Search

Selenoprotein P (SeP; encoded by SELENOP in humans, Selenop in rodents) is a hepatokine that is upregulated in the liver of humans with type 2 diabetes. Excess SeP contributes to the onset of insulin resistance and various type 2 diabetes-related complications. We have previously reported that the long-chain saturated fatty acid, palmitic acid, upregulates Selenop expression, whereas the polyunsaturated fatty acids (PUFAs) downregulate it in hepatocytes. However, the effect of medium-chain fatty acids (MCFAs) on Selenop is unknown. Here we report novel mechanisms that underlie the lauric acid-mediated Selenop gene regulation in hepatocytes. Lauric acid upregulated Selenop expression in Hepa1-6 hepatocytes and mice liver. A luciferase promoter assay and computational analysis of transcription factor-binding sites identified the hepatic nuclear factor 4α (HNF4α) binding site in the SELENOP promoter. A chromatin immunoprecipitation (ChIP) assay showed that lauric acid increased the binding of HNF4α to the SELENOP promoter. The knockdown of Hnf4α using siRNA canceled the upregulation of lauric acid-induced Selenop. Thus, the lauric acid-induced impairment of Akt phosphorylation brought about by insulin was rescued by the knockdown of either Hnf4α or Selenop. These results provide new insights into the regulation of SeP by fatty acids and suggest that SeP may mediate MCFA-induced hepatic insulin signal reduction.

Muscle atrophy is the cause and consequence of obesity. Proteasome dysfunction mediates obesity-induced endoplasmic reticulum (ER) stress and insulin resistance in the liver and adipose tissues. However, obesity-associated regulation of proteasome function and its role in the skeletal muscles remains underinvestigated. Here, we established skeletal muscle-specific 20S proteasome assembly chaperone-1 (PAC1) knockout (mPAC1KO) mice. A high-fat diet (HFD) activated proteasome function by ∼8-fold in the skeletal muscles, which was reduced by 50% in mPAC1KO mice. mPAC1KO induced unfolded protein responses in the skeletal muscles, which were reduced by HFD. Although the skeletal muscle mass and functions were not different between the genotypes, genes involved in the ubiquitin proteasome complex, immune response, endoplasmic stress, and myogenesis were coordinately upregulated in the skeletal muscles of mPAC1KO mice. Therefore, we introduced an immobilization-induced muscle atrophy model in obesity by combining HFD and immobilization. mPAC1KO downregulated atrogin-1 and MuRF1, together with their upstream Foxo1 and Klf15, and protected against disused skeletal muscle mass reduction. In conclusion, obesity elevates proteasome functions in the skeletal muscles. PAC1 deficiency protects mice from immobilization-induced muscle atrophy in obesity. These findings suggest obesity-induced proteasome activation as a possible therapeutic target for immobilization-induced muscle atrophy.

AIM Selenoprotein P (SeP, encoded by SELENOP in humans) is a hepatokine that causes insulin resistance in the liver and skeletal muscle. It was found that polyunsaturated fatty acid eicosapentaenoic acid (EPA) downregulates Selenop expression by inactivating SREBP-1c. The present study aimed to examine the effect of EPA for 12 weeks on circulating SeP levels and insulin sensitivity in humans with type 2 diabetes. METHODS A total of 20 participants with dyslipidemia and type 2 diabetes were randomly assigned to an EPA (900 mg, twice daily) group and a control group. The primary endpoint was a change in serum SeP levels. Organ-specific insulin sensitivity in the liver (HGP and %HGP), skeletal muscle (Rd), and adipose tissue (FFA and %FFA) were assessed using a hyperinsulinemic-euglycemic clamp study with stable isotope-labeled glucose infusion. RESULTS Serum SeP levels were not changed in either group at the end of the study. In the EPA group, the changes in SeP levels were positively correlated with the change in serum EPA levels (r = 0.709, P = 0.022). Treatment with EPA significantly enhanced %FFA but not %HGP and Rd. The change in serum EPA levels was significantly positively correlated with the change in %HGP, and negatively correlated with changes in Rd. CONCLUSIONS The change in serum EPA levels was positively correlated with serum SeP levels, hepatic insulin sensitivity, and negatively with skeletal muscle insulin sensitivity in humans with type 2 diabetes. The EPA-induced enhancement of hepatic insulin sensitivity might be associated with a mechanism independent of serum SeP levels.

Selenoprotein P (SeP; encoded by SEPP1 in humans) is a hepatokine that causes impaired insulin secretion and insulin resistance. Metformin downregulates SELENOP promoter activity through an adenosine monophosphate-activated kinase-forkhead box protein O3a pathway in hepatocytes. This study aimed to test our hypothesis that circulating SeP levels are associated with the glucose-lowering effect of metformin in humans.A total of 84 participants with poorly controlled type 2 diabetes were randomly assigned to receive metformin (1,000 mg, twice daily) or a dipeptidyl peptidase-4 inhibitor, alogliptin (25 mg, once daily) for 12 weeks. We tested metformin and alogliptin on SeP levels and factors associated therewith as a post-hoc analysis.Both metformin and aloglipitin did not change the SeP levels. Although metformin significantly increased the insulin secretory index secretory units of islets in transplantation only in participants with higher baseline SeP (3.87), both agents similarly reduced fasting plasma glucose and glycated hemoglobin. SeP levels at baseline were correlated negatively with changes in SeP (r = -0.484, P = 0.004) and fasting plasma glucose (r = -0.433, P = 0.011), and positively with changes in C-peptide immunoreactivity (r = 0.420, P = 0.017) and secretory units of islets in transplantation (r = 0.388, P = 0.028) in the metformin, but not alogliptin, group.Higher baseline levels of SeP significantly predicted metformin-mediated, but not alogliptin-mediated, glucose-lowering and insulinotropic effects. Serum SeP levels might be a novel biomarker for predicting the outcomes of metformin therapy, which might be helpful in tailoring diabetes medication.

Cyclosporine A (CsA) is a worldwide applied immunosuppressant for preventing graft rejection and autoimmune diseases. However, CsA elevates oxidative stress leading to liver injuries. The present study aimed to clarify the mechanisms underlying the CsA-mediated oxidative stress. Among the redox proteins, CsA concentration-dependently downregulated

It remains unclear how hepatic steatosis links to inflammation. Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine that senses fat in the liver and is upregulated prior to weight gain. The aim of this study was to investigate the significance of LECT2 in the development of nonalcoholic steatohepatitis (NASH). In human liver biopsy samples, elevated LECT2 mRNA levels were positively correlated with body mass index (BMI) and increased in patients who have steatosis and inflammation in the liver. LECT2 mRNA levels were also positively correlated with the mRNA levels of the inflammatory genes CCR2 and TLR4. In C57BL/6J mice fed with a high-fat diet, mRNA levels of the inflammatory cytokines Tnfa and Nos2 were significantly lower in Lect2 KO mice. In flow cytometry analyses, the number of M1-like macrophages and M1/M2 ratio were significantly lower in Lect2 KO mice than in WT mice. In KUP5, mouse kupffer cell line, LECT2 selectively enhanced the LPS-induced phosphorylation of JNK, but not that of ERK and p38. Consistently, LECT2 enhanced the LPS-induced phosphorylation of MKK4 and TAB2, upstream activators of JNK. Hepatic expression of LECT2 is upregulated in association with the inflammatory signature in human liver tissues. The elevation of LECT2 shifts liver residual macrophage to the M1-like phenotype, and contributes to the development of liver inflammation. These findings shed light on the hepatokine LECT2 as a potential therapeutic target that can dissociate liver steatosis from inflammation.

Selenoprotein P is upregulated in type 2 diabetes, causing insulin and exercise resistance. We have previously reported that eicosapentaenoic acid (EPA) negatively regulates Selenop expression by suppressing Srebf1 in H4IIEC3 hepatocytes. However, EPA downregulated Srebf1 long before downregulating Selenop. Here, we report additional novel mechanisms for the Selenop gene regulation by EPA. EPA upregulated Foxo1 mRNA expression, which was canceled with the ERK1/2 inhibitor, but not with the PKA inhibitor. Foxo1 knockdown by siRNA initiated early suppression of Selenop, but not Srebf1, by EPA. However, EPA did not affect the nuclear translocation of the FoxO1 protein. Neither ERK1/2 nor PKA inhibitor affected FoxO1 nuclear translocation. In summary, FoxO1 knockdown accelerates the EPA-mediated Selenop downregulation independent of SREBP-1c in hepatocytes. EPA upregulates Foxo1 mRNA via the ERK1/2 pathway without altering its protein and nuclear translocation. These findings suggest redundant and conflicting transcriptional networks in the lipid-induced redox regulation.

Selenoprotein P is a hepatokine with antioxidative properties that eliminate a physiologic burst of reactive oxygen species required for intracellular signal transduction. Serum levels of selenoprotein P are elevated during aging and in people with type 2 diabetes, non-alcoholic fatty liver disease, and hepatitis C. However, how serum levels of full-length selenoprotein P are regulated largely remains unknown, especially in the general population. To understand the significance of serum selenoprotein P levels in the general population, we evaluated intrinsic and environmental factors associated with serum levels of full-length selenoprotein P in 1,183 subjects participating in the Shika-health checkup cohort. Serum levels of selenium were positively correlated with liver enzymes and alcohol intake and negatively correlated with body mass index. Serum levels of selenoprotein P were positively correlated with age, liver enzymes, and alcohol intake. In multiple regression analyses, alcohol intake was positively correlated with serum levels of both selenium and selenoprotein P independently of age, gender, liver enzymes, and fatty liver on ultrasonography. In conclusion, alcohol intake is associated with elevated serum levels of selenium and selenoprotein P independently of liver enzyme levels and liver fat in the general population. Moderate alcohol intake may exert beneficial or harmful effects on health, at least partly by upregulating selenoprotein P. These findings increase our understanding of alcohol-mediated redox regulation and form the basis for the adoption of appropriate drinking guidelines.

Bovine viral diarrhea (BVD) is a chronic disease of cattle caused by infection with BVD virus (BVDV) and can result in economic losses within the livestock industry. In Japan, the test and culling policy is a basic control measure, and implementation of an adequate vaccination program is recommended as a national policy. In addition, optional control measures, including compulsory testing of introduced animals and bulk tank milk (BTM) testing as a mass screening method, are used in several provinces, but their efficacy has not been completely assessed. We evaluated these control measures using the scenario tree model of BVD in Japan, developed in the previous study. The model outputs indicated that compulsory testing of all introduced cattle, rather than only heifers and/or non-vaccinated cattle, was cost effective and reduced the risk of BVDV introduction due to animal movement and that BTM testing could effectively monitor most part of the cattle population. Vaccination coverage and BVDV prevalence among introduced cattle could also affect the cost effectiveness of compulsory testing of targeted cattle, particularly under low vaccination coverage or high BVDV prevalence. However, even with the implementation of a highly effective monitoring scheme for many years, BVD risk could not be eliminated; it instead converged at a very low level (0.02%). Disease models with a cost-effective output could be a powerful tool in developing a control scheme for chronic animal diseases, including BVD, with the consent of relevant stakeholders.

We aimed to test the hypothesis that selenoprotein P (SELENOP), a hepatokine involved in the development of both insulin resistance and impaired insulin production in mice, is related to future onset of hyperglycemia in humans. 76 healthy non-pregnant human subjects without diabetes underwent oral glucose tolerance test (OGTT) at baseline and 4-years follow-up. Nine subjects developed either impaired glucose tolerance or type 2 diabetes at follow-up. At baseline, SELENOP concentrations correlated negatively with insulinogenic index, but not with homeostasis model assessment-estimated insulin resistance (HOMA-IR). Multivariate analysis showed that baseline SELENOP predicted fasting plasma glucose at follow-up independently of the other parameters. The receiver operating characteristic (ROC) curve analysis showed that baseline concentrations of serum SELENOP, but not of selenium, were a reliable test to predict future onset of glucose intolerance. In conclusion, elevation of circulating SELENOP, but not of circulating selenium, was positively and independently associated with future onset of glucose intolerance in a general Japanese population.

Reactive oxygen species (ROS) oxidize and activate the uncoupler protein 1 (UCP1) in brown adipose tissue (BAT) under physiological cold exposure and noradrenaline (NA) stimulation to increase thermogenesis. However, pathological significance and the endogenous regulator of the ROS-mediated BAT activation remain unclear. Here, we show that serum levels of selenoprotein P (SeP, encoded by Selenop) are negatively correlated with BAT activity in humans. SeP impairs UCP1 activity and thermogenesis. Physiological cold exposure downregulates Selenop in BAT. BAT-specific Selenop-deficient (BAT-Selenop KO) mice presented elevated NA-induced mitochondrial ROS production, sulfenylated UCP1, and enhanced thermogenesis and glucose uptake in BAT during cold exposure. SeP inhibits mitochondrial ROS by upregulating the expression of the antioxidant enzyme, glutathione peroxidase 4, and impairs glucose uptake in brown adipocytes. High fat/high sucrose diet upregulates Selenop in the liver and inhibits the NA-induced BAT thermogenesis in BAT-Selenop KO mice. Our data indicate that SeP, as the hepatokine and BATkine, is the first identified intrinsic factor inducing reductive stress that impairs UCP1 activation and thermogenesis in BAT, and therefore may be a potential therapeutic target for obesity and diabetes.

Reactive oxygen species (ROS) activate uncoupler protein 1 (UCP1) in brown adipose tissue (BAT) under physiological cold exposure and noradrenaline (NA) stimulation to increase thermogenesis. However, the endogenous regulator of ROS in activated BAT and its role in pathological conditions remain unclear. We show that serum levels of selenoprotein P (SeP; encoded by SELENOP) negatively correlate with BAT activity in humans. Physiological cold exposure downregulates Selenop in BAT. Selenop knockout mice show higher rectal temperatures and UCP1 sulfenylation during cold exposure. SeP treatment to brown adipocytes eliminated the NA-induced mitochondrial ROS by upregulating glutathione peroxidase 4 and impaired cellular thermogenesis. A high-fat/high-sucrose diet elevates serum SeP levels and diminishes the elevated NA-induced thermogenesis in BAT-Selenop KO mice. Therefore, SeP is the intrinsic factor inducing reductive stress that impairs thermogenesis in BAT and may be a potential therapeutic target for obesity and diabetes.

Selenoprotein P (SeP) is a selenium (Se)-rich extracellular protein. SeP is identified as a hepatokine, causing insulin resistance in type 2 diabetes. Thus, the measurement of SeP in serum has received much attention, and several enzyme-linked immunosorbent assay (ELISA) kits for SeP determination are now commercially available. In the present study, we determined the serum SeP levels by our original ELISA and sol particle homogeneous immunoassay (SPIA) methods and also by commercially available kits, and these determinants were compared. We found a kit-dependent correlation of the determinants with our methods. These results suggest that the selection of kit is critical for comparison with our previous reports and for discussing the relationship between the serum SeP levels and disease condition.

A scenario tree model was developed to propose efficient bovine viral diarrhea (BVD) control measures. The model used field data in eastern Hokkaido where the risk of BVDV infection in cattle has been reduced by an eradication program including mass vaccination, individual tests prior to communal pasture grazing, herd screening tests using bulk milk, and outbreak investigations of newly infected herds. These four activities were then used as hypothesized control measures in the simulation. In each simulation, the numbers of cattle infected persistently and transiently with BVDV detected by clinical manifestations and diagnosis tests and of missed by all of the diagnosis tests were calculated, and the numbers were used as indicators to be compared for the efficacy of the control measures. The model outputs indicated that the adoption of mass vaccination decreased the number of missed BVD cattle, although it did not increase the number of detected BVD cattle. Under implementation of mass vaccination, the efficacy of individual tests on selected 20% of the young and adult cattle was equal to that of the herd screening test performed in all the herds. When the virus prevalence or the number of sensitive animals becomes low, the efficacy of herd screening test was superior to one of individual tests. Considering the model outputs together, the scenario tree model developed in the present study was useful to compare the efficacy of the control measures for BVD.

Selenoprotein P (encoded by SELENOP in humans, Selenop in rat), a liver-derived secretory protein, induces resistance to insulin and vascular endothelial growth factor (VEGF) in type 2 diabetes. Suppression of selenoprotein P may provide a novel therapeutic approach to treating type 2 diabetes; however, few drugs inhibiting SELENOP expression in hepatocytes have been identified. The present findings demonstrate that eicosapentaenoic acid (EPA) suppresses SELENOP expression by inactivating sterol regulatory element-binding protein-1c (SREBP-1c, encoded by Srebf1 in rat) in H4IIEC3 hepatocytes. Treatment with EPA caused concentration- and time-dependent reduction in SELENOP promoter activity. EPA activated AMP-activated protein kinase (AMPK); however, the inhibitory effect of EPA on SELENOP promoter activity was not canceled with an AMPK inhibitor compound C and dominant-negative AMPK transfection. Deletion mutant promoter assays and computational analysis of transcription factor-binding sites conserved among the species resulted in identification of a sterol regulatory element (SRE)-like site in the SELENOP promoter. A chromatin immunoprecipitation (ChIP) assay revealed that EPA decreases binding of SREBP-1c to the SELENOP promoter. Knockdown of Srebf1 resulted in a significant down-regulation of Selenop expression. Conversely, SREBP-1c overexpression inhibited the suppressive effect of EPA. These data provide a novel mechanism of action for EPA involving improvement of systemic insulin sensitivity through the regulation of selenoprotein P production independently of the AMPK pathway and suggest an additional approach to developing anti-diabetic drugs.

Background Hepatokines such as fibroblast growth factor 21 (FGF21), leukocyte cell–derived chemotaxin 2 (LECT2), fetuin-A, fetuin-B, and selenoprotein P (SeP) are liver-derived proteins that are modulated by chronic energy status and metabolic disease. Emerging data from rodent and cell models indicate that hepatokines may be sensitive to acute nutritional manipulation; however, data in humans are lacking. Objective The aim was to investigate the influence of hyperenergetic, high-fat feeding on circulating hepatokine concentrations, including the time course of responses. Methods In a randomized, crossover design, 12 healthy men [mean ± SD: age, 24 ± 4 y; BMI (kg/m2), 24.1 ± 1.5] consumed a 7-d hyperenergetic, high-fat diet [HE-HFD; +50% energy, 65% total energy as fat (32% saturated, 26% monounsaturated, 8% polyunsaturated)] and control diet (36% total energy as fat), separated by 3 wk. Whole-body insulin sensitivity was assessed before and after each diet using oral-glucose-tolerance tests. Fasting plasma concentrations of FGF21 (primary outcome), LECT2, fetuin-A, fetuin-B, SeP, and related metabolites were measured after 1, 3, and 7 d of each diet. Hepatokine responses were analyzed using 2-factor repeated-measures ANOVA and subsequent pairwise comparisons. Results Compared with the control, the HE-HFD increased circulating FGF21 at 1 d (105%) and 3 d (121%; P ≤ 0.040), LECT2 at 3 d (17%) and 7 d (32%; P ≤ 0.004), and fetuin-A at 7 d (7%; P = 0.028). Plasma fetuin-B and SeP did not respond to the HE-HFD. Whole-body insulin sensitivity was reduced after the HE-HFD by 31% (P = 0.021). Conclusions Acute high-fat overfeeding augments circulating concentrations of FGF21, LECT2, and fetuin-A in healthy men. Notably, the time course of response varies between proteins and is transient for FGF21. These findings provide further insight into the nutritional regulation of hepatokines in humans and their interaction with metabolic homeostasis. This study was registered at clinicaltrials.gov as NCT03369145.

A hepatokine is a collective term for liver-derived secretory factors whose previously-unrecognized functions have been recently elucidated. We have rediscovered selenoprotein P (SeP) and leukocyte cell-derived chemotaxin 2 (LECT2) as hepatokines that are involved in the development of insulin resistance and hyperglycemia. The aim of this study was to determine whether and, if so, how oral glucose loading alters the two hepatokines in humans. We measured concentrations of serum SeP and plasma LECT2 during 75 g oral glucose tolerance test (OGTT) (n = 20) in people with various degrees of glucose tolerance. In OGTT, concentrations of both serum SeP and plasma LECT2 decreased at 120 min compared with the baseline values, irrespective of the severity of glucose intolerance. Decrement of serum SeP during OGTT showed no correlations to the clinical parameters associated with insulin resistance or insulin secretion. In multiple stepwise regression analyses, plasma cortisol was selected as the variable to explain the changes in plasma concentrations of LECT2. The current data reveal the acute inhibitory actions of oral intake of glucose on circulating SeP and LECT2 in humans, irrespective of the severity of glucose intolerance. This study suggests that circulating SeP is regulated by the unknown clinical factors other than insulin and glucose during OGTT.

Dieting often leads to body weight cycling involving repeated weight loss and regain. However, little information is available regarding rapid-response serum markers of overnutrition that predict body weight alterations during weight cycling. Here, we report the rapid response of serum leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine that induces insulin resistance in skeletal muscle, during diet-induced weight cycling in mice. A switch from a high-fat diet (HFD) to a regular diet (RD) in obese mice gradually decreased body weight but rapidly decreased serum LECT2 levels within 10 days. In contrast, a switch from a RD to a HFD rapidly elevated serum LECT2 levels. Serum LECT2 levels showed a positive correlation with liver triglyceride contents but not with adipose tissue weight. This study demonstrates the rapid response of LECT2 preceding body weight alterations during weight cycling in mice and suggests that measurement of serum LECT2 may be clinically useful in the management of obesity.

金沢大学疾患モデル総合研究センター, Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine that causes skeletal muscle insulin resistance. The circulating levels of LECT2 are a possible biomarker that can predict weight cycling because they reflect liver fat and precede the onset of weight loss or gain. Herein, to clarify the dynamics of this rapid change in serum LECT2 levels, we investigated the in vivo kinetics of LECT2, including its plasma half-life and tissue distribution, by injecting 125I-labelled LECT2 into ICR mice and radioactivity tracing. The injected LECT2 was eliminated from the bloodstream within 10min (approximate half-life, 5min). In the kidneys, the radioactivity accumulated within 10min after injection and declined thereafter. Conversely, the radioactivity in urine increased after 30min of injection, indicating that LECT2 is mainly excreted by the kidneys into the urine. Finally, LECT2 accumulated in the skeletal muscle and liver until 30min and 2min after injection, respectively. LECT2 accumulation was not observed in the adipose tissue. These findings are in agreement with LECT2 action on the skeletal muscle. The present study indicates that LECT2 is a rapid-turnover protein, which renders the circulating level of LECT2 a useful rapid-response biomarker to predict body weight alterations. © 2020, The Author(s)., CC-BY 4.0

Hepatokines are liver-secreted proteins with potential to influence glucose regulation and other metabolic parameters. This study investigated differences in adiposity status on 5 novel hepatokines and characterised their response to acute moderate-intensity exercise in groups of normal-weight and overweight/obese men. Twenty-two men were recruited into normal-weight and overweight/obese groups (body mass index: 18.5 to 24.9 and 25.0 to 34.9 kg·m−2). Each completed 2 experimental trials, exercise and control. During exercise trials, participants performed 60 min of moderate-intensity treadmill exercise (∼60% peak oxygen uptake) and then rested for 6 h. Participants rested throughout control trials. Circulating fibroblast growth factor-21 (FGF21), follistatin, leukocyte cell-derived chemotaxin 2 (LECT2), fetuin-A, and selenoprotein-P (SeP) were measured throughout. Fasted (resting) FGF21 and LECT2 were higher in overweight/obese individuals (129% and 55%; P ≤ 0.01) and correlated with indices of adiposity and insulin resistance; whereas circulating follistatin was lower in overweight/obese individuals throughout trial days (17%, P < 0.05). In both groups, circulating concentrations of FGF21 and follistatin were transiently elevated after exercise for up to 6 h (P ≤ 0.02). Circulating fetuin-A and SeP were no different between groups (P ≥ 0.19) and, along with LECT2, were unaffected by exercise (P ≥ 0.06). These findings show that increased adiposity is associated with a modified hepatokine profile, which may represent a novel mechanism linking excess adiposity to metabolic health. Furthermore, acute perturbations in circulating FGF21 and follistatin after exercise may contribute to the health benefits of an active lifestyle.

Aims/hypothesis Impaired angiogenesis induced by vascular endothelial growth factor (VEGF) resistance is a hallmark of vascular complications in type 2 diabetes; however, its molecular mechanism is not fully understood. We have previously identified selenoprotein P (SeP, encoded by the SEPP1 gene in humans) as a liver-derived secretory protein that induces insulin resistance. Levels of serum SeP and hepatic expression of SEPP1 are elevated in type 2 diabetes. Here, we investigated the effects of SeP on VEGF signalling and angiogenesis. Methods We assessed the action of glucose on Sepp1 expression in cultured hepatocytes. We examined the actions of SeP on VEGF signalling and VEGF-induced angiogenesis in HUVECs. We assessed wound healing in mice with hepatic SeP overexpression or SeP deletion. The blood flow recovery after ischaemia was also examined by using hindlimb ischaemia model with Sepp1-heterozygous-knockout mice. Results Treatment with glucose increased gene expression and transcriptional activity for Sepp1 in H4IIEC hepatocytes. Physiological concentrations of SeP inhibited VEGF-stimulated cell proliferation, tubule formation and migration in HUVECs. SeP suppressed VEGF-induced reactive oxygen species (ROS) generation and phosphorylation of VEGF receptor 2 (VEGFR2) and extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs. Wound closure was impaired in the mice overexpressing Sepp1, whereas it was improved in SeP-/-mice. SeP+/-mice showed an increase in blood flow recovery and vascular endothelial cells after hindlimb ischaemia. Conclusions/interpretation The hepatokine SeP may be a novel therapeutic target for impaired angiogenesis in type 2 diabetes. © 2014 Springer-Verlag Berlin Heidelberg.

Selenoprotein P (SeP; encoded by SEPP1 in humans) is a liver-derived secretory protein that induces insulin resistance in type 2 diabetes. Suppression of SeP might provide a novel therapeutic approach to treating type 2 diabetes, but few drugs that inhibit SEPP1 expression in hepatocytes have been identified to date. The present findings demonstrate that metformin suppresses SEPP1 expression by activating AMP-activated kinase (AMPK) and subsequently inactivating FoxO3a in H4IIEC3 hepatocytes. Treatment with metformin reduced SEPP1 promoter activity in a concentration- and time-dependent manner; this effect was cancelled by co-administration of an AMPK inhibitor. Metformin also suppressed Sepp1 gene expression in the liver of mice. Computational analysis of transcription factor binding sites conserved among the species resulted in identification of the FoxO-binding site in the metformin-response element of the SEPP1 promoter. A luciferase reporter assay showed that metformin suppresses Forkhead-response element activity, and a ChIP assay revealed that metformin decreases binding of FoxO3a, a direct target of AMPK, to the SEPP1 promoter. Transfection with siRNAs for Foxo3a, but not for Foxo1, cancelled metformin-induced luciferase activity suppression of the metformin-response element of the SEPP1 promoter. The overexpression of FoxO3a stimulated SEPP1 promoter activity and rescued the suppressive effect of metformin. Metformin did not affect FoxO3a expression, but it increased its phosphorylation and decreased its nuclear localization. These data provide a novel mechanism of action for metformin involving improvement of systemic insulin sensitivity through the regulation of SeP production and suggest an additional approach to the development of anti-diabetic drugs.

Exercise has numerous health-promoting effects in humans; however, individual responsiveness to exercise with regard to endurance or metabolic health differs markedly. This 'exercise resistance' is considered to be congenital, with no evident acquired causative factors. Here we show that the anti-oxidative hepatokine selenoprotein P (SeP) causes exercise resistance through its muscle receptor low-density lipoprotein receptor-related protein 1 (LRP1). SeP-deficient mice showed a 'super-endurance' phenotype after exercise training, as well as enhanced reactive oxygen species (ROS) production, AMP-activated protein kinase (AMPK) phosphorylation and peroxisome proliferative activated receptor γ coactivator (Ppargc)-1α (also known as PGC-1α; encoded by Ppargc1a) expression in skeletal muscle. Supplementation with the anti-oxidant N-acetylcysteine (NAC) reduced ROS production and the endurance capacity in SeP-deficient mice. SeP treatment impaired hydrogen-peroxide-induced adaptations through LRP1 in cultured myotubes and suppressed exercise-induced AMPK phosphorylation and Ppargc1a gene expression in mouse skeletal muscle-effects which were blunted in mice with a muscle-specific LRP1 deficiency. Furthermore, we found that increased amounts of circulating SeP predicted the ineffectiveness of training on endurance capacity in humans. Our study suggests that inhibitors of the SeP-LRP1 axis may function as exercise-enhancing drugs to treat diseases associated with a sedentary lifestyle.

Two cytologic variants of γδ T cell lymphoma are described. Case 1 represented a giant cell variant found in a 5-year-old Holstein cow, which had large tumor masses in the pelvic cavity. This variant consisted of very large lymphoid cells with round to oval nuclei, medium-sized nucleoli and abundant cytoplasm. Case 2 was an aborted 7-month-old female Holstein fetus, which represented an immature cell variant. Most of the neoplastic lesions were located in the skin and pleural and peritoneal submesothelial tissues. The neoplastic tissues were composed of homogeneous growth of lymphoma cells characterized by inconspicuous nucleoli and finely dispersed chromatin. Both cases demonstrated CD3, CD8 and WC1 immunoreactivity. The current study revealed that there are 4 cytologic variants (common, giant cell, hypergranular and immature cell) in bovine γδ T cell lymphomas.

Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH2-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated β-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

A concise and efficient synthetic approach to 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D(3) (4a-c), including 2-epi-ED-71, was developed starting from D-glucose as a chiral template for the construction of the 2 alpha-modified A-ring precursors (11a-c). It was found that the best ligand for the bovine thymus vitamin D receptor (VDR) in this series is 4b, which has 1.8 times greater binding affinity for the bovine thymus VDR than that of the natural hormone 1. Interestingly, potency in the induction of HL-60 cell differentiation for 4a-c was almost the same or weaker than that of 1 despite the strong binding affinity for the VDR. Next, we were interested in the "double modification"of 1 based on 4a-c with C20-epimerization, affording 2 alpha-(omega-hydroxyalkoxy)-20-epi-1 alpha,25-dihydroxyvitamin D(3) (20-epi-4a-c). All three 2 alpha-substituted 20-epi analogues of 1 (20-epi-4a-c) exhibited stronger binding affinities for the VDR, and their conformations in the ligand binding domain of VDR were analyzed by molecular modeling. Double-modified analogues of 20-epi-4a-c showed marked HL-60 cell differentiation activity, and 20-epi-4a possesses an activity 58-fold higher than that of the natural hormone 1.

The immunohistochemical features of tumors from 7 Holstein cows showing fibroblastic, myofibroblastic, smooth muscle or pericytic differentiation are described. In cases 1 and 2, the tumors were characterized by neoplastic fibroblasts immunostained with vimentin alone, but some cells appeared to transiently express alpha smooth muscle actin (SMA) during tumor development. Cutaneous (case 3) and nasal (case 4) myofibroblastomas, which were characterized by SMA and fibronectin positivity, with a minority of cells showing positive reactivity for desmin, were readily distinguishable from tumors of fibroblasts, smooth muscle cells and pericytes by fibronectin staining. The same staining pattern was seen in a vulvar myofibroblastoma (case 5), which differed from a vaginal leiomyoma (case 6) coexpressing SMA and desmin in nearly all neoplastic cells. Both neoplasms, however, had estrogen receptor (ER), and may be derived from immature stromal cells distributed in the vulvovaginal region. A hemangiopericytoma (case 7) resembled myofibroblastomas in cytoskeletal immunophenotypes, but lacked fibronectin. Benign or locally invasive soft tissue tumors with spindle cell morphology and expression of SMA could be divided into several types by immunostaining for SMA, desmin, fibronectin and BR.

Our studies on creation of functional organic compounds and their applications, have focused on three areas, namely, (A) organic chemical studies on VD (vitamin D) analogues, (B) studies on solitary wasp venoms, and (C) studies on functional building blocks for organic synthesis. In the first area, several novel and important vitamin D analogues were synthesized and biologically evaluated, and their high VDR (vitamin D receptor) binding affinities were discussed on the basis of conformational analysis and docking study by Molecular Mechanics Calculation to the LBD (ligand binding domain) of VDR: These compounds include 24,24-difluoro-1 alpha,25-dihydroxy-VD3 (2) (an antimetabolism agent, the first VD analogue having higher potency than the natural hormone (1)), 2 alpha-methyl-1 alpha,25-dihydroxy-VD3 (42b) (the first A-ring-modified VD analogue exhibiting stronger VDR affinity than 1) and its 20-epimer (43b) (a VD analogue having a highest HL-60 cell differentiation inducing activity with a relatively low calcemic effect), and 2 alpha-(omega-hydroxypropyl)-1 alpha,25-dihydroxy-VD3 (exceptionally high calcemic effect). In the second area, we isolated and determined the structure of pompilidotoxins (76, 77), novel peptide neurotoxins in solitary wasp venoms. In the third area, we created furan-fused 3-sulfolene, 4H, 6H-thieno [3,4-c]-furan 5,5-dioxide and pyrrole-fused 3-sulfolene (96), 3,5-dihydro-1H-thieno[3,4-c] pyrrole 2,2-dioxide (125), and studied their inter- and intramolecular Diels-Alder reactions.

We have systematically synthesized 2α-functionalized 1α, 25-dihydroxyvitamin D3 [1α, 25 (OH) 2D3] 1 based on a convergent method using Pd-catalyzed alkylative cyclization with the A-ring precursor enynes and the CD-ring bromoolefin 5 in the following three categories : 2α-alkyl, 2α-hydroxyalkyl, and 2α-hydroxyalkoxyl derivatives, in order to study the structure-activity relationships of the natural hormone 1α, 25 (OH) 2D3, particularly on the A-ring.First, the 2-methyl analogues 2 were designed and all eight possible diastereomers on the A-ring stereochemistry at C 1, C 2, and C 3 were synthesized from the A-ring precursor enynes 22a-h, which were prepared from methyl (S) -and (R) -3-hydroxy-2-methylpropionate. Biological activities including affinities to vitamin D receptor (VDR) and vitamin D binding protein (DBP), elevation of serum Ca level, induction of HL-60 cell differentiation, and apoptosis are discussed.Next, on the basis of biological activities of the 2α-methyl derivative, the other types of 2α-functional groups were introduced stereoselectively into the 1α, 25 (OH) 2D3 skeleton using the A-ring precursor enynes derived from D-xylose or D-glucose.Five of these synthetic 2α-modified analogues showed higher VDR binding affinity than that of the natural hormone. Docking studies of the synthetic ligands to VDR based on Moras' X-ray results and remarkably high biological activities are described.

Several novel A-ring modified analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] have been synthesized in order to investigate the structure-function relationships of 1alpha,25(OH)2D3. We synthesized A-ring modified analogs which contain a methyl group on C-2 of the A-ring. There are eight 2-methyl diastereomers, which differ in the stereochemistry of the methyl group on C-2 and the hydroxyl groups on C-1 and C-3. Further our biological activity studies of the 2-methyl diastereomers indicated that the potency of each analog is highly dependent on the stereochemistry of the A-ring substituents [Konno et al., Biorg. Med. Chem. Letts. 8(2), 151-156 (1998); Nakagawa et al., Biochem. Pharmacol. 60(12), 1937-1947 (2000)]. For example, the VDR binding affinities exhibited by the 1alpha-isomers are significantly higher than those exhibited by the 1beta-isomers. Furthermore, out of all the 1alpha-isomers, the 2alpha-methyl isomers, when compared to the corresponding 2beta-methyl isomers, showed much higher potency in inducing cell differentiation of HL-60 cells, but failed to stimulate apoptosis. In contrast the 2beta-methyl isomers strongly stimulated apoptosis. At present it is unknown how the addition of the 2-methyl modification to the hormone, 1alpha,25(OH)2D3 alters its metabolism in target tissues. Previously, we reported that 1alpha,25(OH)2D3 is metabolized in rat osteosarcoma (UMR 106) cells via both the C-24 oxidation and the C-3 epimerization pathways. Therefore, we studied the metabolism of the four 1alpha,2-methyl diastereomers in UMR 106 cells. Our results indicated that in UMR 106 cells, all four diastereomers were metabolized into several polar metabolites via the C-24 oxidation pathway. Thus, the presence of the 2-methyl group on the A-ring did not inhibit the metabolism of the analogs via the C-24 oxidation pathway. However, it is significant to note that the 2-methyl group prevented the metabolism of the analogs via the C-3 epimerization pathway. In summary, we report that the 2-methyl group interferes with the action of the enzyme(s) involved in C-3 epimerization, but not with the enzyme 1alpha,25(OH)2D3-24-hydroxylase, which is responsible for C-24 oxidation pathway.

Recent articles have reported an association between fatty liver disease and systemic insulin resistance in humans, but the causal relationship remains unclear. The liver may contribute to muscle insulin resistance by releasing secretory proteins called hepatokines. Here we demonstrate that leukocyte cell-derived chemotaxin 2 (LECT2), an energy-sensing hepatokine, is a link between obesity and skeletal muscle insulin resistance. Circulating LECT2 positively correlated with the severity of both obesity and insulin resistance in humans. LECT2 expression was negatively regulated by starvation-sensing kinase adenosine monophosphate-activated protein kinase in H4IIEC hepatocytes. Genetic deletion of LECT2 in mice increased insulin sensitivity in the skeletal muscle. Treatment with recombinant LECT2 protein impaired insulin signaling via phosphorylation of Jun NH2-terminal kinase in C2C12 myocytes. These results demonstrate the involvement of LECT2 in glucose metabolism and suggest that LECT2 may be a therapeutic target for obesity-associated insulin resistance. © 2014 by the American Diabetes Association.

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108–15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nm, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nm, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.

The syntheses and biological evaluations of (23R)- and (23S)-24,24-difluoro-1 alpha,23,25-trithydroxyvitamin D3 (3a and 3b), new C-24 fluorinated analogs of 1 alpha,25-dihydroxyvitamin D3, are described. The syntheses of these compounds were achieved in 3 steps from (5Z,7E,20R)-1 alpha,3 beta-bis-[(tert-butyldimethylsilyl)oxy]-20-formylmethyl-9,10-seco- 5,7,10(19) pregnatriene (5) which is derived from vitamin D2. The absolute configuration at the C-23 position of 3a and 3b was determined by the modified Mosher method. The relative affinities of 3a and 3b to the vitamin D receptor were both 10 and 14 times lower than that of 1 alpha,25-dihydroxyvitamin D3 (1), and to vitamin D binding protein were also both 130 and 40 times lower. The HL-60 cell differentiating activity of 3a was 6 times more potent than that of 1, while there was no remarkable difference in activity between 3b and 1.

The sequential oxidation and cleavage of the side chain of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha,25(OH)2D3, 24, 24-difluoro-1alpha,25-dihydroxyvitamin D3 (F2-1alpha,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha,25(OH)2D3-supplemented rats were incubated with F2-1alpha,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha,25,26-trihydroxyvitamin D3 (F2-1alpha,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha,25,26(OH)3D3. Metabolism of F2-1alpha,25(OH)2D3 into F2-1alpha,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha,25(OH)2D3 into rats. The C-24 oxidation of 1alpha,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha,25(OH)2D3 but also at C-26 of F2-1alpha,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.

The first synthesis of the title compound 4, a novel analog of 1α-hydroxyvitamin D (α-calcidiol) that might be therapeutically useful, is described. A key feature in the synthesis is the radical deoxygenation of the diol 21, which was prepared according to the previously reported procedure starting from 17. The resulting deoxygenated product 22 with the desired side-chain moiety was finally led to 4 in a conventional manner. Some aspects of the unusual reactivity of 25-OH on radical deoxygenation are also discussed.

Two cytologic variants of γδ T cell lymphoma are described. Case 1 represented a giant cell variant found in a 5-year-old Holstein cow, which had large tumor masses in the pelvic cavity. This variant consisted of very large lymphoid cells with round to oval nuclei, medium-sized nucleoli and abundant cytoplasm. Case 2 was an aborted 7-month-old female Holstein fetus, which represented an immature cell variant. Most of the neoplastic lesions were located in the skin and pleural and peritoneal submesothelial tissues. The neoplastic tissues were composed of homogeneous growth of lymphoma cells characterized by inconspicuous nucleoli and finely dispersed chromatin. Both cases demonstrated CD3, CD8 and WC1 immunoreactivity. The current study revealed that there are 4 cytologic variants (common, giant cell, hypergranular and immature cell) in bovine γδ T cell lymphomas.

An alternative synthesis of the title compound 2, a highly potent analog of 1α, 25-dihydroxyvitamin D3 (1), is described. Starting with 1α, 3β-bis[(tert-butyldimethylsilyl)oxy]androst-5-ene (8), 2 was obtained in 3.8% total yield through 10 steps. This method provides compound 2 in much higher yield than that reported previously.

Recent advances in the methodology for the synthesis of vitamin D analogs are reviewed. The synthetic methods of vitamin D are classified into three types of the approaches ; (1) conversions from steroid compounds (2) convergent synthesis (3) partial conversions from readily available vitamin D2 or D3. Their merits and demerits are discussed by using some examples from recent literatures (mainly after 1985). Modifications of vitamin D by fluorine atom (s) are also discussed briefly.