Importance of endothelial NF-κB signalling in vascular remodelling and aortic aneurysm formation.

AIMS: Vascular remodelling and aortic aneurysm formation are induced mainly by inflammatory responses in the adventitia and media. However, relatively little is known about the mechanistic significance of endothelium in the pathogenesis of these vascular disorders. The transcription factor nuclear factor-kappa B (NF-κB) regulates the expressions of numerous genes, including those related to pro-inflammatory responses. Therefore, to investigate the roles of endothelial pro-inflammatory responses, we examined the impact of blocking endothelial NF-κB signalling on intimal hyperplasia and aneurysm formation. METHODS AND RESULTS: To block endothelial NF-κB signalling, we used transgenic mice expressing dominant-negative IκBα selectively in endothelial cells (E-DNIκB mice). E-DNIκB mice were protected from the development of cuff injury-induced neointimal formation, in association with suppressed arterial expressions of cellular adhesion molecules, a macrophage marker, and inflammatory factors. In addition, the blockade of endothelial NF-κB signalling prevented abdominal aortic aneurysm formation in an experimental model, hypercholesterolaemic apolipoprotein E-deficient mice with angiotensin II infusion. In this aneurysm model as well, aortic expressions of an adhesion molecule, a macrophage marker, and inflammatory factors were suppressed with the inhibited expression and activity of matrix metalloproteinases in the aorta. CONCLUSION: Endothelial NF-κB activation up-regulates adhesion molecule expression, which may trigger macrophage infiltration and inflammation in the adventitia and media. Thus, the endothelium plays important roles in vascular remodelling and aneurysm formation through its intracellular NF-κB signalling.

Function of adrenomedullin in inflammatory response of liver against LPS-induced endotoxemia.

Adrenomedullin (AM) is a hypotension-causing peptide that was originally isolated from human pheochromocytoma cells, and it has been found to be expressed in various organs, including the liver. As the individual physiological and pathophysiological properties of AM peptide in the liver during endotoxemia in vivo has not yet been examined, we investigated this in experimental endotoxemia using heterozygote AM-deficient (AM(+/-)) mice. The AM concentration of AM(+/-) mice was significantly lesser than that of wild-type (WT) mice in lipopolysaccharide (LPS)-induced endotoxemia. After administering LPS, the survival rate for AM(+/-) mice was significantly lower than that for WT mice. Also, expressions of IL-1ß mRNA, and TNF-α mRNA, and NF-κB p65 in the liver were markedly increased and serum ALT greatly elevated in comparison with WT mice. However, supplementation of exogenous AM reversed the deteriorations in mortality and inflammatory responses. Therefore, we conclude that AM plays an important role in regulating systemic inflammation and may be an important intrinsic factor for protecting against liver damage in LPS-induced endotoxemia.

Blockade of the nuclear factor-κB pathway in the endothelium prevents insulin resistance and prolongs life spans.

BACKGROUND: Nuclear factor-κB (NF-κB) signaling plays critical roles in physiological and pathological processes such as responses to inflammation and oxidative stress. METHODS AND RESULTS: To examine the role of endothelial NF-κB signaling in vivo, we generated transgenic mice expressing dominant-negative IκB under the Tie2 promoter/enhancer (E-DNIκB mice). These mice exhibited functional inhibition of NF-κB signaling specifically in endothelial cells. Although E-DNIκB mice displayed no overt phenotypic changes when young and lean, they were protected from the development of insulin resistance associated with obesity, whether diet- or genetics-induced. Obesity-induced macrophage infiltration into adipose tissue and plasma oxidative stress markers were decreased and blood flow and mitochondrial content in muscle and active-phase locomotor activity were increased in E-DNIκB mice. In addition to inhibition of obesity-related metabolic deteriorations, blockade of endothelial NF-κB signaling prevented age-related insulin resistance and vascular senescence and, notably, prolonged life span. These antiaging phenotypes were also associated with decreased oxidative stress markers, increased muscle blood flow, enhanced active-phase locomotor activity, and aortic upregulation of mitochondrial sirtuin-related proteins. CONCLUSIONS: The endothelium plays important roles in obesity- and age-related disorders through intracellular NF-κB signaling, thereby ultimately affecting life span. Endothelial NF-κB signaling is a potential target for treating the metabolic syndrome and for antiaging strategies.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis.

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.

Fasting tests of insulin secretion and sensitivity predict future prediabetes in Japanese with normal glucose tolerance.

UNLABELLED: Aims/Introduction: Reduced insulin sensitivity and secretion are important in the pathogenesis of type 2 diabetes. Their relationships to prediabetes, impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) have been previously studied with the oral glucose tolerance test (OGTT). We investigated whether or not baseline measures of insulin secretion and sensitivity obtained from fasting blood specimens were related to the development of prediabetes and how these measures compared with those based on the OGTT. MATERIALS AND METHODS: In 152 Japanese subjects with normal glucose tolerance, we measured baseline plasma glucose and insulin after an overnight fast and during a 75 g OGTT, insulin resistance index (homeostasis model assessment [HOMA-IR]), and insulin secretion (insulinogenic index [30 min insulin - fasting insulin] ÷ [30 min glucose - fasting glucose] or HOMA-ß). RESULTS: At a 5-6 year (mean 5.7 years) follow-up examination, we confirmed 36 cases of prediabetes. After adjusting for age, sex, family history of diabetes, body mass index, and 2-h plasma glucose, the odds ratio comparing the lowest tertile (≤0.82) of insulinogenic index with the highest tertile (≥1.43) was 6.98 (95% confidence interval, 1.96-24.85) and was 10.72 (2.08-55.3) comparing the lowest tertile (≤76.3) of HOMA-ß with the highest tertile (≥122.1), whereas the respective odds ratios of HOMA-IR were 3.74 (1.03-13.57) and 10.89 (1.93-61.41) comparing the highest tertile (≥1.95) with the lowest tertile (≤1.25). CONCLUSIONS: Lower insulin secretion and sensitivity are independent risk factors for prediabetes. Clinically practical identification of those at risk for prediabetes is obtainable from HOMA-ß and HOMA-IR, both of which are measured in fasting state. (J Diabetes Invest, doi: 10.1111.j.2040-1124.2010.00041.x, 2010).

Peginterferon (PEG-IFN) plus ribavirin combination therapy, but neither interferon nor PGE-IFN alone, induced type 1 diabetes in a patient with chronic hepatitis C.

Interferon (IFN) therapies, including IFN, peginterferon (PEG-IFN) and ribavirin (RBV) plus PEG-IFN combination, are widely used for patients with chronic hepatitis C. We encountered a patient with chronic hepatitis C in whom previous IFN or PEG-IFN alone had not induced type 1 diabetes (T1D), while the addition of RBV to PEG-IFN did induce T1D. The patient had HLA types conferring highly susceptibility to T1D. Thus, adding RBV to PEG-IFN may render chronic hepatitis C patients, with T1D-susceptible HLA types, more prone to developing T1D than IFN or PEG-IFN alone. To prevent T1D development, we recommend HLA typing prior to initiating RBV plus PEG-IFN administration.

Impact of plasma oxidized low-density lipoprotein removal on atherosclerosis.

BACKGROUND: Several clinical studies of statin therapy have demonstrated that lowering low-density lipoprotein (LDL) cholesterol prevents atherosclerotic progression and decreases cardiovascular mortality. In addition, oxidized LDL (oxLDL) is suggested to play roles in the formation and progression of atherosclerosis. However, whether lowering oxLDL alone, rather than total LDL, affects atherogenesis remains unclear. METHODS AND RESULTS: To clarify the atherogenic impact of oxLDL, lectin-like oxLDL receptor 1 (LOX-1), an oxLDL receptor, was expressed ectopically in the liver with adenovirus administration in apolipoprotein E-deficient mice at 46 weeks of age. Hepatic LOX-1 expression enhanced hepatic oxLDL uptake, indicating functional expression of LOX-1 in the liver. Although plasma total cholesterol, triglyceride, and LDL cholesterol levels were unaffected, plasma oxLDL was markedly and transiently decreased in LOX-1 mice. In controls, atherosclerotic lesions, detected by Oil Red O staining, were markedly increased (by 38%) during the 4-week period after adenoviral administration. In contrast, atherosclerotic progression was almost completely inhibited by hepatic LOX-1 expression. In addition, plasma monocyte chemotactic protein-1 and lipid peroxide levels were decreased, whereas adiponectin was increased, suggesting decreased systemic oxidative stress. Thus, LOX1 expressed in the livers of apolipoprotein E-deficient mice transiently removes oxLDL from circulating blood and possibly decreases systemic oxidative stress, resulting in complete prevention of atherosclerotic progression despite the persistence of severe LDL hypercholesterolemia and hypertriglyceridemia. CONCLUSIONS: OxLDL has a major atherogenic impact, and oxLDL removal is a promising therapeutic strategy against atherosclerosis.

Regulation of gut-derived resistin-like molecule beta expression by nutrients.

Resistin was initially identified as a protein, secreted by adipocytes, which inhibits insulin action and adipose differentiation. The three proteins homologous to resistin were identified and given the names resistin-like molecules (RELM) alpha, beta and gamma. Resistin and RELMalpha are abundantly expressed in adipose, but RELMbeta and RELMgamma are secreted mainly from the gut. Since nutrient composition greatly affects insulin sensitivity, we investigated the regulatory effects of various nutritional factors in food on the expressions of resistin family proteins. First, mice were given diets with different nutritional compositions (high-carbohydrate, high-protein and high-fat) for 2 weeks. RELMbeta mRNA expression in the intestines was markedly suppressed by the high-protein and high-carbohydrate diets, while slightly but not significantly upregulated by the high-fat diet. In the epididymal fat, resistin expression was unchanged, while RELMalpha expression was markedly decreased by the high-carbohydrate diet. Taking into consideration that humans have neither RELMalpha nor RELMgamma, our subsequent studies focused on RELMbeta expression. We used the human colon cancer cell line LS174T. Treatments with insulin and TNFalpha as well as stearic acid, a saturated free fatty acid, upregulated RELMbeta expression, while d-glucose downregulated RELMbeta. These results suggest RELMbeta expression to be regulated directly by nutrients such as glucose and saturated free fatty acids including stearic acid, as well as by hormones including insulin and TNFalpha. These regulations may play an important role in the nutrient-associated induction of insulin resistance.

A novel method for evaluating human carotid artery elasticity: possible detection of early stage atherosclerosis in subjects with type 2 diabetes.

We recently developed a novel method for evaluating the elasticity of arterial walls, the phased tracking method. Herein, we evaluated atherosclerosis of the carotid artery with this method in 242 individuals with type 2 diabetes. In multiple regression analysis of subject status, age, systolic blood pressure and hyperlipidemia were found to be independently associated with carotid artery elasticity values. We also measured currently established values for atherosclerosis, carotid artery IMT and baPWV, in these subjects. Carotid artery elasticity correlated with max IMT (r=0.291, p<0.01), plaque score (PS) (r=0.220, p<0.01) and baPWV (r=0.345, p<0.01). Elasticity, max IMT and plaque score, all correlated with the number of risk factors for atherosclerosis, i.e. hypertension, hyperlipidemia and smoking, in addition to diabetes, consistent with the view that these values reflect atherosclerosis. Importantly, however, in subjects with IMT <1.1mm, who are classified as not having atherosclerosis as defined by IMT criteria, only carotid artery elasticity correlated with the number of risk factors (p<0.05). These results suggest that (1) the measured carotid artery elasticity values reflect atherosclerosis and (2) our novel method has potential for detecting atherosclerosis in its early stage.

Carboxy-terminal modulator protein induces Akt phosphorylation and activation, thereby enhancing antiapoptotic, glycogen synthetic, and glucose uptake pathways.

Carboxy-terminal modulator protein (CTMP) was identified as binding to the carboxy terminus of Akt and inhibiting the phosphorylation and activation of Akt. In contrast to a previous study, we found CTMP overexpression to significantly enhance Akt phosphorylation at both Thr(308) and Ser(473) as well as the kinase activity of Akt, while phosphatidylinositol 3-kinase (PI3-kinase) activity was unaffected. Translocation of Akt to the membrane fraction was also markedly increased in response to overexpression of CTMP, with no change in the whole cellular content of Akt. Furthermore, the phosphorylations of GSK-3beta and Foxo1, well-known substrates of Akt, were increased by CTMP overexpression. On the other hand, suppression of CTMP with small interfering RNA partially but significantly attenuated this Akt phosphorylation. The cellular activities reportedly mediated by Akt activation were also enhanced by CTMP overexpression. UV-B-induced apoptosis of HeLa cells was significantly reversed not only by overexpression of the active mutant of Akt (myr-Akt) but also by that of CTMP. Increases in glucose transport activity and glycogen synthesis were also induced by overexpression of either myr-Akt or CTMP in 3T3-L1 adipocytes. Taking these results into consideration, it can be concluded that CTMP induces translocation of Akt to the membrane and thereby increases the level of Akt phosphorylation. As a result, CTMP enhances various cellular activities that are principally mediated by the PI3-kinase/Akt pathway.

Bone marrow (BM) transplantation promotes beta-cell regeneration after acute injury through BM cell mobilization.

There is controversy regarding the roles of bone marrow (BM)-derived cells in pancreatic beta-cell regeneration. To examine these roles in vivo, mice were treated with streptozotocin (STZ), followed by bone marrow transplantation (BMT; lethal irradiation and subsequent BM cell infusion) from green fluorescence protein transgenic mice. BMT improved STZ-induced hyperglycemia, nearly normalizing glucose levels, with partially restored pancreatic islet number and size, whereas simple BM cell infusion without preirradiation had no effects. In post-BMT mice, most islets were located near pancreatic ducts and substantial numbers of bromodeoxyuridine-positive cells were detected in islets and ducts. Importantly, green fluorescence protein-positive, i.e. BM-derived, cells were detected around islets and were CD45 positive but not insulin positive. Then to examine whether BM-derived cell mobilization contributes to this process, we used Nos3(-/-) mice as a model of impaired BM-derived cell mobilization. In streptozotocin-treated Nos3(-/-) mice, the effects of BMT on blood glucose, islet number, bromodeoxyuridine-positive cells in islets, and CD45-positive cells around islets were much smaller than those in streptozotocin-treated Nos3(+/+) controls. A series of BMT experiments using Nos3(+/+) and Nos3(-/-) mice showed hyperglycemia-improving effects of BMT to correlate inversely with the severity of myelosuppression and delay of peripheral white blood cell recovery. Thus, mobilization of BM-derived cells is critical for BMT-induced beta-cell regeneration after injury. The present results suggest that homing of donor BM-derived cells in BM and subsequent mobilization into the injured periphery are required for BMT-induced regeneration of recipient pancreatic beta-cells.

Involvement of apolipoprotein E in excess fat accumulation and insulin resistance.

Although apolipoprotein E (apoE) is well known to play a major role in lipid metabolism, its role in glucose and energy homeostasis remains unclear. Herein, we established apoE-deficient genetically obese Ay (apoE(-/-);Ay/+) mice. ApoE deficiency in Ay mice prevented the development of obesity, with decreased fat accumulation in the liver and adipose tissues. ApoE(-/-);Ay/+ mice exhibited better glucose tolerance than apoE(+/+);Ay/+ mice. Insulin tolerance testing and hyperinsulinemic-euglycemic clamp study revealed marked improvement of insulin sensitivity, despite increased plasma free fatty acid levels. These metabolic phenotypes were reversed by adenoviral replenishment of apoE protein, indicating circulating apoE to be involved in increased adiposity and obesity-related metabolic disorders. Uptake of apoE-lacking VLDL into the liver and adipocytes was markedly inhibited, but adipocytes in apoE(-/-);Ay/+ mice exhibited normal differentiation, suggesting that apoE-dependent VLDL transport is involved in the development of obesity, i.e., surplus fat accumulation. Interestingly, apoE(-/-);Ay/+ mice exhibited decreased food intake and increased energy expenditure. Pair-feeding experiments indicate these phenomena to both contribute to the obesity-resistant phenotypes associated with apoE deficiency. Thus, apoE is involved in maintaining energy homeostasis. ApoE-dependent excess fat accumulation is a promising therapeutic target for the metabolic syndrome.

Cold exposure suppresses serum adiponectin levels through sympathetic nerve activation in mice.

OBJECTIVE: Several lines of evidence suggest important roles for adiponectin in glucose and lipid metabolism and atherosclerosis. However, the mechanisms regulating serum adiponectin levels and adiponectin production are still not completely understood. Our aim was to determine whether adiponectin synthesis is physiologically regulated by the sympathetic nervous system (SNS). RESEARCH METHODS AND PROCEDURES: Mice were exposed to cold (4 degrees C) for 12 hours and for 24 hours with or without inhibition of noradrenaline synthesis or pan-beta adrenergic function, followed by measurement of serum adiponectin concentrations and levels of adiponectin and uncoupling protein (UCP) 1 expressions in various white adipose tissues (WATs). RESULTS: Cold exposure significantly reduced serum adiponectin concentrations without changing body weights or WAT sizes in either subcutaneous or intra-abdominal fat tissues. The serum adiponectin reduction was associated with a decrease in adiponectin mRNA expression in subcutaneous, epididymal, and mesenteric fat tissues. In these adipose tissues, UCP1 expression was markedly enhanced, suggesting SNS activation in these tissues. Administration of alpha-methyl-p-tyrosine or a combination of SR59230A and propranolol reversed the cold-exposure-induced decreases in serum adiponectin concentrations and adiponectin mRNA expression in these tissues. In contrast, in retroperitoneal fat, the effects of cold exposure on adiponectin and UCP1 expressions were strikingly weak but were not reversed by SNS inhibitors. DISCUSSION: SNS physiologically regulates serum adiponectin levels and adiponectin synthesis in WATs in vivo, although responsiveness to SNS stimulation differs markedly among WATs. Sympathetic activation might be involved in development of the metabolic syndrome by modulation of serum adiponectin concentrations.

Neuronal pathway from the liver modulates energy expenditure and systemic insulin sensitivity.

Coordinated control of energy metabolism and glucose homeostasis requires communication between organs and tissues. We identified a neuronal pathway that participates in the cross talk between the liver and adipose tissue. By studying a mouse model, we showed that adenovirus-mediated expression of peroxisome proliferator-activated receptor (PPAR)-g2 in the liver induces acute hepatic steatosis while markedly decreasing peripheral adiposity. These changes were accompanied by increased energy expenditure and improved systemic insulin sensitivity. Hepatic vagotomy and selective afferent blockage of the hepatic vagus revealed that the effects on peripheral tissues involve the afferent vagal nerve. Furthermore, an antidiabetic thiazolidinedione, a PPARg agonist, enhanced this pathway. This neuronal pathway from the liver may function to protect against metabolic perturbation induced by excessive energy storage.

Signals from intra-abdominal fat modulate insulin and leptin sensitivity through different mechanisms: neuronal involvement in food-intake regulation.

Intra-abdominal fat accumulation is involved in development of the metabolic syndrome, which is associated with insulin and leptin resistance. We show here that ectopic expression of very low levels of uncoupling protein 1 (UCP1) in epididymal fat (Epi) reverses both insulin and leptin resistance. UCP1 expression in Epi improved glucose tolerance and decreased food intake in both diet-induced and genetically obese mouse models. In contrast, UCP1 expression in Epi of leptin-receptor mutant mice did not alter food intake, though it significantly decreased blood glucose and insulin levels. Thus, hypophagia induction requires a leptin signal, while the improved insulin sensitivity appears to be leptin independent. In wild-type mice, local-nerve dissection in the epididymis or pharmacological afferent blockade blunted the decrease in food intake, suggesting that afferent-nerve signals from intra-abdominal fat tissue regulate food intake by modulating hypothalamic leptin sensitivity. These novel signals are potential therapeutic targets for the metabolic syndrome.

Physiological significance of resistin and resistin-like molecules in the inflammatory process and insulin resistance.

Resistin was initially identified as a protein, secreted by adipocytes, which inhibits insulin action and adipose differentiation. The three proteins homologous to resistin were termed resistin-like molecules (RELM) alpha, beta and gamma. Resistin and RELMalpha are abundantly expressed in adipose, but RELMbeta and RELMgamma are secreted mainly from the gut. Recently, resistin and RELMs were reported to be associated with inflammation. For example, RELMalpha, viewed as an inflammation-related protein, was originally identified in broncho-alveolar lavage fluid obtained from animals with experimentally induced pulmonary inflammation. RELMbeta is also related to bacterial colonization, but RELMbeta injection or hepatic overexpression of RELMbeta induced insulin resistance. RELMgamma isolated from rat nasal respiratory epithelium was found to be altered by cigarette smoke. Thus, resistin and RELMs could be useful for assessing the inflammatory condition in vivo. On the other hand, whether the serum resistin or RELM concentration is strongly related to insulin resistance remains unclear. However, taking recent studies showing a close relationship between inflammation and insulin resistance in diabetes into consideration, these proteins may have interactive roles linking inflammation and insulin resistance, both of which major involvement in the progression of atherosclerosis. If so, the serum resistin or RELM concentration may be a good marker of atherosclerotic risk. In addition, these proteins or unidentified receptors are potential therapeutic targets for the treatment of diabetes and prevention of atherosclerosis. These possibilities merit further study.

Resistin-like molecule beta activates MAPKs, suppresses insulin signaling in hepatocytes, and induces diabetes, hyperlipidemia, and fatty liver in transgenic mice on a high fat diet.

Resistin and resistin-like molecules (RELMs) are a family of proteins reportedly related to insulin resistance and inflammation. Because the serum concentration and intestinal expression level of RELMbeta were elevated in insulin-resistant rodent models, in this study we investigated the effect of RELMbeta on insulin signaling and metabolism using transgenic mice and primary cultured hepatocytes. First, transgenic mice with hepatic RELMbeta overexpression were shown to exhibit significant hyperglycemia, hyperlipidemia, fatty liver, and pancreatic islet enlargement when fed a high fat diet. Hyperinsulinemic glucose clamp showed a decreased glucose infusion rate due to increased hepatic glucose production. In addition, the expression levels of IRS-1 and IRS-2 proteins as well as the degrees of insulin-induced phosphatidylinositol 3-kinase and Akt activations were attenuated in RELMbeta transgenic mice. Similar down-regulations of IRS-1 and IRS-2 proteins were observed in primary cultured hepatocytes chronically treated (for 24 h) with RELMbeta, suggesting the insulin resistance-inducing effect of RELMbeta to be direct. Furthermore, it was shown that RELMbeta acutely and markedly activates ERK and p38, while weakly activating JNK, in primary cultured hepatocytes. This increased basal p38 phosphorylation level was also observed in the livers of RELMbeta transgenic mice. In conclusion, RELMbeta, a gut-derived hormone, impairs insulin signaling probably via the activations of classic MAPKs, and increased expression of RELMbeta may be involved in the pathogenesis of glucose intolerance and hyperlipidemia in some insulin-resistant models. Thus, RELMbeta is a potentially useful marker for assessing insulin resistance and may also be a target for future novel anti-diabetic agents.

Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity.

AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.

A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis.

Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast two-hybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APE-induced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.