Metformin regulates the incretin receptor axis via a pathway dependent on peroxisome proliferator-activated receptor-α in mice

Aims/hypothesis  

Metformin is widely used for the treatment of type 2 diabetes. Although it reduces hepatic glucose production, clinical studies show that metformin may reduce plasma dipeptidyl peptidase-4 activity and increase circulating levels of glucagon-like peptide 1 (GLP-1). We examined whether metformin exerts glucoregulatory actions via modulation of the incretin axis.     

Long-term exposure to TNF-α leads human skin fibroblasts to a p38 MAPK- and ROS-mediated premature senescence

Tumor necrosis factor α (TNF-α) is an inflammatory mediator overexpressed in the skin as a response to ultraviolet radiation, as well as in chronic non-healing wounds. On the other hand, senescent fibroblasts have been shown to accumulate in the skin under these stressful conditions. Accordingly, here we assessed the putative implication of TNF-α in the induction of premature senescence of human adult dermal fibroblasts. We showed that TNF-α led to a rapid transient p38 MAPK activation, while elevation of reactive oxygen species (ROS) only occurred after a chronic exposure to TNF-α. Furthermore, in contrast to the majority of previous reports using various cell models and experimental settings, it was a long-term treatment with TNF-α that resulted in the premature senescence of human dermal fibroblasts, as shown by the reduced proliferative potential and the increased senescence associated β-galactosidase staining of the cells. TNF-α-senescent cells displayed a permanent phosphorylation of p38 MAPK and an inflammatory and catabolic phenotype. Increased ROS levels were also observed, possibly attributed to the weakened anti-oxidative response evidenced by the underexpression of the Nrf2-regulated genes encoding HO-1 and NQO1. These traits and the overall senescent phenotype were significantly reversed using the known anti-oxidant N-acetyl-l-cysteine or a specific p38 MAPK inhibitor, suggesting the participation of oxidative stress and of the p38 MAPK pathway in TNF-α-triggered premature senescence. Even more, the observed blockade of ROS accumulation in senescent skin fibroblasts by p38 MAPK inhibition indicates a possible link between these two separate events during the manifestation of TNF-α-induced senescence.     

Rac1 mediates sex difference in cardiac tumor necrosis factor-α expression via NADPH oxidase-ERK1/2/p38 MAPK pathway in endotoxemia

The purpose of this study was to investigate the role of Rac1 and estrogen in sex difference of cardiac tumor necrosis factor-alpha (TNF-α) expression during endotoxemia. Endotoxemia was induced in male and female mice by peritoneal injection of lipopolysaccharide (LPS, 4 mg/kg). Compared with female mice, male mice produced more TNF-α in the heart 4 h after LPS treatment, which were correlated with higher Rac1 and NADPH oxidase activity, more phosphorylation of ERK1/2 and p38 MAPK, and up-regulation of toll-like receptor-4 (TLR-4) expression in male mice. Cardiac specific Rac1 knockout or administration of 17β-estradiol down-regulated Rac1 expression, attenuated gp91^phox-NADPH oxidase expression and activity, decreased phosphorylation of ERK1/2/p38 MAPK and inhibited cardiac TNF-α expression induced by LPS, suggesting an important role of Rac1 and estrogen in LPS-stimulated TNF-α expression in the heart. More importantly, the sex difference in TNF-α expression was abrogated by Rac1 knockout or gp91^phox knockout and by administration of apocynin or N-acetylcysteine in LPS-stimulated mice. To investigate the functional significance of sex difference in endotoxemia, heart function was measured in isolated hearts with a Langendorff system. Male mice exhibited worse myocardial dysfunction compared with female in endotoxemia. Treatment of male mice with 17β-estradiol attenuated myocardial dysfunction during endotoxemia. In conclusion, LPS induces Rac1 activation, which contributes to NADPH oxidase activity and phosphorylation of ERK1/2/p38 MAPK, leading to TNF-α expression in the heart. The sex difference in TNF-α expression is estrogen-dependent and mediated via Rac1 dependent NADPH oxidase/ERK1/2 and p38 MAPK pathway in LPS-stimulated hearts.     

Metallothionein downregulation in very old age: a phenomenon associated with cellular senescence?

It is known that metallothionein (MT) mRNA expression first increases with age, but then decreases again in the very elderly. Here we report that MT protein levels also decrease in very old age, and that this is independent of dietary zinc intake. Age-related changes of MT, as well as alterations of zinc homeostasis (intracellular labile zinc and NO-induced zinc release), occur both in human PBMCs ex vivo and also in CD4+ T cell clones progressing through their finite life span in vitro. These results suggest that phenomena observed in very old people can be at least partially attributed to diminished cell proliferation.     

DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.Metazoan life is dependent upon the use of molecular oxygen for an array of metabolic processes. Tissue hypoxia occurs during periods of imbalance between oxygen supply and consumption. One of the primary cellular responses to hypoxia is the activation of the hypoxia-inducible factor (HIF) program (1–4). HIF consists of oxygen-regulated α-subunits HIF-1α and HIF-2α and a constitutively expressed β-subunit (HIF-β). In the presence of oxygen, a series of nonheme Fe(II)- and 2-oxoglutarate–dependent dioxygenase oxygen sensors, referred to as HIF prolylhydroxylases (HIF PHDs), promote the hydroxylation of key proline residues on the HIF-α subunits (5, 6). This serves as a recognition site for the von Hippel-Lindau (VHL) tumor-suppressor protein, which mediates ubiquitination and proteasomal degradation of HIF-1α and HIF-2α (7–9). Hypoxia inhibits HIF PHDs, allowing HIF-1α and HIF-2α to evade VHL recognition and assemble with HIF-β to produce the active heterodimeric HIF factor. Once activated, HIF-1α and HIF-2α cooperate through common and distinct pathways to regulate hypoxic gene expression and cellular adaptation to hypoxia (10).A notable feature of the HIF response is the differential expression pattern of HIF-1α and HIF-2α in normal tissues. HIF-1α mRNA is ubiquitous and constitutively expressed in adult cells. In stark contrast, HIF-2α mRNA is detected in a few cell types of adult tissues and is typically not expressed by epithelia (11). This suggests a physiological necessity to fine-tune the HIF program depending upon the cellular settings by negatively regulating the HIF-2α gene (EPAS1) upstream of the HIF oxygen-sensing enzymes. The negative regulation of EPAS1 is often compromised in cancers, as HIF-2α mRNA is observed in the vast majority of overt tumors (11–13). This is particularly evident in renal cancer. Elegant studies by the Maxwell group (13) and others (14) revealed that HIF-2α mRNA is absent in human kidney tubule epithelia but present in dysplastic foci of the nephron. In these incipient renal tumor cells, HIF-2α may function as an oncoprotein (15), collaborating with, or activating, multiple growth-promoting pathways including cancer stewards c-myc (16), ras (17), and EGFR (18, 19). Silencing of HIF-2α suppresses tumorigenesis of various genetically diverse cancers, further highlighting its central role in malignancy (16, 17, 20, 21), although this depends on the experimental context (22). Therefore, EPAS1 is silent in adult epithelia but undergoes unscheduled activation in several malignancies, driving proliferation in the hypoxic tumor microenvironment (23).A clue to the mechanisms involved in the unscheduled activation of EPAS1 during early tumorigenesis may reside in its promoter, which harbors an enrichment of cytosine and guanine bases that often serve as sites of DNA methylation and epigenetic gene silencing (24–27). Cytosine methylation is catalyzed by a family of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a, and DNMT3b. DNMT1 maintains the methylation pattern from the template strand to the newly synthesized strand during DNA replication (28). DNMT3a and DNMT3b are de novo methyltransferases that establish postreplicative methylation patterns (29). Alterations in DNA methylation patterns are common in tumors and likely play a central role in aberrant gene expression that characterizes the malignant phenotype (26, 30, 31). This is particularly evident for DNMT3a, as recent studies have identified mutations in DNMT3a in patients with acute myeloid leukemia (32, 33) or down-regulation of DNMT3a mRNA in a variety of solid tumors (34). It is suggested that DNMT3a is a tumor-suppressor gene and that its mutation, or mRNA down-regulation, contributes to reducing global DNMT3a methyltransferase activity (35, 36). Currently, a key challenge is to link aberrant methylation profiles commonly observed in malignant lesions, including alterations in the DNMT3a epigenetic program, to genes that directly promote the tumorigenic phenotype.Here we show that DNMT3a methylates and silences EPAS1 in normal cells. Loss of DNMT3a observed in primary tumors and malignant cells causes unscheduled EPAS1 activation. This allows emerging cancer cells to exploit the HIF-2α program that facilitates cancer cell traverse of the hypoxic barrier and formation of tumors larger than the diffusion limit of oxygen. We suggest that the DNMT3a epigenetic program is a gatekeeper of the hypoxic cancer cell phenotype.     

AMPA receptors are exocytosed in stimulated spines and adjacent dendrites in a Ras-ERK–dependent manner during long-term potentiation

The exocytosis of AMPA receptors is a key step in long-term potentiation (LTP), yet the timing and location of exocytosis and the signaling pathways involved in exocytosis during synaptic plasticity are not fully understood. Here we combine two-photon uncaging with two-photon imaging of a fluorescent label of surface AMPA receptors to monitor individual AMPA receptor exocytosis events near spines undergoing LTP. AMPA receptors that reached the stimulated spine came from a combination of preexisting surface receptors (70–90%) and newly exocytosed receptors (10–30%). We observed exocytosis in both the dendrite and spine under basal conditions. The rate of AMPA receptor exocytosis increased ∼5-fold during LTP induction and decayed to the basal level within ∼1 min, both in the stimulated spine and in the dendrite within ∼3 μm of the stimulated spine. AMPA receptors inserted in the spine were trapped in the spine in an activity-dependent manner. The activity-dependent exocytosis required the Ras-ERK pathway, but not CaMKII. Thus, diffusive Ras-ERK signaling presumably serves as an important means for signaling from synapses to dendritic shafts to recruit AMPA receptors into synapses during LTP.     

Postlesion estradiol treatment increases cortical cholinergic innervations via estrogen receptor-α dependent nonclassical estrogen signaling in vivo

17β-Estradiol (E2) treatment exerts rapid, nonclassical actions via intracellular signal transduction system in basal forebrain cholinergic (BFC) neurons in vivo. Here we examined the effect of E2 treatment on lesioned BFC neurons in ovariectomized mice and the role of E2-induced nonclassical action in this treatment. Mice given an N-methyl-d-aspartic acid (NMDA) injection into the substantia innominata-nucleus basalis magnocellularis complex (SI-NBM) exhibited cholinergic cell loss in the SI-NBM and ipsilateral cholinergic fiber loss in the cortex. A single injection of E2 after NMDA lesion did not have an effect on cholinergic cell loss in the SI-NBM, but it restored the ipsilateral cholinergic fiber density in the cortex in a time- and dose-dependent manner. The most effective cholinergic fiber restoration was observed with 33 ng/g E2 treatment at 1 h after NMDA lesion. The E2-induced cholinergic fiber restoration was absent in neuron-specific estrogen receptor-α knockout mice in vivo. Selective activation of nonclassical estrogen signaling in vivo by estren induced E2-like restorative actions. Selective blockade of the MAPK or protein kinase A pathway in vivo prevented E2's ability to restore cholinergic fiber loss. Finally, studies in intact female mice revealed an E2-induced restorative effect that was similar to that of E2-treated ovariectomized mice. These observations demonstrate that a single E2 treatment restores the BFC fiber loss in the cortex, regardless of endogenous E2 levels. They also reveal the critical role of nonclassical estrogen signaling via estrogen receptor-α and protein kinase A-MAPK pathways in E2-induced restorative action in the cholinergic system in vivo.     

Human mesenchymal stem cells are recruited to injured liver in a β1-integrin and CD44 dependent manner

Human bone marrow mesenchymal stem cells (hMSCs) have shown benefit in clinical trials of patients with liver disease. Efficient delivery of cells to target organs is critical to improving their effectiveness. This requires an understanding of the mechanisms governing cellular engraftment into the liver. Binding of hMSCs to normal/injured liver tissue, purified extracellular matrices, and human hepatic sinusoidal endothelial cells (HSECs) were quantified in static and flow conditions. To define the mechanisms underpinning hMSC interactions, neutralizing adhesion molecule antibodies were used. Fluorescently labelled hMSCs were infused intraportally into CCl(4) -injured mice with and without neutralizing antibodies. hMSCs expressed high levels of CD29/β1-integrin and CD44. Using liver tissue binding assays, hMSC adhesion was greatest in diseased human liver versus normal liver (32.2 cells/field versus 20.5 cells/field [P = 0.048]). Neutralizing antibodies against CD29 and CD44 reduced hMSC binding to diseased liver by 34% and 35%, respectively (P = 0.05). hMSCs rolled at 528 μm/second on HSECs in flow assays. This rolling was abolished by CD29 blockade on hMSCs and vascular cell adhesion molecule-1 (VCAM-1) blockade on HSECs. Firm adhesion to HSECs was reduced by CD29 (55% [P = 0.002]) and CD44 (51% [P = 0.04]) blockade. Neutralizing antibodies to CD29 and CD44 reduced hepatic engraftment of hMSCs in murine liver from 4.45 cells/field to 2.88 cells/field (P = 0.025) and 2.35 cells/field (P = 0.03), respectively. hMSCs expressed modest levels of chemokine receptors including CCR4, CCR5, and CXCR3, but these made little contribution to hMSC adhesion in this setting. Conclusion: hMSCs bind preferentially to injured liver. Rolling of hMSCs is regulated by CD29/VCAM-1, whereas CD29/CD44 interactions with VCAM-1, fibronectin, and hyaluronan on HSECs determine firm adhesion both in vitro and in vivo as demonstrated using a murine model of liver injury. (HEPATOLOGY 2012;56:1063-1073).     

Anti-aging effect of DL-β-hydroxybutyrate against hepatic cellular senescence induced by D-galactose or γ-irradiation via autophagic flux stimulation in male rats

The present study aims to shed new light on anti-aging effect of DL-β-hydroxybutyrate (βOHB) against hepatic cellular senescence induced by d-galactose or γ-irradiation. The rats divided into 6 groups. Group 1, control, group 2, exposed to γ-ray (5 GY), group 3, injected by d-galactose (150 mg/kg) daily for consecutive 6 weeks, which regarded to induce the aging, group 4, injected intraperitoneal by β-hydroxybutyrate (βOHB) (72.8 mg/kg) daily for consecutive 14 days, group 5, exposed to γ-ray then treated with βOHB daily for consecutive 14 days, group 6, injected daily with d-galactose for consecutive 6 weeks, then treated with βOHB daily at the last two weeks of d-galactose. Aspartate amino transferase (AST), alanine amino transferase (ALT), Insulin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were estimated in serum. Moreover, protein expression of Microtubule-associated proteins 1A/1B light chain 3B (LC3-II/LC3-I) ratio, mechanistic target of rapamycin (mTOR), pAMPK, mRNA gene expression of 5′ AMP-activated protein kinase (AMPK), Nucleoporin p62 (p62), cyclin-dependent kinase inhibitor 1(P21^CIP1), cyclin-dependent kinase inhibitor 2A (p16^INK4a) and DNA fragmentation percentage were measured in liver tissue as a biomarker of cellular senescence. The results confirmed that βOHB modulated serum level of AST, ALT, insulin, IL-6 and TNF-α, protein expression of mTOR and LC3-II/LC3-I ratio, pAMPK and p62 in liver aging model induced by d-galactose or γ-irradiation. Histopathological examination results of liver tissue indicated coincidence with those recorded by molecular biochemical inspection. Taken together, these findings suggest that βOHB may be useful in combating hepatic cellular senescence induced by d-galactose or γ-irradiation via autophagy dependent mechanisms.