Marcin Buler

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. FASEB J. 28, 3225–3237 (2014). www.fasebj.org

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

Background: Metabolic disorders are associated with chronic inflammation. Results: Energy-sensing factor PGC-1α regulates cytokine expression in hepatocytes. PGC-1α, AMPK, and metformin induce expression of interleukin 1 receptor antagonist. Conclusion: PGC-1α and AMPK mediate effects of fasting, physical exercise, and antidiabetic drug metformin on hepatic inflammatory gene expression. Significance: PGC-1α and AMPK are regulatory interlinks between energy homeostasis and inflammation. 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 1β-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.

Pregnane X Receptor Agonists Impair Postprandial Glucose Tolerance

We conducted a randomized, open, placebo‐controlled crossover trial to investigate the effects of the pregnane X receptor (PXR) agonist rifampin on an oral glucose tolerance test (OGTT) in 12 healthy volunteers. The subjects were administered 600 mg rifampin or placebo once daily for 7 days, and OGTT was performed on the eighth day. The mean incremental glucose and insulin areas under the plasma concentration–time curves (AUCincr) increased by 192% (P = 0.008) and 45% (P = 0.031), respectively. The fasting glucose, insulin, and C‐peptide, and the homeostasis model assessment for insulin resistance, were not affected. The glucose AUCincr during OGTT was significantly increased in rats after 4‐day treatment with pregnenolone 16α‐carbonitrile (PCN), an agonist of the rat PXR. The hepatic level of glucose transporter 2 (Glut2) mRNA was downregulated by PCN. In conclusion, both human and rat PXR agonists elicited postprandial hyperglycemia, suggesting a detrimental role of PXR activation on glucose tolerance.

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.

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 andmetabolic, cardiac, andneurodegerativediseases. Theyare extensivelyregulatedinresponse 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 themajor points of regulation, including transcriptional and post‐translationalmechanisms 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 themechanismsmediating thephysiologic andpathologic regulation of sirtuin expressionandactivity. We alsodiscuss 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. FASEB J. 30, 3942–3960 (2016). www.fasebj.org

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

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.

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.

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.

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

Graphical abstract Figure. No caption available. ABSTRACT 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‐16&agr;‐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.