The regulatory role of PGC1α-related coactivator in response to drug-induced liver injury.

PGC1α-Related Coactivator (PRC) is a transcriptional coactivator promoting cytokine expression in vitro in response to mitochondrial injury and oxidative stress, however, its physiological role has remained elusive. Herein we investigate aspects of the immune response function of PRC, first in an in vivo thioacetamide (TAA)-induced mouse model of drug-induced liver injury (DILI), and subsequently in vitro in human monocytes, HepG2, and dendritic (DC) cells. TAA treatment resulted in the dose-dependent induction of PRC mRNA and protein, both of which were shown to correlate with liver injury markers. Conversely, an adenovirus-mediated knockdown of PRC attenuated this response, thereby reducing hepatic cytokine mRNA expression and monocyte infiltration. Subsequent in vitro studies with conditioned media from HepG2 cells overexpressing PRC, activated human monocytes and monocyte-derived DC, demonstrated up to 20% elevated expression of CD86, CD40, and HLA-DR. Similarly, siRNA-mediated knockdown of PRC abolished this response in oligomycin stressed HepG2 cells. A putative mechanism was suggested by the co-immunoprecipitation of Signal Transducer and Activator of Transcription 1 (STAT1) with PRC, and induction of a STAT-dependent reporter. Furthermore, PRC co-activated an NF-κB-dependent reporter, indicating interaction with known major inflammatory factors. In summary, our study indicates PRC as a novel factor modulating inflammation in DILI.

Fasting-Induced Transcription Factors Repress Vitamin D Bioactivation, a Mechanism for Vitamin D Deficiency in Diabetes.

Low 25-hydroxyvitamin D levels correlate with the prevalence of diabetes; however, the mechanisms remain uncertain. Here, we show that nutritional deprivation-responsive mechanisms regulate vitamin D metabolism. Both fasting and diabetes suppressed hepatic cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase responsible for the first bioactivation step. Overexpression of coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), induced physiologically by fasting and pathologically in diabetes, resulted in dramatic downregulation of CYP2R1 in mouse hepatocytes in an estrogen-related receptor α (ERRα)-dependent manner. However, PGC-1α knockout did not prevent fasting-induced suppression of CYP2R1 in the liver, indicating that additional factors contribute to the CYP2R1 repression. Furthermore, glucocorticoid receptor (GR) activation repressed the liver CYP2R1, suggesting GR involvement in the regulation of CYP2R1. GR antagonist mifepristone partially prevented CYP2R1 repression during fasting, suggesting that glucocorticoids and GR contribute to the CYP2R1 repression during fasting. Moreover, fasting upregulated the vitamin D catabolizing CYP24A1 in the kidney through the PGC-1α-ERRα pathway. Our study uncovers a molecular mechanism for vitamin D deficiency in diabetes and reveals a novel negative feedback mechanism that controls crosstalk between energy homeostasis and the vitamin D pathway.

Activation of nuclear receptor PXR impairs glucose tolerance and dysregulates GLUT2 expression and subcellular localization in liver.

Pregnane X receptor (PXR) is a nuclear receptor that senses chemical environment and is activated by numerous clinically used drugs and environmental contaminants. Previous studies have indicated that several drugs known to activate PXR appear to induce glucose intolerance. We now aimed to reveal the role of PXR in drug-induced glucose intolerance and characterize the mechanisms involved. We used PXR knockout mice model to investigate the significance of this nuclear receptor in the regulation of glucose tolerance. PXR ligand pregnenolone-16ɑ-carbonitrile (PCN) impaired glucose tolerance in the wildtype mice but not in the PXR knockout mice. Furthermore, DNA microarray and bioinformatics analysis of differentially expressed genes and glucose metabolism relevant pathways in PCN treated primary hepatocytes indicated that PXR regulates genes involved in glucose uptake. PCN decreased the expression of glucose transporter 2 (GLUT2) in mouse liver and in the wildtype mouse hepatocytes but not in the PXR knockout cells. Data mining of published chromatin immunoprecipitation-sequencing results indicate that Glut2 gene is a direct PXR target. Furthermore, PCN induced internalization of GLUT2 protein from the plasma membrane to the cytosol in the liver in vivo and repressed glucose uptake in the primary hepatocytes. Our results indicate that the activation of PXR impairs glucose tolerance and thus PXR represents a novel diabetogenic pathway. PXR activation dysregulates GLUT2 function by two different mechanisms. These findings may partly explain the diabetogenic effects of medications and environmental contaminants.

Who watches the watchmen? Regulation of the expression and activity of sirtuins.

Sirtuins (SIRT1-7) are a family of nicotine adenine dinucleotide (NAD+)-dependent enzymes that catalyze post-translational modifications of proteins. Together, they regulate crucial cellular functions and are traditionally associated with aging and longevity. Dysregulation of sirtuins plays an important role in major diseases, including cancer and metabolic, cardiac, and neurodegerative diseases. They are extensively regulated in response to a wide range of stimuli, including nutritional and metabolic challenges, inflammatory signals or hypoxic and oxidative stress. Each sirtuin is regulated individually in a tissue- and cell-specific manner. The control of sirtuin expression involves all the major points of regulation, including transcriptional and post-translational mechanisms and microRNAs. Collectively, these mechanisms control the protein levels, localization, and enzymatic activity of sirtuins. In many cases, the regulators of sirtuin expression are also their substrates, which lead to formation of intricate regulatory networks and extensive feedback loops. In this review, we highlight the mechanisms mediating the physiologic and pathologic regulation of sirtuin expression and activity. We also discuss the consequences of this regulation on sirtuin function and cellular physiology.-Buler, M., Andersson, U., Hakkola, J. Who watches the watchmen? Regulation of the expression and activity of sirtuins.

The peroxisome proliferator-activated receptor α agonist, AZD4619, induces alanine aminotransferase-1 gene and protein expression in human, but not in rat hepatocytes: Correlation with serum ALT levels.

Alanine aminotransferase (ALT) in serum is the standard biomarker for liver injury. We have previously described a clinical trial with a novel selective peroxisome proliferator-activated receptor α (PPARα) agonist (AZD4619), which unexpectedly caused increased serum levels of ALT in treated individuals without any other evidence of liver injury. We pinpointed a plausible mechanism through which AZD4619 could increase serum ALT levels; namely through the PPARα-specific activation of the human ALT1 gene at the transcriptional level. In the present study, we present data from the preceding rat toxicity study, demonstrating that AZD4619 had no effect on rat serum ALT activity levels, and further experiments were performed to elucidate the mechanisms responsible for this species-related difference. Our results revealed that AZD4619 increased ALT1 protein expression in a dose-dependent manner in human, but not in rat primary hepatocytes. Cloning of the human and rat ALT1 promoters into luciferase vectors confirmed that AZD4619 induced only the human, but not the rat ALT1 gene promoter in a dose-dependent manner. In PPARα-GAL4 reporter gene assays, AZD4619 was >100-fold more potent on the human vs. rat PPARα levels, explaining the differences in induction of the ALT1 gene between the species at the concentration range tested. These data demonstrate the usefulness of the human and rat ALT1 reporter gene assays for testing future drug candidates at the preclinical stage. In drug discovery projects, these assays elucidate whether elevations in ALT levels observed in vivo or in the clinic are due to metabolic effects rather than a toxic event in the liver.

SIRT5 is under the control of PGC-1α and AMPK and is involved in regulation of mitochondrial energy metabolism.

The sirtuins (SIRTs; SIRT1-7) are a family of NAD(+)-dependent enzymes that dynamically regulate cellular physiology. Apart from SIRT1, the functions and regulatory mechanisms of the SIRTs are poorly defined. We explored regulation of the SIRT family by 2 energy metabolism-controlling factors: peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK). Overexpression of PGC-1α in mouse primary hepatocytes increased SIRT5 mRNA expression 4-fold and also the protein in a peroxisome proliferator-activated receptor α (PPARα)- and estrogen-related receptor α (ERRα)-dependent manner. Furthermore, food withdrawal increased SIRT5 mRNA 1.3-fold in rat liver. Overexpression of AMPK in mouse hepatocytes increased expression of SIRT1, SIRT2, SIRT3, and SIRT6 <2-fold. In contrast, SIRT5 mRNA was down-regulated by 58%. The antidiabetes drug metformin (1 mM), an established AMPK activator, reduced the mouse SIRT5 protein level by 44% in cultured hepatocytes and by 31% in liver in vivo (300 mg/kg, 7 d). Metformin also induced hypersuccinylation of mitochondrial proteins. Moreover, SIRT5 overexpression increased ATP synthesis and oxygen consumption in HepG2 cells, but did not affect mitochondrial biogenesis. In summary, our results identified SIRT5 as a novel factor that controls mitochondrial function. Moreover, SIRT5 levels are regulated by PGC-1α and AMPK, which have opposite effects on its expression.-Buler, M., Aatsinki, S.-M., Izzi, V., Uusimaa, J., Hakkola, J. SIRT5 is under the control of PGC-1α and AMPK and is involved in regulation of mitochondrial energy metabolism.

Metformin induces PGC-1α expression and selectively affects hepatic PGC-1α functions.

BACKGROUND AND PURPOSE: The objective of this study was to determine how the AMPK activating antidiabetic drug metformin affects the major activator of hepatic gluconeogenesis, PPARγ coactivator 1α (PGC-1α) and liver functions regulated by PGC-1α. EXPERIMENTAL APPROACH: Mouse and human primary hepatocytes and mice in vivo were treated with metformin. Adenoviral overexpression, siRNA and reporter gene constructs were used for mechanistic studies. KEY RESULTS: Metformin increased PGC-1α mRNA and protein expression in mouse primary hepatocytes. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) (another AMPK activator) had the opposite effect. Metformin also increased PGC-1α in human primary hepatocytes; this effect of metformin was abolished by AMPK inhibitor compound C and sirtuin 1 siRNA. AMPK overexpression by AMPK-Ad also increased PGC-1α. Whereas metformin increased PGC-1α, it down-regulated gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Furthermore, metformin attenuated the increase in PEPCK and G6Pase mRNAs induced by PGC-1α overexpression, but did not affect PGC-1α-mediated induction of mitochondrial genes. Metformin down-regulated several key transcription factors that mediate the effect of PGC-1α on gluconeogenic genes including Krüppel-like factor 15, forkhead box protein O1 and hepatocyte NF 4α, whereas it increased nuclear respiratory factor 1, which is involved in PGC-1α-mediated regulation of mitochondrial proteins. CONCLUSIONS AND IMPLICATIONS: Down-regulation of PGC-1α is not necessary for suppression of gluconeogenic genes by metformin. Importantly, metformin selectively affects hepatic PGC-1α-mediated gene regulation and prevents activation of gluconeogenesis, but does not influence its regulation of mitochondrial genes. These results identify selective modulation of hepatic PGC-1α functions as a novel mechanism involved in the therapeutic action of metformin.

Poxvirus-based vaccines for cancer immunotherapy: new insights from combined cytokines/co-stimulatory molecules delivery and "uncommon" strains.

Poxvirus-based vaccines have a long record of efficacy as both anti-tumour agents and vectors for gene therapy in different human tumour models. Interestingly, several studies of these vaccines have now entered the clinical evaluation phase for safety and effectiveness. A desirable outcome of antigen specific cancer immunotherapy is the disruption of host self-tolerance against endogenous tumour-associated antigens (TAAs). Nonetheless, recent studies have found reductions in vaccine efficacy due to host anti-vaccine immune reactions. Thus, newer approaches bringing together poxvirus-based vaccination and immunostimulation are being developed, and new poxvirus strains are being examined in tumour therapy studies. Our review summarizes the current knowledge on the efficacy of poxvirus-based vaccination on human tumours, with a particular focus on approaches aimed at increasing innate and specific immune responses. Special attention will be devoted to the new poxvirus strains that are currently under consideration for tumour therapy; the current knowledge on clinical trials and outcomes will also be reviewed.

Metformin reduces hepatic expression of SIRT3, the mitochondrial deacetylase controlling energy metabolism.

Metformin inhibits ATP production in mitochondria and this may be involved in the anti-hyperglycemic effects of the drug. Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates the function of the electron transport chain and maintains basal ATP yield. We hypothesized that metformin treatment could diminish mitochondrial ATP production through downregulation of SIRT3 expression. Glucagon and cAMP induced SIRT3 mRNA in mouse primary hepatocytes. Metformin prevented SIRT3 induction by glucagon. Moreover, metformin downregulated constitutive expression of SIRT3 in primary hepatocytes and in the liver in vivo. Estrogen related receptor alpha (ERRα) mediates regulation of Sirt3 gene by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). ERRα mRNA expression was regulated in a similar manner as SIRT3 mRNA by glucagon, cAMP and metformin. However, a higher metformin concentration was required for downregulation of ERRα than SIRT3. ERRα siRNA attenuated PGC-1α mediated induction of SIRT3, but did not affect constitutive expression. Overexpression of the constitutively active form of AMP-activated protein kinase (AMPK) induced SIRT3 mRNA, indicating that the SIRT3 downregulation by metformin is not mediated by AMPK. Metformin reduced the hepatocyte ATP level. This effect was partially counteracted by SIRT3 overexpression. Furthermore, metformin decreased mitochondrial SIRT3 protein levels and this was associated with enhanced acetylation of several mitochondrial proteins. However, metformin increased mitochondrial mass in hepatocytes. Altogether, our results indicate that metformin attenuates mitochondrial expression of SIRT3 and suggest that this mechanism is involved in regulation of energy metabolism by metformin in the liver and may contribute to the therapeutic action of metformin.

Energy-sensing factors coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase control expression of inflammatory mediators in liver: induction of interleukin 1 receptor antagonist.

Obesity and insulin resistance are associated with chronic, low grade inflammation. Moreover, regulation of energy metabolism and immunity are highly integrated. We hypothesized that energy-sensitive coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK) may modulate inflammatory gene expression in liver. Microarray analysis revealed that PGC-1α up-regulated expression of several cytokines and cytokine receptors, including interleukin 15 receptor α (IL15Rα) and, even more importantly, anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). Overexpression of PGC-1α and induction of PGC-1α by fasting, physical exercise, glucagon, or cAMP was associated with increased IL1Rn mRNA and protein expression in hepatocytes. Knockdown of PGC-1α by siRNA down-regulated cAMP-induced expression of IL1Rn in mouse hepatocytes. Furthermore, knockdown of peroxisome proliferator-activated receptor α (PPARα) attenuated IL1Rn induction by PGC-1α. Overexpression of PGC-1α, at least partially through IL1Rn, suppressed interleukin 1ß-induced expression of acute phase proteins, C-reactive protein, and haptoglobin. Fasting and exercise also induced IL15Rα expression, whereas glucagon and cAMP resulted in reduction in IL15Rα mRNA levels. Finally, AMPK activator metformin and adenoviral overexpression of AMPK up-regulated IL1Rn and down-regulated IL15Rα in primary hepatocytes. We conclude that PGC-1α and AMPK alter inflammatory gene expression in liver and thus integrate energy homeostasis and inflammation. Induction of IL1Rn by PGC-1α and AMPK may be involved in the beneficial effects of exercise and caloric restriction and putative anti-inflammatory effects of metformin.

Energy sensing factors PGC-1α and SIRT1 modulate PXR expression and function.

The pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor plays a major role in regulation of drug metabolism but also modulates hepatic energy metabolism. PXR interacts with and represses several important transcription factors and coactivators regulating key enzymes in energy metabolism. Much less is known about how energy sensing cellular factors regulate PXR function. In this study we have investigated the effect of two major regulators of hepatic energy homeostasis, the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) and the NAD-dependent deacetylase protein, sirtuin 1 (SIRT1) on PXR expression and function. Fasting induces PXR expression in liver. Furthermore, glucagon and PGC-1α overexpression upregulate PXR expression level in mouse primary hepatocytes suggesting that PGC-1α, in addition to coactivation of PXR, also transcriptionally regulates PXR gene. Knockdown of peroxisome proliferator-activated receptor α by siRNA attenuates PGC-1α mediated induction of PXR mRNA. PGC-1α overexpression alone has no effect on cytochrome P450 (CYP) 3A11 expression but potentiates induction by pregnenolone-16α-carbonitrile (PCN). Pyruvate, a nutrient signal activating SIRT1 abolishes synergistic induction of CYP3A11 by PCN and PGC-1α. Knockdown of SIRT1 prevented this effect of pyruvate. Downregulation of CYP7A1 by PCN was not affected by PGC-1α or pyruvate. Mammalian two hydrid assays indicate that pyruvate and SIRT1 interfere with interaction of PXR and PGC-1α. This may be mediated by well established PGC-1α deacetylation by SIRT1. However, we show by immunoprecipitation that SIRT1 also interacts with PXR. Thus we show that two fasting activated pathways PGC-1α and SIRT1 differentially modify PXR expression and function.

Functional TLR9 modulates bone marrow B cells from rheumatoid arthritis patients.

TLR9 recognizes unmethylated CpG-rich, pathogen-derived DNA sequences and represents the component of the innate immune system that heavily influences adaptive immunity and may contribute to the immunological disturbances in rheumatoid arthritis (RA). Accumulating data indicate that BM of RA patients participates in the pathogenesis of this disease as a site of proinflammatory cytokines overproduction and lymphocytes activation. Here, we investigated the functionality of TLR9 and its role in the modulation of RA BM B-cell functions. We report that BM B cells isolated from RA patients express TLR9 at the mRNA and protein levels acquired at the stage of preB/immature B-cell maturation. Stimulation of BM CD20(+) B cells by CpG-containing oligodeoxynucleotide-enhanced expression of activation markers (CD86 and CD54) triggered IL-6 and TNF-alpha secretion and cell proliferation. Significantly higher levels of eubacterial DNA encoding 16S-rRNA were found in BM samples from RA than osteoarthritis patients. Moreover, RA BM B cells exerted higher expression of CD86 than their osteoarthritis counterparts, suggesting their in situ activation via TLR9. Thus, our data indicate that TLR9 may participate in direct activation and proliferation of B cells in BM, and therefore could play a role in the pathogenesis of RA.