bone marrow-derived macrophages promotes IL-1β secretion by enhancing ROS generation, which is an important intracellular signal for NLRP3 inflammasome activation. Pharmacological inhibition of intracellular and mitochondrial ROS successfully abrogated the upregulation of IL-1β secretion in APPL1 deficient macrophages. In this study, we demonstrated the novel role of APPL1 in alleviating macrophage-mediated adipose tissue inflammation and insulin resistance. Lack of APPL1 in macrophages promotes ROS generation and NLRP3 inflammasome activation, which causes enhanced IL-1β secretion in adipose tissue and the development of insulin resistance. Disclosure K.K.L. Wu: None. A. Xu: None. K.K. Cheng: None. Funding Research Grants Council of Hong Kong (171018/15M)

Activating transcriptional factor 4 correlated with obesity and insulin resistance in polycystic ovary syndrome

PCOS is a systemic disorder that is commonly characterized by insulin resistance (IR). ATF4 participates in the regulation of energy homeostasis and glucose metabolism, but its role in PCOS remains unclear. In this study, we found that ATF4 was highly expressed in human granulosa cells (hGCs) of PCOS patients with obesity and IR. Thus, we performed Spearman's correlation analysis to further investigate the correlation between ATF4 expression and obesity, lipometabolic disorders, or IR in PCOS. We found that increased ATF4 was an important trigger for lipid accumulation and abnormal insulin signal transduction in PCOS. In cultured KGN cells, insulin positively regulated the mRNA and protein abundance of ATF4. Overexpression of ATF4 significantly impaired insulin-stimulated phosphorylation of AKT. Collectively, our findings provided a novel insight into the molecular mechanisms underlying the occurrence and development of PCOS, implying that ATF4 may be a new molecular target for PCOS therapy.

Signal crosstalk between cytokine RANKL and AhR signaling in osteoclasts controls bone homeostasis

Abstract The nuclear receptor aryl hydrocarbon receptor (AhR) plays a crucial role in receptor activator of nuclear factor (NF)-kB ligand (RANKL)-mediated osteoclastogenesis. A considerable amount of evidence reveals that AhR is also a key modulator of skeletal remodeling. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than the expression of signature osteoclast genes such as NFATc1, c-Src, integrin avb3, and cathepsin K. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and mitogen-activated protein kinase as well as NF-kB, while their response to the macrophage colony-stimulating factor remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in a RANKL/c-Fos-dependent manner. Further, ligand activation of AhR by the smoke toxin benzo [a] pyrene (BaP) accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest the new role that the RANKL-AhR-c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone.

Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses

Triggering receptor expressed on myeloid cells (TREM)-1 on polymorphonuclear neutrophils (PMN) regulates innate immune activation in infectious and non-infectious conditions. TREM-1 ligation activates phosphatidyl-inositol 3 kinase (PI3K) triggering all neutrophil effector functions. As idelalisib is a PI3K inhibitor in clinical use for the treatment of non-Hodgkin lymphomas, we asked whether this inhibitor affects PMN functionalities. We analyzed PMNs from healthy donors or lymphoma patients for oxidative burst, phagocytosis, activation markers and IL-8 release upon TREM-1 or TLR ligation ex vivo. In addition, we performed western blot analyses to characterize the signaling events inhibited by idelalisib and other PI3K inhibitors. Upon TREM-1 ligation, the oxidative burst, degranulation, L-selectin shedding and cytokine release were all strongly reduced in the presence of idelalisib along impaired phosphorylation of P38, AKT and ERK by western blot analyses. In line with this, PMNs from patients receiving idelalisib also displayed an impaired TREM-1 mediated PMN activation ex vivo. In conclusion, PI3K inhibitors might cause a neutropenia-like susceptibility to infections in patients by leading to impaired PMN functionality. This should be considered when evaluating patients for infections treated with such inhibitors in daily clinical routine.

Impact of diabetes on gingival wound healing via oxidative stress.

The aim of this study is to investigate the mechanisms linking high glucose to gingival wound healing. Bilateral wounds were created in the palatal gingiva adjacent to maxillary molars of control rats and rats with streptozotocin-induced diabetes. After evaluating postsurgical wound closure by digital imaging, the maxillae including wounds were resected for histological examinations. mRNA expressions of angiogenesis, inflammation, and oxidative stress markers in the surgical sites were quantified by real-time polymerase chain reaction. Primary fibroblast culture from the gingiva of both rats was performed in high glucose and normal medium. In vitro wound healing and cell proliferation assays were performed. Oxidative stress marker mRNA expressions and reactive oxygen species production were measured. Insulin resistance was evaluated via PI3K/Akt and MAPK/Erk signaling following insulin stimulation using Western blotting. To clarify oxidative stress involvement in high glucose culture and cells of diabetic rats, cells underwent N-acetyl-L-cysteine treatment; subsequent Akt activity was measured. Wound healing in diabetic rats was significantly delayed compared with that in control rats. Nox1, Nox2, Nox4, p-47, and tumor necrosis factor-α mRNA levels were significantly higher at baseline in diabetic rats than in control rats. In vitro study showed that cell proliferation and migration significantly decreased in diabetic and high glucose culture groups compared with control groups. Nox1, Nox2, Nox4, and p47 expressions and reactive oxygen species production were significantly higher in diabetic and high glucose culture groups than in control groups. Akt phosphorylation decreased in the high glucose groups compared with the control groups. Erk1/2 phosphorylation increased in the high glucose groups, with or without insulin treatment, compared with the control groups. Impaired Akt phosphorylation partially normalized after antioxidant N-acetyl-L-cysteine treatment. Thus, delayed gingival wound healing in diabetic rats occurred because of impaired fibroblast proliferation and migration. Fibroblast dysfunction may occur owing to high glucose-induced insulin resistance via oxidative stress.

Camel whey protein protects lymphocytes from apoptosis via the PI3K-AKT, NF-κB, ATF-3, and HSP-70 signaling pathways in heat-stressed male mice.

Heat stress (HS) is an environmental factor that depresses the immune systems that mediate dysfunctional immune cells. Camel whey protein (CWP) can scavenge free radicals and enhance immunity. This study investigated the impact of dietary supplementation with CWP on immune dysfunction induced by exposure to HS. Male mice (n = 45) were distributed among 3 groups: control group; HS group; and HS mice that were orally administered CWP (HS + CWP group). The HS group exhibited elevated levels of reactive oxygen species (ROS) and pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, tumor necrosis factor-α) as well as a significant reduction in the IL-2 and IL-4 levels. Exposure to HS resulted in impaired phosphorylation of AKT and IκB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha); increased expression of activating transcription factor 3 (ATF-3) and 70 kDa heat shock proteins (HSP70); and aberrant distribution of CD3+ T cells and CD20+ B cells in the thymus and spleen. Interestingly, HS mice treated with CWP presented significantly restored levels of reactive oxygen species and pro-inflammatory cytokines near the levels observed in the control mice. Furthermore, supplementation of HS mice with CWP enhanced the phosphorylation of AKT and IκB-α; attenuated the expression of ATF-3, HSP70, and HSP90; and improved T and B cell distributions in the thymus and spleen. Our findings reveal a potential immunomodulatory effect of CWP in attenuating immune dysfunction induced by exposure to thermal stress.

ErbB3 drives mammary epithelial survival and differentiation during pregnancy and lactation

BackgroundDuring pregnancy, as the mammary gland prepares for synthesis and delivery of milk to newborns, a luminal mammary epithelial cell (MEC) subpopulation proliferates rapidly in response to systemic hormonal cues that activate STAT5A. While the receptor tyrosine kinase ErbB4 is required for STAT5A activation in MECs during pregnancy, it is unclear how ErbB3, a heterodimeric partner of ErbB4 and activator of phosphatidyl inositol-3 kinase (PI3K) signaling, contributes to lactogenic expansion of the mammary gland.MethodsWe assessed mRNA expression levels by expression microarray of mouse mammary glands harvested throughout pregnancy and lactation. To study the role of ErbB3 in mammary gland lactogenesis, we used transgenic mice expressing WAP-driven Cre recombinase to generate a mouse model in which conditional ErbB3 ablation occurred specifically in alveolar mammary epithelial cells (aMECs).ResultsProfiling of RNA from mouse MECs isolated throughout pregnancy revealed robust Erbb3 induction during mid-to-late pregnancy, a time point when aMECs proliferate rapidly and undergo differentiation to support milk production. Litters nursed by ErbB3KO dams weighed significantly less when compared to litters nursed by ErbB3WT dams. Further analysis revealed substantially reduced epithelial content, decreased aMEC proliferation, and increased aMEC cell death during late pregnancy. Consistent with the potent ability of ErbB3 to activate cell survival through the PI3K/Akt pathway, we found impaired Akt phosphorylation in ErbB3KO samples, as well as impaired expression of STAT5A, a master regulator of lactogenesis. Constitutively active Akt rescued cell survival in ErbB3-depleted aMECs, but failed to restore STAT5A expression or activity. Interestingly, defects in growth and survival of ErbB3KO aMECs as well as Akt phosphorylation, STAT5A activity, and expression of milk-encoding genes observed in ErbB3KO MECs progressively improved between late pregnancy and lactation day 5. We found a compensatory upregulation of ErbB4 activity in ErbB3KO mammary glands. Enforced ErbB4 expression alleviated the consequences of ErbB3 ablation in aMECs, while combined ablation of both ErbB3 and ErbB4 exaggerated the phenotype.ConclusionsThese studies demonstrate that ErbB3, like ErbB4, enhances lactogenic expansion and differentiation of the mammary gland during pregnancy, through activation of Akt and STAT5A, two targets crucial for lactation.

Hyperandrogenemia Induced by Letrozole Treatment of Pubertal Female Mice Results in Hyperinsulinemia Prior to Weight Gain and Insulin Resistance.

Women with polycystic ovary syndrome (PCOS) diagnosed with hyperandrogenism and ovulatory dysfunction have an increased risk of developing metabolic disorders, including type 2 diabetes and cardiovascular disease. We previously developed a model that uses letrozole to elevate endogenous testosterone levels in female mice. This model has hallmarks of PCOS, including hyperandrogenism, anovulation, and polycystic ovaries, as well as increased abdominal adiposity and glucose intolerance. In the current study, we further characterized the metabolic dysfunction that occurs after letrozole treatment to determine whether this model represents a PCOS-like metabolic phenotype. We focused on whether letrozole treatment results in altered pancreatic or liver function as well as insulin resistance. We also investigated whether hyperinsulinemia occurs secondary to weight gain and insulin resistance in this model or if it can occur independently. Our study demonstrated that letrozole-treated mice developed hyperinsulinemia after 1 week of treatment and without evidence of insulin resistance. After 2 weeks of letrozole treatment, mice became significantly heavier than placebo mice, demonstrating that weight gain was not required to develop hyperinsulinemia. After 5 weeks of letrozole treatment, mice exhibited blunted glucose-stimulated insulin secretion, insulin resistance, and impaired insulin-induced phosphorylation of AKT in skeletal muscle. Moreover, letrozole-treated mice exhibited dyslipidemia after 5 weeks of treatment but no evidence of hepatic disease. Our study demonstrated that the letrozole-induced PCOS mouse model exhibits multiple features of the metabolic dysregulation observed in obese, hyperandrogenic women with PCOS. This model will be useful for mechanistic studies investigating how hyperandrogenemia affects metabolism in females.

Abstract 3971: MTA-mediated inhibition of human T cells: Mechanism and MTAP overexpression as putative overcoming strategy

Abstract Metabolic changes of malignant cells lead to the secretion of tumor metabolites which contribute to the shaping of a favorable milieu for tumor immune escape facilitating cancer development and resistance to anti-tumor immunotherapy. Recently, disrupted methylthioadenosine metabolism drew interest as further putative immunoinhibitory metabolic dysregulation. A broad spectrum of tumor entities was reported to lack methylthioadenosine phosphorylase (MTAP) expression leading to elevated levels of its substrate 5’-deoxy-5’-methylthioadenosine (MTA). MTAP deficiency is reported to correlate with worse response to adjuvant interferon-alpha therapy and a higher risk for metastatic disease in malignant melanoma providing evidence for immunobiological relevance. We therefore examined the effect of the tumor metabolite MTA on human T cell responses. We could show that MTA has a strong inhibitory impact on human T cell function regarding proliferation, viability, activation, differentiation, tumor antigen-specific expansion and cytotoxicity. We therefore aimed to overexpress MTAP in human T cells by retroviral transduction as a putative strategy to overcome MTA-mediated inhibition of T cells. In addition, we investigated the effect of MTA on T cell metabolism and maturation of human monocyte derived mDC. We successfully generated stable MTAP-overexpressing primary human T cells. First studies were performed to examine the resistance to MTA after equipping the T cells with higher levels of its metabolizing enzyme MTAP. MTAPhigh T cells revealed less sensitivity against inhibitory effects of MTA on proliferation, viability or cytotoxicity compared to mock transduced controls suggesting a promising approach to strengthen the performance of T cells facing the MTA-rich milieu of MTAP-deficient tumors. Mechanistically, we found MTA to interfere with several signaling pathways. Of most importance, MTA impaired Akt phosphorylation as well as intracellular protein methylation, both crucial processes for proper T cell function. In addition, we studied the combined effect of MTA and PRMT5 inhibition on T cells. Several metabolic studies focusing on glycolysis and fatty acid metabolism could confirm our findings that MTA keeps T cells in a rather inactive, naïve state. Finally, we found MTA to interfere with DC maturation and their potential to induce cytokine secretion of T cells. Our data emphasizes the crucial role of tumor metabolites such as MTA in the tumor milieu for tumor immune escape. Additionally, the identification of potential molecular mechanisms of MTA-induced immunosuppression offer the opportunity to pharmacologically tackle MTA-mediated immunoinhibition and will help to develop more effective immune-based therapies against MTAP-deficient tumors. Citation Format: Carolin D. Strobl, Frederik Henrich, Katrin Singer, Katrin Peter, Marina Kreutz, Anita Kremer, Andreas Mackensen, Michael Aigner. MTA-mediated inhibition of human T cells: Mechanism and MTAP overexpression as putative overcoming strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2017-3971

The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis.

The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR-/- mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos-dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR-/- mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone.

Glycemic Control with Ipragliflozin, a Novel Selective SGLT2 Inhibitor, Ameliorated Endothelial Dysfunction in Streptozotocin-Induced Diabetic Mouse.

Endothelial dysfunction caused by increased oxidative stress is a critical initiator of macro- and micro-vascular disease development in diabetic patients. Ipragliflozin, a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor, offers a novel approach for the treatment of diabetes by enhancing urinary glucose excretion. The aim of this study was to examine whether ipragliflozin attenuates endothelial dysfunction in diabetic mice. Eight-week-old male C57BL/6 mice were treated with streptozotocin (150 mg/kg) by a single intraperitoneal injection to induce diabetes mellitus. At 3 days of injection, ipragliflozin (3 mg/kg/day) was administered via gavage for 3 weeks. Vascular function was assessed by isometric tension recording. Human umbilical vein endothelial cells (HUVEC) were used for in vitro experiments. RNA and protein expression were examined by quantitative RT-PCR (qPCR) and western blot, respectively. Oxidative stress was determined by measuring urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. Ipragliflozin administration significantly reduced blood glucose level (P < 0.001) and attenuated the impairment of endothelial function in diabetic mice, as determined by acetylcholine-dependent vasodilation (P < 0.001). Ipragliflozin did not alter metabolic parameters, such as body weight and food intake. Ipragliflozin administration ameliorated impaired phosphorylation of Akt and eNOSSer1177 in the abdominal aorta and reduced reactive oxygen species generation as determined by urinary excretion of 8-OHdG in diabetic mice. Furthermore, qPCR analyses demonstrated that ipragliflozin decreased the expression of inflammatory molecules [e.g., monocyte chemoattractant protein-1 (MCP-1) vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule (ICAM)-1] in the abdominal aorta (P < 0.05). In in vitro studies, incubation with methylglyoxal, one of the advanced glycation end products, significantly impaired phosphorylation of Akt and eNOSSer1177 (P < 0.01) and increased the expression of MCP-1, VCAM-1, and ICAM-1 in HUVEC. Ipragliflozin improved hyperglycemia and prevented the development of endothelial dysfunction under a hyperglycemic state, at least partially by attenuation of oxidative stress.

IGF-1 prevents simvastatin-induced myotoxicity in C2C12 myotubes.

Statins are generally well tolerated, but treatment with these drugs may be associated with myopathy. The mechanisms of statin-associated myopathy are not completely understood. Statins inhibit AKT phosphorylation by an unclear mechanism, whereas insulin-like growth factor (IGF-1) activates the IGF-1/AKT signaling pathway and promotes muscle growth. The aims of the study were to investigate mechanisms of impaired AKT phosphorylation by simvastatin and to assess effects of IGF-1 on simvastatin-induced myotoxicity in C2C12 myotubes. C2C12 mouse myotubes were exposed to 10μM simvastatin and/or 10ng/mL IGF-1 for 18h. Simvastatin inhibited the IGF-1/AKT signaling pathway, resulting in increased breakdown of myofibrillar proteins, impaired protein synthesis and increased apoptosis. Simvastatin inhibited AKT S473 phosphorylation, indicating reduced activity of mTORC2. In addition, simvastatin impaired stimulation of AKT T308 phosphorylation by IGF-1, indicating reduced activation of the IGF-1R/PI3K pathway by IGF-1. Nevertheless, simvastatin-induced myotoxicity could be at least partially prevented by IGF-1. The protective effects of IGF-1 were mediated by activation of the IGF-1R/AKT signaling cascade. Treatment with IGF-1 also suppressed muscle atrophy markers, restored protein synthesis and inhibited apoptosis. These results were confirmed by normalization of myotube morphology and protein content of C2C12 cells exposed to simvastatin and treated with IGF-1. In conclusion, impaired activity of AKT can be explained by reduced function of mTORC2 and of the IGF-1R/PI3K pathway. IGF-1 can prevent simvastatin-associated cytotoxicity and metabolic effects on C2C12 cells. The study gives insight into mechanisms of simvastatin-associated myotoxicity and provides potential targets for therapeutic intervention.

Impaired Akt phosphorylation in B-cells of patients with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous group of primary immunodeficiency characterized by recurrent infections. We evaluated whether defective PI3K/Akt/FoxO pathway could influence B-cell fate. Determination of B-cell subsets in CVD patients and healthy donors (HDs) were performed using flow cytometry. We evaluated mRNA and protein expression of PI3K, Akt and FoxO using real-time PCR and flow cytometry, respectively. Moreover, phosphorylated Akt (pAkt) expression in B-cells has been measured by flowcytometry. We identified a significant reduction in the percentage of marginal zone like B-cells, memory B-cells (total, switched and unswitched) and plasmablasts in patients, as these decreased B-cell subsets had a significant negative correlation with increased apoptosis in patients. Surprisingly, we identified decreased pAkt expression in B-cells of patients than HDs. We described for the first time impaired pAkt expression in B-cells of CVID patients that had a significant correlation with antibody response to the vaccine and worse clinical complications.

S100A4 accelerates the proliferation and invasion of endometrioid carcinoma and is associated with the "MELF" pattern.

Endometrioid carcinoma (EC) is one of the most common malignancies of the female genital system. Although the behavior of EC ranges from an excellent prognosis to aggressive disease with a poor outcome, the factors that determine its diversity have not been determined. Here, we show that S100A4, a calcium‐binding protein of the EF‐hand type, is correlated with the proliferation and invasion ability of EC. We demonstrated previously that EC cells with high aldehyde dehydrogenase (ALDH) activity were more tumorigenic than ALDH‐lo cells. Screening by shotgun proteomics demonstrated that the expression level of S100A4 in ALDH‐hi EC cells was significantly higher than that in ALDH‐lo cells. S100A4‐knockout cells generated by the CRISPR/Cas9 system showed reduced proliferation and invasion. These cells showed impaired AKT phosphorylation and matrix metalloproteinase‐2 activation, accounting for their impaired proliferation and invasion, respectively. Furthermore, in clinical EC samples, elevated expression of S100A4 was highly related to myometrial and lymphatic invasion in well to moderately differentiated EC. Notably, strong and diffuse expression of S100A4 was observed in tumor tissues with a microcystic, elongated and fragmented (“MELF”) pattern, which is associated with a highly invasive EC phenotype. Collectively, our results demonstrate not only that high expression of S100A4 contributes to an aggressive phenotype of EC, but also that its elevated expression is closely related to the MELF histopathological pattern.

CD44 induced enhancement of phosphatase activity and calcium influx: Modifications of EGR-1 expression and cell proliferation

The purpose of this study was to investigate how CD44 impaired Akt phosphorylation, EGR-1 expression and cell proliferation. E6.1 Jurkat cells, which lack endogenous CD44 expression, were engineered to express CD44. Previously we showed that Akt is hypophosphorylated, EGR-1 expression is reduced and proliferation is impaired in CD44 expressing E6.1 Jurkat cells. The cell cycle was studied using flow cytometry and the role of calcium (Ca2+) in Akt phosphorylation and EGR-1 expression was investigated using Western blotting. Phosphatase activity was assessed using a commercially available kit. CD44 expressing cells showed disruption at the G1 to S transition. Chelation of Ca2+ from the culture media impaired Akt phosphorylation and EGR-1 expression in both CD44 expressing cells and the open vector control. Moreover, Ni2+ disrupted cell proliferation in both cell types suggesting Ca2+ import through calcium release activated calcium channels (CRAC). Staining of cells with fura-2 AM showed significantly higher Ca2+ in CD44 expressing cells as compared with the vehicle control. Finally, non-calcium mediated phosphatase activity was significantly greater in CD44 expressing cells. We propose that the enhanced phosphatase activity in the CD44 cells increased the dephosphorylation rate of Akt; at the same time, the increased intracellular concentration of Ca2+ in the CD44 cells ensured that the phosphorylation of Akt remains intact albeit at lower concentrations as compared with the vector control. Reduced Akt phosphorylation resulted in lowered expression of EGR-1 and hence, reduced the cell proliferation rate.

Decreased adiponectin links elevated adipose tissue autophagy with adipocyte endocrine dysfunction in obesity.

Adipose tissue (AT) autophagy gene expression is elevated in human obesity, correlating with increased metabolic risk, but mechanistic links between the two remain unclear. Thus, the objective of this study was to assess whether elevated autophagy may cause AT endocrine dysfunction, emphasizing the putative role of adiponectin in fat-liver endocrine communication. We utilized a large (N=186) human AT biobank to assess clinical associations between human visceral AT autophagy genes, adiponectin and leptin, by multivariate models. A broader view of adipocytokines association with elevated autophagy was assessed using adipocytokine array. Finally, to establish causality, ex vivo studies utilizing a murine AT-hepatocyte cell line co-culture system was used. Circulating high-molecular-weight adiponectin and leptin levels were associated with human omental-AT expression of ATG5 mRNA, associations that remained significant (β=-0.197, P=0.011; β=0.267, P<0.001, respectively) in a multivariate model adjusted for age, sex, body mass index and interleukin-6 (IL-6). A similar association was observed with omental-AT LC3A mRNA levels. Bafilomycin-A1 (Baf A) pretreatment of AT explants from high-fat-fed (HFF) mice had no effect on the secretion of some AT-derived endocrine factors, but partially or fully reversed obesity-related changes in secretion of a subset of adipocytokines by >30%, including the obesity-associated upregulation of IL-6, vascular endothelial growth factor, tumor necrosis factor alpha (TNFα) and certain insulin-like growth factor-binding proteins, and the HFF-induced downregulated secretion of IL-10 and adiponectin. Similarly, decreased adiponectin and increased leptin secretion from cultured adipocytes stimulated with TNFα+IL-1β was partially reversed by small interfering RNA-mediated knockdown of ATG7. AT explants from HFF mice co-cultured with Hepa1c hepatoma cells impaired insulin-induced Akt and GSK3 phosphorylation. This effect was significantly reversed by pretreating explants with Baf A, but not if adiponectin was immunodepleted from the conditioned media. Reduced secretion of adiponectin may link obesity-associated elevated AT autophagy/lysosomal activity with adipose endocrine dysfunction.