Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1.

Sirtuin 2 (Sirt2) is known to negatively regulate anoxia-reoxygenation injury in myoblasts. Because protein levels of Sirt2 are increased in ischemia-reperfusion (I/R)-injured liver tissues, we examined whether Sirt2 is protective or detrimental against hepatic I/R injury. We overexpressed Sirt2 in the liver of C57BL/6 mice using a Sirt2 adenovirus. Wild-type and Sirt2 knockout mice were subjected to a partial (70%) hepatic ischemia for 45 minutes, followed by various periods of reperfusion. In another set of experiments, wild-type mice were pretreated intraperitoneally with AGK2, a Sirt2 inhibitor. Isolated hepatocytes and Kupffer cells from wild-type and Sirt2 knockout mice were subjected to hypoxia-reoxygenation injury to determine the in vitro effects of Sirt2. Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Prior injection with Sirt2 adenovirus aggravated liver injury, as demonstrated by increases in serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration relative to control virus-injected mice. Pretreatment with AGK2 resulted in significant improvements in serum aminotransferase levels and histopathologic findings. Similarly, experiments with Sirt2 knockout mice also revealed reduced hepatocellular injury. The molecular mechanism of Sirt2's involvement in this aggravation of hepatic I/R injury includes the deacetylation and inhibition of mitogen-activated protein kinase phosphatase-1 and consequent activation of mitogen-activated protein kinases. CONCLUSION: Sirt2 is an aggravating factor during hepatic I/R injury. (Hepatology 2017;65:225-236).

Hepatocyte-specific sirtuin 6 deletion predisposes to nonalcoholic steatohepatitis by up-regulation of Bach1, an Nrf2 repressor.

Sirtuin (Sirt)6 has been implicated in negative regulation of inflammation and lipid metabolism, although its function in the progression from simple steatosis to nonalcoholic steatohepatitis (NASH) remains to be defined. To explore the role of hepatocyte Sirt6 in NASH development, we generated hepatocyte-specific Sirt6-knockout (KO) mice that were fed a high-fat and high-fructose (HFHF) diet for 16 wk. HFHF-fed KO mice had increased hepatic steatosis and inflammation and aggravated glucose intolerance and insulin resistance compared with wild-type mice. HFHF-induced liver fibrosis and oxidative stress and related gene expression were significantly elevated in KO mice. In the livers of KO mice, nuclear factor erythroid 2-related factor 2 (Nrf2) was down-regulated; conversely, BTB domain and CNC homolog 1 (Bach1), a nuclear repressor of Nrf2, were up-regulated. We discovered that Sirt6, which interacts with Bach1 under basal condition, induces its detachment from the antioxidant response element (ARE) region of heme oxygenase 1 promoter. Furthermore, we found that Sirt6 promotes Nrf2 binding to ARE in response to oxidative stimuli, which leads to the expression of phase II/antioxidant enzymes. Finally, we showed that HFHF-induced steatosis, inflammation, and fibrosis were ameliorated by adenoviral Sirt6 overexpression. Sirt6 may be a useful therapeutic target for amelioration of NASH by curbing inflammation and oxidative stress.-Ka, S.-O, Bang, I. H., Bae, E. J., Park, B.-H. Hepatocyte-specific sirtuin 6 deletion predisposes to nonalcoholic steatohepatitis by up-regulation of Bach1, an Nrf2 repressor.

Metformin and AICAR regulate NANOG expression via the JNK pathway in HepG2 cells independently of AMPK.

NANOG, a marker of stemness, impacts tumor progression and therapeutic resistance in cancer cells. In human hepatocellular carcinoma (HCC), upregulation of NANOG is associated with metastasis and a low survival rate, while its downregulation results in a lower colony formation rate and enhanced chemosensitivity. Metformin, an agent widely used for diabetes treatment, and AICAR, another AMP-activated protein kinase (AMPK) activator, have been reported to inhibit the growth of several types of cancer. Although inhibitory effects of metformin on NANOG in pancreatic cancer cells and of AICAR in mouse embryonic stem cells have been described, the underlying molecular mechanisms remain uncertain in HCC. In this study, we used the HepG2 cell line and found that metformin/AICAR downregulated NANOG expression with decreased cell viability and enhanced chemosensitivity to 5-fluorouracil (5-FU). Moreover, metformin/AICAR inhibited c-Jun N-terminal kinase (JNK) activity, and blockade of either the JNK MAPK pathway or knockdown of JNK1 gene expression reduced NANOG levels. The upregulation of NANOG and phospho-JNK by basic fibroblast growth factor (bFGF) was abrogated by metformin/AICAR. Additionally, although transient upregulation of NANOG within 2 h of treatment with metformin/AICAR was concordant with both JNK and AMPK activation, increased NANOG expression with activation of JNK was also observed following AMPK inhibition with compound C. Taken together, our data suggest that metformin/AICAR regulate NANOG expression via the JNK MAPK pathway in HepG2 cells independently of AMPK, and that this JNK/NANOG signaling pathway may offer new therapeutic strategies for the treatment of HCC.

Glucocorticoid Suppresses Connexin 43 Expression by Inhibiting the Akt/mTOR Signaling Pathway in Osteoblasts.

The inhibition of proliferation or functional alteration of osteoblasts by glucocorticoids (GCs) has been recognized as an important etiology of GC-induced osteoporosis (GIO). Connexin 43 (Cx43) is the most abundant connexin isoform in bone cells and plays important roles in bone remodeling. Despite the important role of Cx43 in bone homeostasis and the prevalence of GIO, the direct action of GCs on Cx43 expression in osteoblasts has been poorly described. The aim of the present study was to evaluate how GCs affect Cx43 expression in osteoblasts. Dexamethasone (Dex) treatment decreased expression of Cx43 RNA and protein in MC3T3-E1 mouse osteoblastic cells. Reduction of Cx43 expression by Dex was dependent on the glucocorticoid receptor (GR), as it was abolished by pretreatment with a GR blocker. Treatment with PTH (1-34), a medication used for GIO management, counteracted the suppression of Cx43 by Dex. Akt or mTOR signaling modulators revealed the involvement of the Akt/mTOR signaling pathway in Dex-induced reduction of Cx43 expression. Moreover, overexpression of Cx43 significantly attenuated Dex-inhibited cell viability and proliferation, as evidenced by MTT and bromodeoxyuridine (BrdU) incorporation assay of MC3T3-E1 cells. To account for possible species or cell type differences, human primary osteoblasts were treated with Dex and similar downregulation of Cx43 by Dex was observed. In addition, immunofluorescent staining for Cx43 further demonstrated an apparent decrease in Dex-treated human osteoblasts, while analysis of lucifer yellow propagation revealed reduced gap junction intercellular communication by Dex. Collectively, these findings indicate that GCs suppress Cx43 expression in osteoblasts via GR and the Akt/mTOR signaling pathway and overexpression of Cx43 may, at least in part, rescue osteoblasts from GC-induced reductions in proliferation.

Overexpression of sirtuin 6 suppresses inflammatory responses and bone destruction in mice with collagen-induced arthritis.

OBJECTIVE: Sirtuin 6 (SIRT-6) is an NAD(+) -dependent deacetylase and mono-ADP-ribosyltransferase. It is known to interfere with the NF-κB signaling pathway and thereby has an antiinflammatory function. Due to the central role of NF-κB in rheumatoid arthritis (RA) development, we undertook this study to test our hypothesis that SIRT-6 could have antiarthritic effects. METHODS: An adenovirus containing SIRT-6 complementary DNA (Ad-SIRT6) was used to deliver SIRT-6 to human RA fibroblast-like synoviocytes in vitro as well as to mice with collagen-induced arthritis (CIA) in vivo via bilateral intraarticular injections into the ankle joints. RESULTS: In vitro experiments demonstrated that SIRT-6 overexpression suppressed NF-κB target gene expression induced by tumor necrosis factor α. SIRT-6 overexpression inhibited osteoclast differentiation induced by macrophage colony-stimulating factor and RANKL in bone marrow-derived macrophages. Mice with CIA had an increased incidence of disease and developed arthritis in the hind paws. In contrast, mice injected with Ad-SIRT6 showed attenuated severity of arthritis based on clinical scores, hind paw thickness, and radiographic and pathologic findings. Moreover, the injection of Ad-SIRT6 down-regulated local and systemic levels of proinflammatory cytokines. After induction of CIA, mice injected with Ad-SIRT6 showed significantly decreased arthritis severity, from the onset of clinical signs to the end of the study. CONCLUSION: These results suggest that blocking the NF-κB pathway by SIRT-6 in rheumatoid joints reduces both the inflammatory response and tissue destruction. Therefore, the development of an immunoregulatory strategy based on SIRT-6 may have therapeutic potential for the treatment of RA.

Silibinin attenuates adipogenesis in 3T3-L1 preadipocytes through a potential upregulation of the insig pathway.

In the past few decades, the use of silibinin, a plant flavonoid extracted from the milk thistle, as a hepatoprotective and chemopreventive agent has gained much attention. In this study, we investigated the effects of silibinin on adipogenesis. Treatment with silibinin suppressed terminal differentiation of 3T3-L1 cells into adipocytes as evidenced by Oil red O staining and TG assay results. Real-time RT-PCR analysis revealed that silibinin decreased the expression of adipogenesis-related genes such as CAAT/enhancer binding protein-alpha, fatty acid synthase, sterol response element binding protein 1c, adipocyte-specific lipid binding protein, peroxisome proliferator-activated receptor gamma and lipoprotein lipase, and increased the expression of preadipocyte factor-1, a preadipocyte marker gene. The anti-adipogenic effect of silibinin was associated with the up-regulation of insig-1 and insig-2. Collectively, these results suggest that silibinin inhibits adipocyte differentiation through a potential up-regulation of insig-1 and insig-2 at an early phase in adipocyte differentiation.

Inhibitory Effect of Sestrin 2 on Hepatic Stellate Cell Activation and Liver Fibrosis.

Aims: Hepatic fibrosis results from chronic liver injury and inflammatory responses. Sestrin 2 (Sesn2), an evolutionarily conserved antioxidant enzyme, reduces the severities of acute hepatitis and metabolic liver diseases. However, the role of Sesn2 in the pathogenesis of liver fibrosis remains obscure. Here, we used cultured hepatic stellate cells (HSCs) and chronic carbon tetrachloride (CCl4) and bile duct ligation (BDL) murine models to investigate the effects of Sesn2 on fibrogenesis. Results: Sesn2 protein and mRNA levels were upregulated in activated primary HSCs, and by increasing transcription, transforming growth factor-ß (TGF-ß) also increased Sesn2 expression in HSCs. Furthermore, Smad activation was primarily initiated by TGF-ß signaling, and Smad3 activation increased Sesn2 luciferase activity. In silico analysis of the 5' upstream region of the Sesn2 gene revealed a putative Smad-binding element (SBE), and its deletion demonstrated that the SBE between -964 and -956 bp within human Sesn2 promoter was critically required for TGF-ß-mediated response. Moreover, ectopic expression of Sesn2 reduced gene expressions associated with HSC activation, and this was accompanied by marked decreases in SBE luciferase activity and Smad phosphorylation. Infection of recombinant adenovirus Sesn2 reduced hepatic injury severity, as evidenced by reductions in CCl4- or BDL-induced alanine aminotransferase and aspartate aminotransferase, and inhibited collagen accumulation. Furthermore, HSC-specific lentiviral delivery of Sesn2 prevented CCl4-induced liver fibrosis. Finally, Sesn2 expression was downregulated in the livers of patients with liver cirrhosis and in mouse models of hepatic fibrosis. Innovation and Conclusion: Our findings suggest that Sesn2 has the potential to inhibit HSC activation and hepatic fibrosis.

Dihydropyranoaurone compound damaurone D inhibits LPS-induced inflammation and liver injury by inhibiting NF-κB and MAPK signaling independent of AMPK.

Recently, we reported the synthesis of damaurone D (DD), originally derived from Rosa damascene, and its anti-inflammatory effect in macrophages. Here, we investigated the molecular mechanism underlying the anti-inflammatory effect of DD in macrophages and further tested whether DD is protective against lipopolysaccharide (LPS)-induced liver injury. DD inhibited LPS-stimulated expression of pro-inflammatory genes and cytokine/chemokine secretion in a concentration-dependent manner in RAW 264.7 cells and thioglycolate-elicited mouse peritoneal macrophages. DD suppressed LPS-stimulated nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, as demonstrated by reduction in IκB kinase α/ß phosphorylation, IκBα degradation, and levels of phosphorylated ERK, JNK, and p38 MAPK. The luciferase reporter activity of NF-κB and activator protein 1 was also attenuated by DD pretreatment. Furthermore, DD treatment induced AMP-activated protein kinase (AMPK) activation in cells and mouse liver, although the anti-inflammatory effect of DD was similar in dominant-negative AMPK-overexpressing cells. Lastly, DD-treated mice were protected against LPS-induced acute liver injury, based on morphologic and immunohistochemical observations; reduction in the plasma levels of aspartate aminotransferase, TNF-α, and MCP-1; and a decrease in inflammatory gene expression. In summary, our findings indicate that DD can protect against LPS-stimulated inflammation and liver injury at least partly by suppression of NF-κB and MAPK signaling pathways.

Genistein protects pancreatic beta cells against cytokine-mediated toxicity.

In the past few decades, the use of genistein as an anti-inflammatory agent has gained much attention. Our current study focuses on the preventive effects of genistein on cytokine-induced pancreatic beta-cell damage. Treatment of RINm5F (RIN) rat insulinoma cells with interleukin (IL)-1beta and interferon (IFN)-gamma induced cell damage, which was correlated with nitric oxide (NO) production. Genistein completely prevented cytokine-mediated cytotoxicity and NO production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism of genistein inhibition of iNOS gene expression appeared to involve the inhibition of NFkappaB activation. The cytokine induced increases in NFkappaB binding activity, nuclear p50 and p65 subunit levels, and IkappaBalpha degradation in cytosol compared to unstimulated cells; genistein abolished all of these parameters. The cytoprotective effects of genistein are also mediated through the suppression of ERK-1/2 and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. In a second set of experiments, rat islets were used. The findings on beta-cell protective effects of genistein were essentially the same as for the RIN cell data, namely genistein prevented cytokine-induced NO production, iNOS expression, ERK-1/2 activation, JAK/STAT activation, and impairment of glucose-stimulated insulin secretion. Collectively, these results suggest that genistein might be used to preserve functional beta-cell mass.

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic beta-cells through suppression of NF-kappaB activation.

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in nucleus, and IkappaB alpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.

Diallyl disulfide accelerates adipogenesis in 3T3-L1 cells.

Adipocyte differentiation is regulated by the sequential activation of transcription factors such as the CAAT/enhancer binding protein and peroxisome proliferator- activated receptor-gamma (PPAR-gamma). Several recent studies have shown that regulators of chromatin structure are also involved in adipocyte differentiation. Here we investigated the effects of diallyl disulfide (DADS), an oil-soluble sulfur compound found in processed garlic and an inhibitor of histone deacetylase (HDAC), on adipogenesis. Treatment with DADS accelerated terminal differentiation of 3T3-L1 cells into adipocytes as evidenced by Oil red O staining and cellular triglyceride assay results. Notably, the inhibition of HDAC during the first 2 days was sufficient to stimulate adipogenesis. Western blot analysis revealed that DADS increased the level of acetylated histones H3 and H4. In addition, DADS increased the expression of adipogenesis-related genes; LPL, FAS, SREBP1c, aP2 and PPAR-gamma, and decreased the expression of pref-1, a preadipocyte marker gene. Taken together, our results suggest that DADS affects adipocyte differentiation through histone acetylation at an early phase of adipocyte differentiation.

Butein induction of HO-1 by p38 MAPK/Nrf2 pathway in adipocytes attenuates high-fat diet induced adipose hypertrophy in mice.

Adipose tissue inflammation and oxidative stress are key components in the development of obesity and insulin resistance. Heme oxygenase (HO)-1 in adipocytes protects against obesity and adipose dysfunction. In this study, we report the identification of butein, a flavonoid chalcone, as a novel inducer of HO-1 expression in adipocytes in vitro and in vivo. Butein upregulated HO-1 mRNA and protein expression in 3T3-L1 adipocytes, accompanied by Kelch-Like ECH-Associated Protein (Keap) 1 degradation and increase in the nuclear level of nuclear factor erythroid 2-related factor 2 (Nrf2). Butein modulation of Keap1 and Nrf2 as well as HO-1 upregulation was reversed by pretreatment with p38 MAPK inhibitor SB203580, indicating the involvement of p38 MAPK in butein activation of Nrf2 in adipocytes. In addition, HO-1 activation by butein led to the inhibitions of reactive oxygen species and adipocyte differentiation, as evidenced by the fact that butein repression of reactive oxygen species and adipogenesis was reversed by pretreatment with HO-1 inhibitor SnPP. Induction of HO-1 expression by butein was also demonstrated in the adipose tissue of C57BL/6 mice fed a high-fat diet administered along with butein for three weeks, and correlated with the inhibitions of adiposity and adipose tissue inflammation, which were reversed by co-administration of SnPP. Altogether, our results demonstrate that butein activates the p38 MAPK/Nrf2/HO-1 pathway to act as a potent inhibitor of adipose hypertrophy and inflammation in a diet-induced obesity model and thus has potential for suppressing obesity-linked metabolic syndrome.

Myeloid Sirtuin 6 Deficiency Causes Insulin Resistance in High-Fat Diet-Fed Mice by Eliciting Macrophage Polarization Toward an M1 Phenotype.

Obesity-related insulin resistance is closely associated with macrophage accumulation and subsequent cytokine release in local tissues. Sirtuin 6 (Sirt6) is known to exert an anti-inflammatory function, but its role in macrophages in the context of obesity has not been investigated. We generated myeloid-specific Sirt6 knockout (mS6KO) mice and investigated the metabolic characteristics after high-fat diet (HFD) feeding for 16 weeks. Compared with their wild-type littermates, HFD-fed mS6KO mice exhibited greater increases in body weight, fasting blood glucose and insulin levels, hepatic steatosis, glucose intolerance, and insulin resistance. Gene expression, histology, and flow cytometric analyses demonstrated that liver and adipose tissue inflammation were elevated in HFD-fed mS6KO mice relative to wild type, with a greater accumulation of F4/80+CD11b+CD11c+ adipose tissue macrophages. Myeloid Sirt6 deletion facilitated proinflammatory M1 polarization of bone marrow macrophages and augmented the migration potential of macrophages toward adipose-derived chemoattractants. Mechanistically, Sirt6 deletion in macrophages promoted the activation of nuclear factor-κB (NF-κB) and endogenous production of interleukin-6, which led to STAT3 activation and the positive feedback circuits for NF-κB stimulation; this cross talk expedited an M1 polarization. We conclude that Sirt6 in macrophages is required for the prevention of obesity-associated tissue inflammation and insulin resistance.

Insulin secretion impairment in Sirt6 knockout pancreatic ß cells is mediated by suppression of the FoxO1-Pdx1-Glut2 pathway.

Sirtuin 6 (Sirt6), a chromatin associated class III deacetylase, controls whole-body energy homeostasis and has a critical role in glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells. However, its underlying molecular mechanism remains poorly understood. To gain further insights, we studied the pathway by which Sirt6 regulates GSIS utilizing mice lacking Sirt6 in their ß cells (ßS6KO). Further, we overexpressed wild type or deacetylase-inactive mutant Sirt6 in isolated islets as well as in MIN6 cells. We confirmed that ßS6KO mice developed glucose intolerance with severely impaired GSIS. Gene expression analysis of knockout islets and overexpression studies demonstrated that Sirt6 deacetylates forkhead box protein O1 (FoxO1) to trigger its nuclear export and releases its transcriptional repression of key glucose sensing genes such as Pdx1 and Glut2. Ectopic overexpression of Sirt6 in knockout islets resulted in rescue of the defective insulin secretion and restoration of the expression of Pdx1 and Glut2. These results show that Sirt6 in pancreatic ß cells deacetylates FoxO1 and subsequently increases the expression of Pdx1 and Glut2 to maintain the glucose-sensing ability of pancreatic ß cells and systemic glucose tolerance.

Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability.

Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-ß signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4Δ(Os) mice, in which Smad4 was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4Δ(Os) mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5'-bromo-2'deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4Δ(Os) mice. Apoptosis in isolated calvaria cells from Smad4Δ(Os) mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4Δ(Os) mice. Conversely, osteoblast cells overexpressing Smad4 showed increased apoptosis. In an apoptosis induction model of Smad4Δ(Os) mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.

Hepatic role in an early glucose-lowering effect by a novel dipeptidyl peptidase 4 inhibitor, evogliptin, in a rodent model of type 2 diabetes.

Although multiple dipeptidyl peptidase 4 (DPP4) inhibitors have shown glucose-lowering effects by preserving pancreatic cells in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, the hepatic role in regulation of glucose homeostasis by DPP4 inhibitors in HFD/STZ mice remains elusive. In herein study, parallel comparison of effects on the liver (expression of gluconeogenic genes and the linked signaling molecules) and pancreas (islet morphology and relative area of alpha or beta cells) in combination with glucose-lowering effects were made at the end of 2- and 10-week of evogliptin treatment in HFD/STZ mice. Significant control of hyperglycemia was observed from the second week and persisted during 10-week treatment of 0.3% evogliptin in HFD/STZ mice. This effect was accompanied by increased level of plasma glucagon-like peptide-1 and preserved pancreas islet structure. Furthermore, the hepatic increases in gluconeogenic gene expression in HFD/STZ mice was significantly reduced by evogliptin treatment, which was accompanied by the suppression of cAMP response element-binding protein (CREB) phosphorylation and expression of transducer of regulated CREB protein 2. This hepatic effect of evogliptin treatment was reproduced in 2-week study, however, pancreatic beta-cell area was not altered yet although the expression of pancreatic and duodenal homeobox protein 1 was increased. We conclude that the suppression of hepatic gluconeogenesis by evogliptin is followed by preservation of pancreatic islet, leading to remarkable and persistent glucose-lowering effect in HFD/STZ mice. Our findings provide further insight for the hepatic role in DPP4 inhibitor-mediated glucose control in diabetes.

Myeloid SIRT1 regulates macrophage infiltration and insulin sensitivity in mice fed a high-fat diet.

Inflammation is an important factor in the development of insulin resistance. SIRT1, a class 3 histone/protein deacetylase, has anti-inflammatory functions. Myeloid-specific deletion of Sirt1 promotes macrophage infiltration into insulin-sensitive organs and aggravates tissue inflammation. In this study, we investigated how SIRT1 in macrophages alters tissue inflammation in the pancreas as well as liver and adipose tissue, and further explored the role of SIRT1 in locomotion of macrophages. Myeloid-specific Sirt1-deleted mice (mS1KO) and WT littermates were fed a 60% calorie high-fat diet (HFD) for 16 weeks. Tissue inflammation and metabolic phenotypes were compared. Bone marrow macrophages (BMMs) from WT or mS1KO mice were used in in vitro chemotaxis assays and macrophage polarization studies. mS1KO mice fed a HFD exhibited glucose intolerance, reduced insulin secretion, and insulin sensitivity with a slight decrease in body weight. Consistent with these results, pancreatic islets of mS1KO mice fed a HFD displayed decreased mass with profound apoptotic cell damage and increased macrophage infiltration and inflammation. Liver and adipose tissues from mS1KO HFD mice also showed greater accumulation of macrophages and tissue inflammation. Results from in vitro experiments indicated that deletion of myeloid Sirt1 stimulated proinflammatory M1-like polarization of BMMs and augmented the adipocyte-mediated macrophage chemotaxis. The latter effect was accompanied by increased expression and acetylation of focal adhesion kinase, as well as nuclear factor kappa B. Our results indicate that myeloid SIRT1 plays a crucial role in macrophage polarization and chemotaxis, and thus regulates the development of HFD-induced pancreatic inflammation and insulin secretion, and metabolic derangements in liver and adipose tissue.

The new 4-O-methylhonokiol analog GS12021 inhibits inflammation and macrophage chemotaxis: role of AMP-activated protein kinase α activation.

Preventing pathologic tissue inflammation is key to treating obesity-induced insulin resistance and type 2 diabetes. Previously, we synthesized a series of methylhonokiol analogs and reported that compounds with a carbamate structure had inhibitory function against cyclooxygenase-2 in a cell-free enzyme assay. However, whether these compounds could inhibit the expression of inflammatory genes in macrophages has not been investigated. Here, we found that a new 4-O-methylhonokiol analog, 3',5-diallyl-4'-methoxy-[1,1'-biphenyl]-2-yl morpholine-4-carboxylate (GS12021) inhibited LPS- or TNFα-stimulated inflammation in macrophages and adipocytes, respectively. LPS-induced phosphorylation of nuclear factor-kappa B (NF-κB)/p65 was significantly decreased, whereas NF-κB luciferase activities were slightly inhibited, by GS12021 treatment in RAW 264.7 cells. Either mitogen-activated protein kinase phosphorylation or AP-1 luciferase activity was not altered by GS12021. GS12021 increased the phosphorylation of AMP-activated protein kinase (AMPK) α and the expression of sirtuin (SIRT) 1. Inhibition of mRNA expression of inflammatory genes by GS12021 was abolished in AMPKα1-knockdown cells, but not in SIRT1 knockout cells, demonstrating that GS12021 exerts anti-inflammatory effects through AMPKα activation. The transwell migration assay results showed that GS12021 treatment of macrophages prevented the cell migration promoted by incubation with conditioned medium obtained from adipocytes. GS12021 suppression of p65 phosphorylation and macrophage chemotaxis were preserved in AMPKα1-knockdown cells, indicating AMPK is not required for these functions of GS12021. Identification of this novel methylhonokiol analog could enable studies of the structure-activity relationship of this class of compounds and further evaluation of its in vivo potential for the treatment of insulin-resistant states and other chronic inflammatory diseases.

The protein kinase 2 inhibitor tetrabromobenzotriazole protects against renal ischemia reperfusion injury.

Protein kinase 2 (CK2) activation was reported to enhance reactive oxygen species production and activate the nuclear factor κB (NF-κB) pathway. Because oxidative stress and inflammation are critical events for tissue destruction during ischemia reperfusion (I/R), we sought to determine whether CK2 was important in the renal response to I/R. Mice underwent 25 min of renal ischemia and were then reperfused. We confirmed an increased expression of CK2α during the reperfusion period, while expression of CK2ß remained consistent. We administered tetrabromobenzotriazole (TBBt), a selective CK2α inhibitor before inducing I/R injury. Mice subjected to I/R injury showed typical patterns of acute kidney injury; blood urea nitrogen and serum creatinine levels, tubular necrosis and apoptosis, inflammatory cell infiltration and proinflammatory cytokine production, and oxidative stress were markedly increased when compared to sham mice. However, pretreatment with TBBt abolished these changes and improved renal function and architecture. Similar renoprotective effects of CK2α inhibition were observed for emodin. Renoprotective effects of CK2α inhibition were associated with suppression of NF-κB and mitogen activated protein kinase (MAPK) pathways. Taken together, these results suggest that CK2α mediates proapoptotic and proinflammatory signaling, thus the CK2α inhibitor may be used to prevent renal I/R injuries observed in clinical settings.