Pioglitazone ameliorates hepatic damage in irradiated rats via regulating anti-inflammatory and antifibrogenic signalling pathways

Hepatic irradiation during radiotherapy is associated with liver damage. The current study was designed to investigate the possible modulatory effects of pioglitazone against γ irradiation-induced hepatic damage in rats. Animals were exposed to a single dose of 6?Gy and received pioglitazone (10?mg/kg/day) orally for 4 weeks starting on the same day of irradiation. Results showed that irradiation increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities as well as serum triglyceride (TG) and total cholesterol (TC) levels. Furthermore, it elevated inflammatory mediators; tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6); nuclear factor kappa B (NF-κB) and inducible nitric oxide synthase (iNOS) in hepatic tissues. Moreover, it increased levels of serum fibrotic markers; hyaluronic acid (HA), laminin (LN), and type III procollagen (PCIII). Additionally, hepatic fibrotic markers; transforming growth factor-β1 (TGF-β1) and hydroxyproline (HP) levels were elevated. Histological analysis of H&E and MT staining of liver sections exhibited cellular infiltration and fibrous deposition in irradiated rats. It was observed that pioglitazone modulated the described deviations. In conclusion, pioglitazone could serve as a promising therapeutic tool for attenuating radiation-induced liver injury in patients with radiotherapy which might be attributed to its anti-inflammatory and antifibrotic activities.     

Nonoxynol-9 induces apoptosis of endometrial explants by both caspase-dependent and -independent apoptotic pathways

Contraceptive microbicides formulated as vaginal gels offer the possibility of women-controlled contraception and prevention of HIV infection. The effects of these gels on the upper reproductive tract are largely unknown. The purpose of this study was to determine whether nonoxynol-9 (N-9) induces apoptosis in human endometrium using endometrial explant as a model. Apoptosis was determined by gel electrophoresis for the detection of DNA fragmentation and by immunohistochemistry using the M30 CytoDEATH and anti-cleaved caspase-3 (CASP3) antibodies for the detection of caspase activity. The ability of the broad-spectrum caspase inhibitor and CASP3-specific inhibitor to prevent N-9-induced cell death was measured. Expression of apoptosis-related genes such as BCL2, BAX, Fas receptor (FAS), and Fas ligand (FASLG) was quantified using real-time polymerase chain reaction (PCR) analysis. This study demonstrated that N-9 induced DNA fragmentation and caspase activity in endometrial explants in a dose- and time-dependent manner. Caspase inhibitors did not fully prevent the N-9-induced DNA fragmentation. Real-time PCR analysis revealed that FAS and FASLG were largely increased following N-9 treatment. Together, these results suggested that apoptosis triggered by N-9 in endometrial explants is mediated upstream via FAS and FASLG, followed by CASP3 activation leading to final cell death. It appears that other factors besides caspases are also involved in the N-9-induced apoptosis.     

Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways

Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.     

Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways

Leptin controls body weight by activating the long form of the leptin receptor (LEPRb). Janus kinase 2 (JAK2) is associated with LEPRb and autophosphorylates in response to leptin. JAK2 also phosphorylates LEPRb, STAT3, and multiple other downstream molecules. Surprisingly, here we show that JAK2 is not required for leptin stimulation of STAT3 phosphorylation. Leptin time- and dose-dependently stimulated tyrosine phosphorylation of STAT3 in both human and mouse JAK2-null cells. Leptin also increased the viability of JAK2-null cells. Overexpression of c-Src or Fyn, two Src family members, promoted STAT3 phosphorylation, whereas inhibition of the endogenous Src family members by either pharmacological inhibitors or dominant negative Src(K298M) decreased the ability of leptin to stimulate the phosphorylation of STAT3 and ERK1/2. Leptin also stimulated tyrosine phosphorylation of kinase-inactive JAK2(K882E) in JAK2-null cells. Overexpression of JAK2(K882E) enhanced the ability of leptin to stimulate STAT3 phosphorylation in JAK2-null cells. Tyr1138 in LEPRb was required for leptin-stimulated phosphorylation of STAT3 but not JAK2(K882E). These data suggest that leptin stimulates non-JAK2 tyrosine kinase(s), including the Src family members, which phosphorylate JAK2, STAT3, and other molecules downstream of LEPRb. JAK2 mediates leptin signaling by both phosphorylating its substrates and forming a signaling complex as a scaffolding/adaptor protein. The non-JAK2 kinase(s) and JAK2 may act coordinately and synergistically to mediate leptin response.     

The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [(3)H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but (3)H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1.     

Exercise ameliorates insulin resistance and improves ASK1-mediated insulin signalling in obese rats

Increasing evidence reveals that physical exercise is an efficient therapeutical approach in the treatment of insulin resistance (IR) and related metabolic diseases. However, the potential beneficial effects of exercise on insulin resistance and its underlying mechanisms remain unclear. Recent findings elucidated the negative role of ASK1 in repressing the glucose uptake through JNK1-IRS1-Akt signalling in liver. Thus, a detailed investigation of the effect of ASK1-mediated insulin signalling on exercise-mediated improvement of insulin sensitivity and its underlying mechanism was implemented in this study. Using a high-fat diet-induced IR rat model of chronic or acute swimming exercise training, we here showed that body weight and visceral fat mass were significantly reduced after chronic exercise. Moreover, chronic exercise reduced serum FFAs levels and hepatic triglyceride content. Both chronic and acute exercise promoted glucose tolerance and insulin sensitivity. Meanwhile, both chronic and acute exercise decreased ASK1 phosphorylation and improved JNK1-IRS1-Akt signalling. Furthermore, exercise training decreased CFLAR, CREG and TRAF1 protein levels in liver of obese rats, which are positive regulator of ASK1 activity. These results suggested that swimming exercise demonstrated to be an effective ameliorator of IR through the regulation of ASK1-mediated insulin signalling and therefore, could present a prospective therapeutic mean towards the treatment of IR and several metabolic diseases based on IR, containing NAFLD and type Ⅱ diabetes.     

Alternative M2 activation of Kupffer cells by PPARdelta ameliorates obesity-induced insulin resistance

Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator-activated receptor delta (PPARdelta) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARdelta(-/-) (Ppard(-/-)) bone marrow into wild-type mice diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned medium from PPARdelta(-/-) macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes.     

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.     

IRS1-independent defects define major nodes of insulin resistance

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.     

Activation of the small G protein Rap1 in dog thyroid cells by both cAMP-dependent and -independent pathways

All sixteen stereoisomeric analogues of endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)) were synthesized by Fmoc-strategy using solid phase methods. Although synthetic endomorphin-2 exhibited similar mu- and delta-opioid receptor-binding activity to the natural compound, endomorphin-2 analogues containing d-amino acid isomers exhibited lower interaction with mu-receptors depending on the particular combination. The data clearly indicated that the three dimensional structure of endomorphin-2 with the natural l-configuration was the most suitable for binding within the mu receptor, but specific residues are important for activity. Circular dichroism studies verified that changes in chirality of amino acids in the endomorphin-2 sequence resulted in structural conformation. These alterations significantly reduced the specificity for mu-receptor-binding sites.     

Nanos3 maintains the germ cell lineage in the mouse by suppressing both Bax-dependent and -independent apoptotic pathways

Cell death in the germ line is controlled by both positive and negative mechanisms that maintain the appropriate number of germ cells and that prevent the possible formation of germ cell tumors. In the mouse embryo, Steel/c-Kit signaling is required to prevent migrating primordial germ cells (PGCs) from undergoing Bax-dependent apoptosis. In our current study, we show that migrating PGCs also undergo apoptosis in Nanos3-null embryos. We assessed whether the Bax-dependent apoptotic pathway is responsible for this cell death by knocking out the Bax gene together with the Nanos3 gene. Differing from Steel-null embryos, however, the Bax elimination did not completely rescue PGC apoptosis in Nanos3-null embryos, and only a portion of the PGCs survived in the double knockout embryo. We further established a mouse line, Nanos3-Cre-pA, to undertake lineage analysis and our results indicate that most of the Nanos3-null PGCs die rather than differentiate into somatic cells, irrespective of the presence or absence of Bax. In addition, a small number of surviving PGCs in Nanos3/Bax-null mice are maintained and differentiate as male and female germ cells in the adult gonads. Our findings thus suggest that heterogeneity exists in the PGC populations and that Nanos3 maintains the germ cell lineage by suppressing both Bax-dependent and Bax-independent apoptotic pathways.     

Involvement of both caspase-dependent and -independent pathways in apoptotic induction by hexaminolevulinate-mediated photodynamic therapy in human lymphoma cells

Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.     

Obesity, diabetes and insulin resistance. Alterations of insulin signalling

Obesity is often associated with diabetes and insulin resistance. This review summarizes evidence obtained in our lab on the role of the serine phosphorylation of the insulin receptor substrate 1 in the down regulation of insulin signalling. The role of the ERK1 isoform in the development of adipose tissue and insulin sensitivity is also presented.     

Allyl isothiocyanate ameliorates insulin resistance through the regulation of mitochondrial function

Mitochondrial dysfunction is associated with the pathophysiology of insulin resistance. Allylisothiocyanate (AITC) is found in many cruciferous vegetables and has been reported to possess anticancer activity. However, the effect of AITC on insulin resistance and mitochondrial function has not yet been investigated. Here, we show that AITC increased glucose uptake in insulin-resistant C2C12 myotubes and augmented glucose transporter 4 (GLUT4) translocation in L6-GLUT4myc cells. AITC recovered the impaired insulin signaling evoked by free fatty acid exposure and increased mitochondrial membrane potential and mitochondrial DNA content. AITC also elevated the rate of oxygen consumption in C2C12 cells. Furthermore, mice that were fed a high-fat diet with AITC for 10 weeks had reduced diet-induced obesity and hepatic steatosis. AITC also inhibited the hyperglycemia and hyperinsulinemia induced by the consumption of a high-fat diet. Glucose and insulin tolerance tests indicated that AITC improved both glucose tolerance and insulin sensitivity. In addition, AITC inhibited hepatic gluconeogenesis and ameliorated high fat diet-induced mitochondrial dysfunction. Collectively, these data suggest that the protective effect of AITC on insulin resistance is partly mediated through the modulation of mitochondrial dysfunction.     

Oxymatrine ameliorates insulin resistance in rats with type 2 diabetes by regulating the expression of KSRP,PETN, and AKT in the liver

Insulin resistance plays a key role in the development and progression of type 2 diabetes mellitus (T2DM). Recent studies found that insulin resistance was associated with the dysfunction of KH-type splicing regulatory protein (KSRP) expression and AKT pathway, and that oxymatrine possesses an antidiabetic effect. The aim of the present study was to investigate whether the protection of oxymatrine against T2DM was associated with the modulation of the KSRP expression and AKT pathway. Sprague-Dawley rats were fed a high-fat diet and injected with streptozotocin intraperitoneally to induce T2DM, which led to an increase in blood glucose levels and insulin resistance, and a decrease in insulin sensitivity and glycogen synthesis concomitant with KSRP downregulation, PTEN upregulation, and AKT phosphorylation deficiency. The administration of oxymatrine decreased blood glucose levels and insulin resistance, increased insulin sensitivity, and improved glycogen synthesis in the liver of T2DM rats, through a reversal in the expression of KSRP, PTEN, and AKT. On the basis of these observations, we concluded that oxymatrine can protect T2DM rats from insulin resistance through the regulation of the KSRP, PETN, and AKT expression in the liver.     

Endoplasmic reticulum stress in insulin resistance and diabetes

The endoplasmic reticulum is the main intracellular Ca²⁺ store for Ca²⁺ release during cell signaling. There are different strategies to avoid ER Ca²⁺ depletion. Release channels utilize first Ca²⁺-bound to proteins and this minimizes the reduction of the free luminal [Ca²⁺]. However, if release channels stay open after exhaustion of Ca²⁺-bound to proteins, then the reduction of the free luminal ER [Ca²⁺] (via STIM proteins) activates Ca²⁺ entry at the plasma membrane to restore the ER Ca²⁺ load, which will work provided that SERCA pump is active. Nevertheless, there are several noxious conditions that result in decreased activity of the SERCA pump such as oxidative stress, inflammatory cytokines, and saturated fatty acids, among others. These conditions result in a deficient restoration of the ER [Ca²⁺] and lead to the ER stress response that should facilitate recovery of the ER. However, if the stressful condition persists then ER stress ends up triggering cell death and the ensuing degenerative process leads to diverse pathologies; particularly insulin resistance, diabetes and several of the complications associated with diabetes. This scenario suggests that limiting ER stress should decrease the incidence of diabetes and the mobility and mortality associated with this illness.     

Altered protein expression in gestational diabetes mellitus placentas provides insight into insulin resistance and coagulation/fibrinolysis pathways

Objective

To investigate the placental proteome differences between pregnant women complicated with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT).

Methods

We used two-dimensional electrophoresis (2DE) to separate and compare placental protein levels from GDM and NGT groups. Differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and further confirmed by Western blotting. The mRNA levels of related proteins were measured by realtime RT-PCR. Immunohistochemistry (IHC) was performed to examine the cellular location of the proteins expressed in placenta villi.

Results

Twenty-one protein spots were differentially expressed between GDM and NGT placenta villi in the tested samples, fifteen of which were successfully identified by mass spectrometry. The molecular functions of these differentially expressed proteins include blood coagulation, signal transduction, anti-apoptosis, ATP binding, phospholipid binding, calcium ion binding, platelet activation, and tryptophan-tRNA ligase activity. Both protein and mRNA levels of Annexin A2, Annexin A5 and 14-3-3 protein ζ/δ were up-regulated, while the expression of the Ras-related protein Rap1A was down-regulated in the GDM placenta group.

Conclusion

Placenta villi derived from GDM pregnant women exhibit significant proteome differences compared to those of NGT mothers. The identified differentially expressed proteins are mainly associated with the development of insulin resistance, transplacental transportation of glucose, hyperglucose-mediated coagulation and fibrinolysis disorders in the GDM placenta villi.