Roberta Piccoletti

Vitamin E dietary supplementation inhibits transforming growth factor β1 gene expression in the rat liver

Overexpression of transforming growth factor β1 (TGFβ1) and increased transcription of pro‐collagen type I, are known to represent major events implicated in the development of liver fibrosis under either experimental or clinical conditions. Here we report that long‐term dietary vitamin E supplementation in animals undergoing an experimental model of liver fibrosis (induced by chronic treatment of rats with carbon tetrachloride) results in a net inhibition of both hepatic TGFβ1 and α2 (I) procollagen mRNA levels. Moreover, of striking interest is the observation that vitamin E supplementation per so down‐modulates basal levels of TGFβ1 mRNA in the liver of untreated animals, suggesting that a dietary regimen rich in vitamin E may potentially interfere with both the initiation and progression of the fibrosclerotic processes.

The MAP kinase cascades are activated during post-ischemic liver reperfusion

We have investigated the involvement of MAP kinase cascades in the response of the liver to post‐ischemic reperfusion. Both JNKs and ERKs are activated but the duration and magnitude of the increase in their activities appear to be different. JNK activation is more marked but shorter than that of ERKs. The increase observed in the phosphotyrosine content of the 52 kDa Shc protein, accompanied by an increased amount of co‐immunoprecipitated Grb2, and the activation of Raf‐1 kinase provide evidence of the involvement of a Ras‐Raf‐dependent pathway, with a time course that is similar to that of ERK activation. The treatment of rats with IL‐1 receptor antagonist modified all of the described effects, suggesting that IL‐1 plays a role in the response of the liver to reperfusion.

The PPAR-γ agonist troglitazone antagonizes survival pathways induced by STAT-3 in recombinant interferon-β treated pancreatic cancer cells.

We have previously shown that cancer cells can protect themselves from apoptosis induced by type I interferons (IFNs) through a ras→MAPK-mediated pathway. In addition, since IFN-mediated signalling components STATs are controlled by PPAR gamma we studied the pharmacological interaction between recombinant IFN-β and the PPAR-γ agonist troglitazone (TGZ). This combination induced a synergistic effect on the growth inhibition of BxPC-3, a pancreatic cancer cell line, through the counteraction of the IFN-β-induced activation of STAT-3, MAPK and AKT and the increase in the binding of both STAT-1 related complexes and PPAR-γ with specific DNA responsive elements. The synergism on cell growth inhibition correlated with a cell cycle arrest in G0/G1 phase, secondary to a long-lasting increase of both p21 and p27 expressions. Blockade of MAPK activation and the effect on p21 and p27 expressions, induced by IFN-β and TGZ combination, were due to the decreased activation of STAT-3 secondary to TGZ. IFN-β alone also increased p21 and p27 expression through STAT-1 phosphorylation and this effect was attenuated by the concomitant activation of IFNbeta-induced STAT-3-activation. The combination induced also an increase in autophagy and a decrease in anti-autophagic bcl-2/beclin-1 complex formation. This effect was mediated by the inactivation of the AKT→mTOR-dependent pathway. To the best of our knowledge this is the first evidence that PPAR-γ activation can counteract STAT-3-dependent escape pathways to IFN-β-induced growth inhibition through cell cycle perturbation and increased autophagic death in pancreatic cancer cells.

Early intracellular events induced by in vivo leptin treatment in mouse skeletal muscle

Experimental evidence suggests that leptin may exert direct effects on peripheral tissues. In this study we investigated some transductional molecules in skeletal muscle, after intraperitoneal leptin injection in wild-type and ob/ob mice. By immunoprecipitation and immunoblotting with anti-phosphotyrosine antibodies, we observed a modified pattern of phosphotyrosine proteins. We then identified an increase in JAK2, IRS1 and IRS2 tyrosine-phosphorylation and in their association with p85, a subunit of PI3K. The increase in PI3K activity in immunoprecipitated p85 did not reach statistical significance, however, both Akt and GSK3 resulted significantly hyper-phosphorylated. Bad, an Akt substrate involved in cell survival, appeared modified in its phosphorylation. ERK1, ERK2 and p38 MAP kinase phosphorylation significantly increased, even if the latter only in wild-type animals. Finally, by EMSA experiments, we documented that leptin increased the DNA binding capacity of Stat3 homodimers and AP-1. Thus, leptin appears to activate, within minutes, some insulin signalling molecules. Stat3 and AP-1 activation by gene expression remodelling could subsequently trigger more leptin-specific effects. Further, leptin might play a still underestimated role in cell survival.

Leptin activates Stat3, Stat1 and AP-1 in mouse adipose tissue

Intraperitoneal leptin administration to wild-type and ob/ob mice caused a prompt activation of Stat1 and Stat3, the former to a lesser extent, in epididymal adipose tissue. Immunoblot experiments showed that tyrosine phosphorylation of Stat3 increased in total cellular extracts and that the phosphorylated protein translocated into the nucleus upon leptin treatment. Tyrosine phosphorylation and nuclear translocation of Stat1 were evident only in ob/ob mice. Gel shift and supershift analyses showed that leptin activated sis-inducible element (SIE) binding activity of adipose nuclear extracts, with Stat3 homodimer as the predominant complex. Stat1/3 heterodimers and Stat1 homodimers take part as well in the response in wild-type and ob/ob mice, although to a lesser degree. AP-1 binding activity was also induced in adipose tissue by in vivo leptin treatment with a time course that suggests a post-transcriptional inductive mechanism. This effect was greater in the ob/ob than in wild-type mice. Our data indicate that leptin operates in vivo directly on adipose tissue by triggering responses that modulate gene expression.

Phosphorylation and redox states in ischemic liver

Abstract Metabolite concentrations, redox and phosphorylation states were studied in ischemic and postischemic rat livers; oxidative phosphorylation and related functional parameters also were investigated with mitochondria derived from these tissues. Time periods were chosen to assess the effects of short-duration (15 min), long-duration reversible (60 min), and necrogenic ischemia (120 min). The energy-charge of liver cells drops promptly and deeply within the first 15 min of ischemia, with a concurrent profound decrease of the [NAD + ] [NADH] ratio both in cytoplasm and mitochondria. Once affected by this initial change, redox and phosphorylation states do not vary appreciably by prolonging blood deprivation for 120 min. The functional capacity of isolated mitochondria declines steadily with increasing duration of ischemia and severe reductions—especially of the ADP O ratios—are attained only at the end of 120 min of ischemia. The reestablishment of the blood supply promotes a good recovery of the energy-charge and redox states (at a slightly different pace) only if previous ischemia did not last more than 60 min. Results with isolated mitochondria correlate well with chemical determinations carried out on postischemic tissue, but respiratory control index does not recover completely over the investigated period following reversible ischemia, thus indicating a less tight coupling of phosphorylation to oxidation during the repair of reversible cellular damage. The relationships between impaired mitochondrial functions, phosphorylation and redox states, and the relevance of their changes to the fate of ischemic cells are discussed.

Intracellular signal transduction pathways induced by leptin in C2C12 cells

As experimental evidence suggests that leptin may have direct effects on peripheral tissues, we investigated some of the transductional molecules induced by leptin in C2C12 cells. In immunoprecipitation experiments using anti‐p85 antibodies (a regulatory subunit of phosphatidylinositol‐3‐kinase; PI3K), we observed a significant increase in PI3K activity. Immunoblot analyses showed that Akt, GSK3, ERK1, ERK2, and p38 mitogen‐activated protein kinase (p38 MAPK) phosphorylation significantly increased after leptin treatment. Protein kinase C (PKC)‐ζ was also activated by leptin, as documented by an immunocomplex kinase assay and immunoblotting experiments. The treatment of C2C12 cells with Wortmannin before leptin administration inhibited induction of the phosphorylation of ERKs (extracellular signal‐regulated kinases) but not that of p38 MAPK, whereas pre‐treatment with a PKC‐ζ inhibitor partially decreased ERK phosphorylation. Taken together, our in vitro results further support the hypothesis that leptin acts acutely on skeletal muscle tissue through some of the components of insulin signalling, including PKC‐ζ.

CELLULAR SIGNALLING AFTER IN VIVO HEAT SHOCK IN THE LIVER

In an experimental model of in vivo hyperthermia, we investigated the involvement of a number of signalling events in rat liver. We report that in vivo heat shock causes a powerful activation of c‐Jun N‐terminal kinase and p38 kinase but does not trigger poly(ADP‐ribose) polymerase cleavage, a signature event of apoptosis. Among the upstream regulators of the kinases, we show that stress‐activated protein kinase/extracellular signal‐regulated kinase/nitrogen‐activated protein kinase kinase 4 SEK1/MKK4 is not involved whereas MKK3 and/or MKK6 are activated. PAK activity displays a transient rise, whereas GCK does not change. PI3‐kinase activity increases in anti‐phosphotyrosine immunoprecipitates, suggesting a tyrosine kinase‐dependent induction mechanism, and the co‐immunoprecipitation of PI3‐kinase with p60 Src kinase supports the involvement of this latter. GSK3, which may act downstream to PI3‐kinase through AKT, undergoes hyperphosphorylation, thus playing a possible role in the protection from apoptosis and in the modulation of heat‐shock transcription factor activity.

Signal transduction pathway of prolactin in rat liver.

We previously reported that a single intraperitoneal injection of prolactin (PRL) in female adult rats rapidly and transiently activates mitogen-activated protein kinase (MAPK) in the liver (Piccoletti et al., (1994) Biochem. J. 303, 429-423). Here we analysed the PRL signalling pathway that accounts for MAPK activation. We found that total liver MAPK kinase-1 phosphorylating activity and Raf-1 activity significantly increase after PRL treatment, following a time course that accounts for the activation of MAPK. We also identified a significant increase in the phosphotyrosine content of the 52 kDa Shc protein, accompanied by an increase in Shc coimmunoprecipitated Grb2, which suggests the Ras involvement by PRL. We found that Janus kinase (JAK)2 tyrosine kinase, which appears constitutively associated with the PRL receptor expressed in the liver, is activated and associated with Shc proteins after in vivo PRL treatment. Taken together our data provide evidence that in vivo PRL activates the Shc Ras Raf MAPK cascade in the liver by the involvement of JAK2 and suggests the possibility that the liver short form of PRL receptor plays a role in triggering this signalling pathway.

Amino acid- and lipid-induced insulin resistance in rat heart: molecular mechanisms

Lipids compete with glucose for utilization by the myocardium. Amino acids are an important energetic substrate in the heart but it is unknown whether they reduce glucose disposal. The molecular mechanisms by which lipids and amino acids impair insulin-mediated glucose disposal in the myocardium are unknown. We evaluated the effect of lipids and amino acids on the insulin stimulated glucose uptake in the isolated rat heart and explored the involved target proteins. The hearts were perfused with 16 mM glucose alone or with 6% lipid or 10% amino acid solutions at the rate of 15 ml/min. After 1 h of perfusion (basal period), insulin (240 nmol/l) was added and maintained for an additional hour. Both lipids and amino acids blocked the insulin effect on glucose uptake (P<0.01) and reduced the activity of the IRSs/PI 3-kinase/Akt/GSK3 axis leading to the activation of glucose transport and glycogen synthesis. Amino acids, but not lipids, increased the activity of the p70 S6 kinase leading to the stimulation of protein synthesis. Amino acids induce myocardial insulin resistance recruiting the same molecular mechanisms as lipids. Amino acids retain an insulin-like stimulatory effect on p70 S6 kinase, which is independent from the PI 3-Kinase downstream effectors.