Valerio Izzi

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

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.

Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition

Significance A previously unrecognized role is described for collagen XVIII—a ubiquitous, structurally complex basement membrane proteoglycan—in supporting preadipocyte differentiation and the maintenance of this differentiated state, and hence the size and lipid-clearing/storage functions of white adipose tissue depots. Specific lack of medium and long isoforms of this nonfibrillar collagen in mice led to reduced adiposity, ectopic deposition of fat in liver, and elevated very low-density lipoprotein-triglyceride levels. The finding of a previously unidentified extracellular mechanism contributing to control of adipogenesis is expected to promote understanding of the molecular and functional bases of human hyperlipidemic syndromes associated with fatty liver. Collagen XVIII is an evolutionary conserved ubiquitously expressed basement membrane proteoglycan produced in three isoforms via two promoters (P). Here, we assess the function of the N-terminal, domain of unknown function/frizzled-like sequences unique to medium/long collagen XVIII by creating P-specific null mice. P2-null mice, which only produce short collagen XVIII, developed reduced bulk-adiposity, hepatic steatosis, and hypertriglyceridemia. These abnormalities did not develop in P1-null mice, which produce medium/long collagen XVIII. White adipose tissue samples from P2-null mice contain larger reserves of a cell population enriched in early adipocyte progenitors; however, their embryonic fibroblasts had ∼50% lower adipocyte differentiation potential. Differentiating 3T3-L1 fibroblasts into mature adipocytes produced striking increases in P2 gene-products and dramatic falls in P1-transcribed mRNA, whereas Wnt3a-induced dedifferentiation of mature adipocytes produced reciprocal changes in P1 and P2 transcript levels. P2-derived gene-products containing frizzled-like sequences bound the potent adipogenic inhibitor, Wnt10b, in vitro. Previously, we have shown that these same sequences bind Wnt3a, inhibiting Wnt3a-mediated signaling. P2-transcript levels in visceral fat were positively correlated with serum free fatty acid levels, suggesting that collagen α1 (XVIII) expression contributes to regulation of adipose tissue metabolism in visceral obesity. Medium/long collagen XVIII is deposited in the Space of Disse, and interaction between hepatic apolipoprotein E and this proteoglycan is lost in P2-null mice. These results describe a previously unidentified extracellular matrix-directed mechanism contributing to the control of the multistep adipogenic program that determines the number of precursors committing to adipocyte differentiation, the maintenance of the differentiated state, and the physiological consequences of its impairment on ectopic fat deposition.

Bone marrow adipocytes support haematopoietic stem cell survival

In bone marrow (BM), hematopoietic elements are mingled with adipocytes (BM‐A), which are the most abundant stromal component in the niche. BM‐A progressively increase with aging, eventually occupying up to 50% of BM cavities. In this work, the role played by BM‐A was explored by studying primary human BM‐A isolated from hip surgery patients at the molecular level, through microarray analysis, and at the functional level, by assessing their relationship with primary human hematopoietic stem cells (HSC) by the long‐term culture initiating cell (LTC‐IC) assay. Findings demonstrated that BM‐A are capable of supporting HSC survival in the LTC‐IC assay, since after 5 weeks of co‐culture, HSC were still able to proliferate and differentiate. Furthermore, critical molecules such as C‐X‐C motif chemokine 12 (CXCL12), interleukin (IL)‐8, colony‐stimulating factor 3 (CSF3), and leukaemia inhibitory factor (LIF), were expressed at similar levels in BM‐A and in primary human BM mesenchymal stromal cells (BM‐MSC), whereas IL‐3 was higher in BM‐A. Interestingly, BM‐A displayed a different gene expression profile compared with subcutaneous adipose tissue adipocytes (AT‐A) collected from abdominal surgery patients, especially in terms of regulation of lipid metabolism, stemness genes, and white‐to‐brown differentiation pathways. Accordingly, analysis of the gene pathways involved in hematopoiesis regulation showed that BM‐A are more closely related to BM‐MSC than to AT‐A. The present data suggest that BM‐A play a supporting role in the hematopoietic niche and directly sustain HSC survival.

Human white adipocytes convert into "rainbow" adipocytes in vitro

White adipocytes are plastic cells able to reversibly transdifferentiate into brown adipocytes and into epithelial glandular cells under physiologic stimuli in vivo. These plastic properties could be used in future for regenerative medicine, but are incompletely explored in their details. Here, we focused on plastic properties of human mature adipocytes (MA) combining gene expression profile through microarray analysis with morphologic data obtained by electron and time lapse microscopy. Primary MA showed the classic morphology and gene expression profile of functional mature adipocytes. Notably, despite their committed status, MA expressed high levels of reprogramming genes. MA from ceiling cultures underwent transdifferentiation toward fibroblast‐like cells with a well‐differentiated morphology and maintaining stem cell gene signatures. The main morphologic aspect of the transdifferentiation process was the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion further supported the liposecretion process. Of note, electron microscope findings suggesting liposecretion phenomena were found also in explants of human fat and rarely in vivo in fat biopsies from obese patients. In conclusion, both MA and post‐liposecretion adipocytes show a well‐differentiated phenotype with stem cell properties in line with the extraordinary plasticity of adipocytes in vivo. J. Cell. Physiol. 232: 2887–2899, 2017.

Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

Hypoxia-inducible factor 1α (HIF1α) induces the expression of several hundred genes in hypoxia aiming at restoration of oxygen homeostasis. HIF prolyl-4-hydroxylases (HIF-P4Hs) regulate the stability of HIF1α in an oxygen-dependent manner. Hypoxia is a common feature in inflammation and cancer and the HIF pathway is closely linked with the inflammatory NF-κB and tumor suppressor p53 pathways. Here we show that genetic inactivation or chemical inhibition of HIF-P4H-1 leads to downregulation of proinflammatory genes, while proapoptotic genes are upregulated. HIF-P4H-1 inactivation reduces the inflammatory response under LPS stimulus in vitro and in an acute skin inflammation model in vivo. Furthermore, HIF-P4H-1 inactivation increases p53 activity and stability and hydroxylation of proline 142 in p53 has an important role in this regulation. Altogether, our data suggest that HIF-P4H-1 inhibition may be a promising therapeutic candidate for inflammatory diseases and cancer, enhancing the reciprocal negative regulation of the NF-κB and p53 pathways.

Notch Downregulation and Extramedullary Erythrocytosis in Hypoxia-Inducible Factor Prolyl 4-Hydroxylase 2-Deficient Mice

ABSTRACT Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.

An extracellular matrix signature in leukemia precursor cells and acute myeloid leukemia

Despite major therapeutic advancements, worldwide death rates of acute myeloid leukemia (AML) remain high, with approximately 20,830 people diagnosed in the USA in 2015, 10,460 (50.21%) of whom were estimated to die from the disease.[1][1] Several studies have shown that leukemia stem cells (LSCs),

Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors

Vascular endothelial growth factor D (VEGF-D) promotes the lymph node metastasis of cancer by inducing the growth of lymphatic vasculature, but its specific roles in tumorigenesis have not been elucidated. We monitored the effects of VEGF-D in cutaneous squamous cell carcinoma (cSCC) by subjecting transgenic mice overexpressing VEGF-D in the skin (K14-mVEGF-D) and VEGF-D knockout mice to a chemical skin carcinogenesis protocol involving 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate treatments. In K14-mVEGF-D mice, tumor lymphangiogenesis was significantly increased and the frequency of lymph node metastasis was elevated in comparison with controls. Most notably, the papillomas regressed more often in K14-mVEGF-D mice than in littermate controls, resulting in a delay in tumor incidence and a remarkable reduction in the total tumor number. Skin tumor growth and metastasis were not obviously affected in the absence of VEGF-D; however, the knockout mice showed a trend for reduced lymphangiogenesis in skin tumors and in the untreated skin. Interestingly, K14-mVEGF-D mice showed an altered immune response in skin tumors. This consisted of the reduced accumulation of macrophages, mast cells, and CD4+ T-cells and an increase of cytotoxic CD8+ T-cells. Cytokine profiling by flow cytometry and quantitative real time PCR revealed that elevated VEGF-D expression results in an attenuated Th2 response and promotes M1/Th1 and Th17 polarization in the early stage of skin carcinogenesis, leading to an anti-tumoral immune environment and the regression of primary tumors. Our data suggest that VEGF-D may be beneficial in early-stage tumors since it suppresses the pro-tumorigenic inflammation, while at later stages VEGF-D-induced tumor lymphatics provide a route for metastasis.

Understanding the extracellular matrix in acute myeloid leukemia

Despite the continuous progress in therapy, acute myeloid leukemia (AML) remains the leading cause of death among hematologic neoplasms.[1][1] Understanding how leukemic clones arise and how they interact with the microenvironment of the bone marrow, especially within the hematopoietic stem cell (