Akihiro Kikuchi

Proteasome Dysfunction Mediates Obesity-Induced Endoplasmic Reticulum Stress and Insulin Resistance in the Liver

Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. However, the molecular link between obesity and ER stress remains to be identified. Proteasomes are important multicatalytic enzyme complexes that degrade misfolded and oxidized proteins. Here, we report that both mouse models of obesity and diabetes and proteasome activator (PA)28-null mice showed 30–40% reduction in proteasome activity and accumulation of polyubiquitinated proteins in the liver. PA28-null mice also showed hepatic steatosis, decreased hepatic insulin signaling, and increased hepatic glucose production. The link between proteasome dysfunction and hepatic insulin resistance involves ER stress leading to hyperactivation of c-Jun NH2-terminal kinase in the liver. Administration of a chemical chaperone, phenylbutyric acid (PBA), partially rescued the phenotypes of PA28-null mice. To confirm part of the results obtained from in vivo experiments, we pretreated rat hepatoma-derived H4IIEC3 cells with bortezomib, a selective inhibitor of the 26S proteasome. Bortezomib causes ER stress and insulin resistance in vitro—responses that are partly blocked by PBA. Taken together, our data suggest that proteasome dysfunction mediates obesity-induced ER stress, leading to insulin resistance in the liver.

LECT2 functions as a hepatokine that links obesity to skeletal muscle insulin resistance

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.

Metformin Suppresses Expression of the Selenoprotein P Gene via an AMP-activated Kinase (AMPK)/FoxO3a Pathway in H4IIEC3 Hepatocytes

Background: The suppression of selenoprotein P production may be a novel therapeutic target for reducing insulin resistance. Results: Selenoprotein P expression was suppressed by metformin treatment, but co-administration of AMPK inhibitor or FoxO3a siRNA cancelled this suppression. Conclusion: Metformin suppresses selenoprotein P expression via the AMPK/FoxO3a pathway. Significance: The AMPK/FoxO3a pathway in the liver may be a therapeutic target for type 2 diabetes. 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.

Automated segmentation of the skeleton in whole-body bone scans: influence of difference in atlas

AimAutomated segmentation of the skeleton is the first step for quantitative analysis and computer-aided diagnosis (CAD) of whole-body bone scans. The purpose of this study was to examine the influence of differences in skeletal atlas on the automated segmentation of skeletons in a Japanese patient group. MethodsThe study was based on a bone scan CAD system that included a skeletal atlas obtained using 10 normal bone scans from European patients and 23 normal bone scans from Japanese patients. These were incorporated into the CAD system. The performance of the skeletal segmentation, based on either the European or the Japanese Atlas, was evaluated independently by three observers in a group of 50 randomly selected bone scans from Japanese patients. ResultsThe skeletal segmentation was classified as correct in 41–44 of the 50 cases by the three observers using the Japanese atlas. The corresponding results were 15–18 of the 50 cases using the European atlas, and this difference was statistically significant (P<0.001). The anatomical areas most commonly classified as not correct were the skull, cervical vertebrae, and ribs. ConclusionAutomated segmentation of the skeleton in a Japanese patient group was more successful when the CAD system based on a Japanese atlas was used than when the corresponding system based on a European atlas was used. The results of this study indicate that it is of value to use a skeletal atlas based on normal Japanese bone scans in a CAD system for Japanese patients.

Rapid response of the steatosis-sensing hepatokine LECT2 during diet-induced weight cycling in mice.

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.

Where does liver fat go? A possible molecular link between fatty liver and diabetes.

An elevation of fatty acid delivery amplifies the TCA cycle flux with a rise in anaplerosis/cataplerosis, leading to a proportional rise in oxidative stress and inflammation in liver.

Eicosapentaenoic Acid Downregulates Expression of the Selenoprotein P Gene by Inhibiting SREBP-1c Independently of the AMPK Pathway in H4IIEC3 Hepatocytes

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.

Inhibin βE (INHBE) is a possible insulin resistance-associated hepatokine identified by comprehensive gene expression analysis in human liver biopsy samples

The liver plays a major role in whole-body energy homeostasis by releasing secretory factors, termed hepatokines. To identify novel target genes associated with insulin resistance, we performed a comprehensive analysis of gene expression profiles using a DNA chip method in liver biopsy samples from humans with varying degrees of insulin resistance. Inhibin βE (INHBE) was identified as a novel putative hepatokine with hepatic gene expression that positively correlated with insulin resistance and body mass index in humans. Quantitative real time-PCR analysis also showed an increase in INHBE gene expression in independent liver samples from insulin-resistant human subjects. Additionally, Inhbe gene expression increased in the livers of db/db mice, a rodent model of type 2 diabetes. To preliminarily screen the role of Inhbe in vivo in whole-body energy metabolic status, hepatic mRNA was knocked down with siRNA for Inhbe (siINHBE) in db/db mice. Treatment with siINHBE suppressed body weight gain during the two-week experimental period, which was attributable to diminished fat rather than lean mass. Additionally, treatment with siINHBE decreased the respiratory quotient and increased plasma total ketone bodies compared with treatment with non-targeting siRNA, both of which suggest enhanced whole-body fat utilization. Our study suggests that INHBE functions as a possible hepatokine to alter the whole-body metabolic status under obese insulin-resistant conditions.

p62-mediated autophagy affects nutrition-dependent insulin receptor substrate 1 dynamics in 3T3-L1 preadipocytes

Previous studies have shown that an organisms nutritional status changes the protein levels of insulin receptor substrate 1 (IRS‐1) in a tissue‐specific manner. Although the mechanisms underlying the regulation of IRS‐1 in the nutrient‐rich conditions associated with diabetes and insulin resistance have been well studied, those under nutrient‐poor conditions remain unknown. The aim of the present study was to investigate how IRS‐1 protein levels change depending on the nutritional status of 3T3‐L1 preadipocytes.