Noboru Furukawa

Diet-induced obesity alters AMP kinase activity in hypothalamus and skeletal muscle

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10–12 weeks. Leptin acutely decreased food intake by ∼30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased α2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited α2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters α2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.

Neuronal Protein Tyrosine Phosphatase 1B Deficiency Results in Inhibition of Hypothalamic AMPK and Isoform-Specific Activation of AMPK in Peripheral Tissues

ABSTRACT PTP1B−/− mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptins metabolic effects. We find that α1 and α2 AMPK activity are elevated and acetyl-coenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B−/− mice. The effects of PTP1B deficiency on α2, but not α1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron- but not muscle-specific PTP1B−/− mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B−/− mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.

Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis

Angiopoietin-like protein family 4 (Angptl 4) has been shown to regulate lipoprotein metabolism through the inhibition of lipoprotein lipase (LPL). We generated ApoE(-/-)Angptl 4(-/-) mice to study the effect of Angptl 4 deficiency on lipid metabolism and atherosclerosis. Fasting and postolive oil-loaded triglyceride (TG) levels were largely decreased in ApoE(-/-)Angptl 4(-/-) mice compared with and ApoE(-/-)Angptl 4(+/+) mice. There was a significant (75+/-12%) reduction in atherosclerotic lesion size in ApoE(-/-)Angptl 4(-/-) mice compared with ApoE(-/-) Angptl 4(+/+) mice. Peritoneal macrophages, isolated from Angptl 4(-/-) mice to investigate the foam cell formation, showed a significant decrease in newly synthesized cholesteryl ester (CE) accumulation induced by acetyl low-density lipoprotein (acLDL) compared with those from Angptl 4(+/+) mice. Thus, genetic knockout of Angptl 4 protects ApoE(-/-) mice against development and progression of atherosclerosis and strongly suppresses the ability of the macrophages to become foam cells in vitro.

Impact of Natural IRS-1 Mutations on Insulin Signals: Mutations of IRS-1 in the PTB Domain and Near SH2 Protein Binding Sites Result in Impaired Function at Different Steps of IRS-1 Signaling

Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and m209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and m209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in ]3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and m209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and m209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and m209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.

Hyperthermia With Mild Electrical Stimulation Protects Pancreatic β-Cells From Cell Stresses and Apoptosis

Induction of heat shock protein (HSP) 72 improves metabolic profiles in diabetic model mice. However, its effect on pancreatic β-cells is not known. The current study investigated whether HSP72 induction can reduce β-cell stress signaling and apoptosis and preserve β-cell mass. MIN6 cells and db/db mice were sham-treated or treated with heat shock (HS) and mild electrical stimulation (MES) (HS+MES) to induce HSP72. Several cellular markers, metabolic parameters, and β-cell mass were evaluated. HS+MES treatment or HSP72 overexpression increased HSP72 protein levels and decreased tumor necrosis factor (TNF)-α–induced Jun NH2-terminal kinase (JNK) phosphorylation, endoplasmic reticulum (ER) stress, and proapoptotic signal in MIN6 cells. In db/db mice, HS+MES treatment for 12 weeks significantly improved insulin sensitivity and glucose homeostasis. Upon glucose challenge, a significant increase in insulin secretion was observed in vivo. Compared with sham treatment, levels of HSP72, insulin, pancreatic duodenal homeobox-1, GLUT2, and insulin receptor substrate-2 were upregulated in the pancreatic islets of HS+MES-treated mice, whereas JNK phosphorylation, nuclear translocation of forkhead box class O-1, and nuclear factor-κB p65 were reduced. Apoptotic signals, ER stress, and oxidative stress markers were attenuated. Thus, HSP72 induction by HS+MES treatment protects β-cells from apoptosis by attenuating JNK activation and cell stresses. HS+MES combination therapy may preserve pancreatic β-cell volume to ameliorate glucose homeostasis in diabetes.

A carboxy-terminal truncation of human alpha-galactosidase A in a heterozygous female with Fabry disease and modification of the enzymatic activity by the carboxy-terminal domain. Increased, reduced, or absent enzyme activity depending on number of amino acid residues deleted.

Fabry disease is an X-linked disorder of glycosphingolipid metabolism caused by a deficiency of alpha-galactosidase A (alpha-Gal A). We identified a novel mutation of alpha-Gal A gene in a family with Fabry disease, which converted a tyrosine at codon 365 to a stop and resulted in a truncation of the carboxy (C) terminus by 65 amino acid (AA) residues. In a heterozygote of this family, although the mutant and normal alleles were equally transcribed in cultured fibroblasts, lymphocyte alpha-Gal A activity was approximately 30% of the normal control and severe clinical symptoms were apparent. COS-1 cells transfected with this mutant cDNA showed a complete loss of its enzymatic activity. Furthermore, those cotransfected with mutant and wildtype cDNAs showed a lower alpha-Gal A activity than those with wild type alone (approximately 30% of wild type alone), which suggested the dominant negative effect of this mutation and implied the importance of the C terminus for its activity. Thus, we generated mutant cDNAs with various deletion of the C terminus, and analyzed. Unexpectedly, alpha-Gal A activity was enhanced by up to sixfold compared with wild-type when from 2 to 10 AA residues were deleted. In contrast, deletion of 12 or more AA acid residues resulted in a complete loss of enzyme activity. Our data suggest that the C-terminal region of alpha-Gal A plays an important role in the regulation of its enzyme activity.

Caloric restriction decreases ER stress in liver and adipose tissue in ob/ob mice

Endoplasmic reticulum (ER) stress plays a crucial role in the development of insulin resistance and diabetes. Although caloric restriction (CR) improves obesity-related disorders, the effects of CR on ER stress in obesity remain unknown. To investigate how CR affects ER stress in obesity, ob/ob mice were assigned to either ad libitum (AL) (ob-AL) or CR (ob-CR) feeding (2 g food/day) for 1-4 weeks. The body weight (BW) of ob-CR mice decreased to the level of lean AL-fed littermates (lean-AL) within 2 weeks. BW of lean-AL and ob-CR mice was less than that of ob-AL mice. The ob-CR mice showed improved glucose tolerance and hepatic insulin action compared with ob-AL mice. Levels of ER stress markers such as phosphorylated PKR-like ER kinase (PERK) and eukaryotic translation initiation factor 2α and the mRNA expression of activating transcription factor 4 were significantly higher in the liver and epididymal fat from ob-AL mice compared with lean-AL mice. CR for 2 and 4 weeks significantly reduced all of these markers to less than 35% and 50%, respectively, of the levels in ob-AL mice. CR also significantly reduced the phosphorylation of insulin receptor substrate (IRS)-1 and c-Jun NH(2)-terminal kinase (JNK) in ob/ob mice. The CR-mediated decrease in PERK phosphorylation was similar to that induced by 4-phenyl butyric acid, which reduces ER stress in vivo. In conclusion, CR reduced ER stress and improved hepatic insulin action by suppressing JNK-mediated IRS-1 serine-phosphorylation in ob/ob mice.

The role of class I histone deacetylase (HDAC) on gluconeogenesis in liver.

Hepatic gluconeogenesis is crucial for glucose homeostasis. Although sirtuin 1 (Sirt1) is implicated in the regulation of gluconeogenesis in the liver, the effects of other histone deacetylases (HDAC) on gluconeogenesis are unclear. The aim of this study was to identify the role of class I HDACs in hepatic gluconeogenesis. In HepG2 cells and the liver of mice, the expressions of phosphoenol pyruvate carboxykinase (PEPCK) and hepatocyte nuclear factor 4α (HNF4α) were significantly decreased by treatment with a newly designed class I HDAC inhibitor, Ky-2. SiRNA knockdown of HDAC1 expression, but not of HDAC2 or HDAC3, in HepG2 cells decreased PEPCK and HNF4α expression. In HepG2 cells, insulin-stimulated phosphorylation of Akt and forkhead box O 1 (FoxO1) was increased by Ky-2. Pyruvate tolerance tests in Ky-2-treated high-fat-diet (HFD)-fed mice showed a marked reduction in blood glucose compared with vehicle-treated HFD mice. These data suggest that class I HDACs increase HNF4α protein expression and the transcriptional activity of FoxO1, followed by the induction of PEPCK mRNA expression and gluconeogenesis in liver.

Cis-acting DNA elements of mouse granulocyte/macrophage colony-stimulating factor gene responsive to oxidized low density lipoprotein.

We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5′-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from −97 to −59 and from −59 to −37 and one negative regulatory site from −120 to −97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from −225 to −120 and one negative responsive site from −97 to −59, which contained the NF-κB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from −169 to −160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-κB binding site. Supershift assay showed that nuclear proteins bound to NF-κB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from −169 to −160 was a positive responsive element to Ox-LDL and that the NF-κB binding site from −91 to −82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.

Cell-Specific Regulation of IRS-1 Gene Expression: Role of E Box and C/EBP Binding Site in HepG2 Cells and CHO Cells

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter–CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from −1645 to −1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region sa fragment from −1645 to −1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from −1605 to −1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] −1635 to −1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt −1599 to −1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.