Hypoxia causes pancreatic ß-cell dysfunction and impairs insulin secretion by activating the transcriptional repressor BHLHE40.

Hypoxia can occur in pancreatic ß-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on ß-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human ß-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or ß-cells of ob/ob mice reverses defects in insulin secretion. Mechanistically, BHLHE40 represses the expression of Mafa, encoding the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating the binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic ß-cells was recovered by MAFA re-expression. Collectively, our work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in ß-cells that inhibit insulin secretion by suppressing MAFA expression.

Roles of ß-Cell Hypoxia in the Progression of Type 2 Diabetes.

Type 2 diabetes is a chronic disease marked by hyperglycemia; impaired insulin secretion by pancreatic ß-cells is a hallmark of this disease. Recent studies have shown that hypoxia occurs in the ß-cells of patients with type 2 diabetes and hypoxia, in turn, contributes to the insulin secretion defect and ß-cell loss through various mechanisms, including the activation of hypoxia-inducible factors, induction of transcriptional repressors, and activation of AMP-activated protein kinase. This review focuses on advances in our understanding of the contribution of ß-cell hypoxia to the development of ß-cell dysfunction in type 2 diabetes. A better understanding of ß-cell hypoxia might be useful in the development of new strategies for treating type 2 diabetes.

SIRT7 Deficiency Protects against Aß42-Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells.

Alzheimer's disease (AD) is an age-related neurodegenerative disease that is characterized by irreversible memory loss and cognitive decline. The deposition of amyloid-ß (Aß), especially aggregation-prone Aß42, is considered to be an early event preceding neurodegeneration in AD. Sirtuins (SIRT1-7 in mammals) are nicotinamide adenine dinucleotide-dependent lysine deacetylases/deacylases, and several sirtuins play important roles in AD. However, the involvement of SIRT7 in AD pathogenesis is not known. Here, we demonstrate that SIRT7 mRNA expression is increased in the cortex, entorhinal cortex, and prefrontal cortex of AD patients. We also found that Aß42 treatment rapidly increased NADPH oxidase 4 (NOX4) expression at the post-transcriptional level, and induced reactive oxygen species (ROS) production and apoptosis in neuronal SH-SY5Y cells. In contrast, SIRT7 knockdown inhibited Aß42-induced ROS production and apoptosis by suppressing the upregulation of NOX4. Collectively, these findings suggest that the inhibition of SIRT7 may play a beneficial role in AD pathogenesis through the regulation of ROS production.

Development of a DELFIA method to detect oncofetal antigen ROR1-positive exosomes.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed in a wide variety of hematological and solid cancers, but is low or absent in adult tissues. Here, we show that ROR1 is released with exosomes from ROR1-positive cancer cells. We also developed a simple dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) to detect cancer-derived ROR1-positive exosomes, which are captured by two anti-ROR1 antibodies and detected by the fluorescence of free chelating europium. This new DELFIA method can detect cancer-derived ROR1-positive exosomes in the cell supernatant and serum with a wide range and rapidly compared with the conventional Western blot assay. This method may be useful as a companion diagnostics for ROR1-positive cancers.

Brown adipocyte-derived exosomal miR-132-3p suppress hepatic Srebf1 expression and thereby attenuate expression of lipogenic genes.

Recent evidence has revealed a novel signaling mechanism through which brown adipose tissue (BAT)-derived exosomal microRNAs (miRNAs) influence hepatic gene expression. Here, we uncover neuronal control of these miRNAs and identify exosomal miR-132-3p as a regulator of hepatic lipogenesis under cold stress conditions. Norepinephrine, a sympathetic nervous system neurotransmitter mediating cold-induced BAT activation, altered the composition of brown adipocyte (BAC)-derived exosomal miRNAs; among them, miR-132-3p was significantly induced. The isolated BAC-derived exosomes suppressed expression of hepatic Srebf1, a predicted target of miR-132-3p. In an indirect co-culture system, BACs suppressed expression of hepatic Srebf1 and its target lipogenic genes; this effect was not seen with miR-132-3p-inhibited BACs. Srebf1 was experimentally validated as an miR-132-3p target. Cold stimuli consistently induced miR-132-3p expression in BAT and attenuated Srebf1 expression in the liver. Our results suggest that BAT-derived exosomal miR-132-3p acts as an endocrine factor that regulates hepatic lipogenesis for cold adaptation.

Hypoxia reduces HNF4α/MODY1 protein expression in pancreatic ß-cells by activating AMP-activated protein kinase.

Hypoxia plays a role in the deterioration of ß-cell function. Hepatocyte nuclear factor 4α (HNF4α) has an important role in pancreatic ß-cells, and mutations of the human HNF4A gene cause a type of maturity-onset diabetes of the young (MODY1). However, it remains unclear whether hypoxia affects the expression of HNF4α in ß-cells. Here, we report that hypoxia reduces HNF4α protein expression in ß-cells. Hypoxia-inducible factor was not involved in the down-regulation of HNF4α under hypoxic conditions. The down-regulation of HNF4α was dependent on the activation of AMP-activated protein kinase (AMPK), and the reduction of HNF4α protein expression by metformin, an AMPK activator, and hypoxia was inhibited by the overexpression of a kinase-dead (KD) form of AMPKα2. In addition, hypoxia decreased the stability of the HNF4α protein, and the down-regulation of HNF4α was sensitive to proteasome inhibitors. Adenovirus-mediated overexpression of KD-AMPKα2 improved insulin secretion in metformin-treated islets, hypoxic islets, and ob/ob mouse islets. These results suggest that down-regulation of HNF4α could be of importance in ß-cell dysfunction by hypoxia.

SIRT7 is an important regulator of cartilage homeostasis and osteoarthritis development.

Sirtuins (SIRT1-7) are NAD+-dependent deacetylase/deacylases that regulate a wide variety of biological functions. Although the roles of sirtuins in cartilage homeostasis and cartilage diseases have been well studied, there is no information on the contribution of SIRT7 to cartilage homeostasis and osteoarthritis (OA) pathologies. Here, we demonstrate that Sirt7 knockout mice are resistant to the development of aging-associated OA and forced exercise-induced OA. Attenuation of Sirt7 in the murine chondrogenic cell line ATDC5 increased the deposition of a glycosaminoglycan-rich extracellular matrix and the mRNA expression of extracellular matrix components such as Col2a1 and Acan. Mechanistically, we found that SIRT7 suppressed the transcriptional activity of SOX9, which is an important transcription factor in chondrocytes, and that the enzymatic activity of SIRT7 was required for its function. Our results indicate that SIRT7 is a novel important regulator of cartilage homeostasis and OA development.

Successful Control of Pain from Malignant Psoas Syndrome by Spinal Opioid with Local Anesthetic Agents.

BACKGROUND: Malignant psoas syndrome (MPS) is a rare but distressing pain syndrome observed in advanced cancer patients. Pain due to MPS is often refractory to multimodal analgesic treatment, including opioid analgesics. As only 1 case demonstrating the efficacy of neuraxial analgesia in managing pain due to MPS has been reported, its role in MPS remains uncertain. CASES: We present 3 cases demonstrating the successful management of pain due to MPS using spinal opioids with local anesthetic agents. All patients were under the care of the palliative care consultation service in an acute care hospital and refractory to multimodal analgesic treatment, including opioid, non-opioid, and adjuvant analgesics. Switching opioid administration to the epidural or intrathecal route with a local anesthetic agent provided good pain control in all 3 patients. Moreover, all patients showed improvements in both Palliative Performance Scale and Functional Independence Measure scores after starting a spinal opioid with a local anesthetic agent. CONCLUSIONS: The findings in the present cases indicate neuraxial analgesia may be of benefit, in terms of managing pain and improving functional status, in MPS patients with insufficient pain control by multimodal analgesic treatment. Physicians should consider the use of neuraxial analgesia in cases of MPS where pain is uncontrolled with multimodal analgesic treatment to provide the best possible quality of life for patients with MPS.

Sirtuin 7-dependent deacetylation of DDB1 regulates the expression of nuclear receptor TR4.

Sirtuin 7 (SIRT7) is an NAD+-dependent deacetylase/deacylase, but only a limited number of SIRT7 substrates have been identified. Recently, we found that Sirt7 knockout mice are resistant to high-fat diet-induced fatty liver, and that SIRT7 positively regulates the protein level of TR4, a nuclear receptor involved in lipid metabolism, by inhibiting the CUL4B/DDB1/DCAF1 E3 ubiquitin ligase complex. However, the mechanism by which SIRT7 inhibits the E3 ubiquitin ligase complex was not identified. Here, we demonstrate that SIRT7 binds directly to DDB1 and deacetylates DDB1 at Lys1121. K1121R-DDB1 (a deacetylation-mimicking mutant) displayed reduced binding with DCAF1. The expression of TR4 protein and TR4 target genes, including Cd36, Cidea, Cidec and Pparg1, was increased in K1121R-DDB1-overexpressing Hepa1-6 cells compared to WT-DDB1-overexpressing cells. Our results indicate that the SIRT7-mediated deacetylation of K1121 attenuates the activity of the CUL4B/DDB1/DCAF1 E3 ubiquitin ligase complex by reducing binding between DDB1 and DCAF1, leading to the increased expression of TR4.

Variants in KCNQ1 are associated with susceptibility to type 2 diabetes mellitus.

We carried out a multistage genome-wide association study of type 2 diabetes mellitus in Japanese individuals, with a total of 1,612 cases and 1,424 controls and 100,000 SNPs. The most significant association was obtained with SNPs in KCNQ1, and dense mapping within the gene revealed that rs2237892 in intron 15 showed the lowest Pvalue (6.7 x 10(-13), odds ratio (OR) = 1.49). The association of KCNQ1 with type 2 diabetes was replicated in populations of Korean, Chinese and European ancestry as well as in two independent Japanese populations, and meta-analysis with a total of 19,930 individuals (9,569 cases and 10,361 controls) yielded a P value of 1.7 x 10(-42) (OR = 1.40; 95% CI = 1.34-1.47) for rs2237892. Among control subjects, the risk allele of this polymorphism was associated with impairment of insulin secretion according to the homeostasis model assessment of beta-cell function or the corrected insulin response. Our data thus implicate KCNQ1 as a diabetes susceptibility gene in groups of different ancestries.

Roles of hepatic glucokinase in intertissue metabolic communication: Examination of novel liver-specific glucokinase knockout mice.

Glucokinase is expressed principally in pancreatic ß-cells and hepatocytes, and catalyzes the phosphorylation of glucose to glucose-6-phosphate, a rate-limiting step of glycolysis. To better understand the roles of hepatic glucokinase, we generated Gck knockout mice by ablating liver-specific exon 1b. The knockout mice exhibited impaired glucose tolerance, decreased hepatic glycogen content, and reduced Pklr and Fas gene expression in the liver, indicating that hepatic glucokinase plays important roles in glucose metabolism. It has also been reported that hepatic glucokinase regulates the expression of thermogenesis-related genes in brown adipose tissue (BAT) and insulin secretion in response to glucose. However, the liver-specific Gck knockout mice displayed neither altered expression of thermogenesis-related genes in BAT nor impaired insulin secretion by ß-cells under a normal chow diet. These results suggest that chronic suppression of hepatic glucokinase has a small influence on intertissue (liver-to-BAT as well as liver-to-ß-cell) metabolic communication.

Interferon stimulated gene 15 has an anti-apoptotic effect on MIN6 cells.

Type 1 diabetes, one of two major forms of diabetes, results from the complete destruction of pancreatic beta cells. Viral infection has been suggested to be a trigger of beta cell destruction, the pathogenesis of type 1 diabetes. The aim of this study was to clarify the role of the protein encoded by intherferon stimulated gene (ISG) 15, an antiviral effector, in the development of this clinical entity. We used the mouse beta cell line MIN6 to investigate the role of ISG15 and paid special attention to apoptosis. Although not detected in native MIN6 cells, free ISG15 and ISG15 conjugated proteins were both present in dose-dependently increased amounts following stimulation with interferon alpha. As assessed both by caspase 3/7 activity and an annexin V assay, the percentage of apoptotic MIN6 cells (after exposure to the inflammatory cytokines of interleukin-1beta plus interferon gamma or tumor necrosis factor alpha) was decreased by pretreatment with adenovirus-expressing ISG15 and increased by expressing a short hairpin RNA directed against ISG15. In conclusion, ISG15 has an anti-apoptotic effect on MIN6 cells. Thus, promoting ISG15 expression in the pancreatic beta cells could be a potential therapeutic approach for patients with type 1 diabetes.

Anks4b, a novel target of HNF4α protein, interacts with GRP78 protein and regulates endoplasmic reticulum stress-induced apoptosis in pancreatic ß-cells.

Mutations of the HNF4A gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of pancreatic ß-cell function. HNF4α is a transcription factor belonging to the nuclear receptor superfamily (NR2A1), but its target genes in pancreatic ß-cells are largely unknown. Here, we report that ankyrin repeat and sterile α motif domain containing 4b (Anks4b) is a target of HNF4α in pancreatic ß-cells. Expression of Anks4b was decreased in both ßHNF4α KO islets and HNF4α knockdown MIN6 ß-cells, and HNF4α activated Anks4b promoter activity. Anks4b bound to glucose-regulated protein 78 (GRP78), a major endoplasmic reticulum (ER) chaperone protein, and overexpression of Anks4b enhanced the ER stress response and ER stress-associated apoptosis of MIN6 cells. Conversely, suppression of Anks4b reduced ß-cell susceptibility to ER stress-induced apoptosis. These results indicate that Anks4b is a HNF4α target gene that regulates ER stress in ß-cells by interacting with GRP78, thus suggesting that HNF4α is involved in maintenance of the ER.

Inhibition of H3K18 deacetylation of Sirt7 by Myb-binding protein 1a (Mybbp1a).

Sirt7 localizes in the nucleus (enriched in the nucleolus) and is an NAD(+)-dependent deacetylase with high selectivity for the acetylated lysine 18 of histone H3 (H3K18Ac). It has been reported that Sirt7 is necessary for maintaining the fundamental properties of the cancer cell phenotype and stabilizing the tumorigenicity of human cancer via deacetylation of H3K18Ac. However, the regulators of Sirt7 deacetylase activity are unknown. Myb-binding protein 1a (Mybbp1a) is reported to interact with and regulate the function of a number of transcription factors. In the present study, we demonstrated that Mybbp1a binds to Sirt7 in vitro and in vivo. Serial deletion studies indicated that N- and C-terminal regions of Sirt7 and C-terminal region of Mybbp1a are important for the binding. Furthermore, transfection experiments showed that Mybbp1a is capable of inhibiting the deacetylation activity of H3K18Ac by Sirt7. Our findings demonstrate that Mybbp1a is a novel negative regulator of Sirt7.

Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro.

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic ß-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic ß-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated ß-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.

Identification of hepatocyte growth factor activator (Hgfac) gene as a target of HNF1α in mouse ß-cells.

HNF1α is a transcription factor that is expressed in pancreatic ß-cells and mutations of the HNF1α gene cause a form of monogenic diabetes. To understand the role of HNF1α in pancreatic ß-cells, we established the MIN6 ß-cell line that stably expressed HNF1α-specific shRNA. Expression of the gene encoding hepatocyte growth factor (HGF) activator (Hgfac), a serine protease that efficiently activates HGF, was decreased in HNF1α KD-MIN6 cells. Down-regulation of Hgfac expression was also found in the islets of HNF1α (+/-) mice. Reporter gene analysis and the chromatin immunoprecipitation assay indicated that HNF1α directly regulates the expression of Hgfac in ß-cells. It has been reported that HGF has an important influence on ß-cell mass and ß-cell function. Thus, HNF1α might regulate ß-cell mass or function at least partly by modulating Hgfac expression.

SIRT7 Deficiency Protects against Aging-Associated Glucose Intolerance and Extends Lifespan in Male Mice.

Sirtuins (SIRT1-7 in mammals) are evolutionarily conserved nicotinamide adenine dinucleotide-dependent lysine deacetylases/deacylases that regulate fundamental biological processes including aging. In this study, we reveal that male Sirt7 knockout (KO) mice exhibited an extension of mean and maximum lifespan and a delay in the age-associated mortality rate. In addition, aged male Sirt7 KO mice displayed better glucose tolerance with improved insulin sensitivity compared with wild-type (WT) mice. Fibroblast growth factor 21 (FGF21) enhances insulin sensitivity and extends lifespan when it is overexpressed. Serum levels of FGF21 were markedly decreased with aging in WT mice. In contrast, this decrease was suppressed in Sirt7 KO mice, and the serum FGF21 levels of aged male Sirt7 KO mice were higher than those of WT mice. Activating transcription factor 4 (ATF4) stimulates Fgf21 transcription, and the hepatic levels of Atf4 mRNA were increased in aged male Sirt7 KO mice compared with WT mice. Our findings indicate that the loss of SIRT7 extends lifespan and improves glucose metabolism in male mice. High serum FGF21 levels might be involved in the beneficial effect of SIRT7 deficiency.

Phosphatase protector alpha4 (α4) is involved in adipocyte maintenance and mitochondrial homeostasis through regulation of insulin signaling.

Insulin signaling is mediated via a network of protein phosphorylation. Dysregulation of this network is central to obesity, type 2 diabetes and metabolic syndrome. Here we investigate the role of phosphatase binding protein Alpha4 (α4) that is essential for the serine/threonine protein phosphatase 2A (PP2A) in insulin action/resistance in adipocytes. Unexpectedly, adipocyte-specific inactivation of α4 impairs insulin-induced Akt-mediated serine/threonine phosphorylation despite a decrease in the protein phosphatase 2A (PP2A) levels. Interestingly, loss of α4 also reduces insulin-induced insulin receptor tyrosine phosphorylation. This occurs through decreased association of α4 with Y-box protein 1, resulting in the enhancement of the tyrosine phosphatase protein tyrosine phosphatase 1B (PTP1B) expression. Moreover, adipocyte-specific knockout of α4 in male mice results in impaired adipogenesis and altered mitochondrial oxidation leading to increased inflammation, systemic insulin resistance, hepatosteatosis, islet hyperplasia, and impaired thermogenesis. Thus, the α4 /Y-box protein 1(YBX1)-mediated pathway of insulin receptor signaling is involved in maintaining insulin sensitivity, normal adipose tissue homeostasis and systemic metabolism.

SIRT7 suppresses energy expenditure and thermogenesis by regulating brown adipose tissue functions in mice.

Brown adipose tissue plays a central role in the regulation of the energy balance by expending energy to produce heat. NAD+-dependent deacylase sirtuins have widely been recognized as positive regulators of brown adipose tissue thermogenesis. However, here we reveal that SIRT7, one of seven mammalian sirtuins, suppresses energy expenditure and thermogenesis by regulating brown adipose tissue functions. Whole-body and brown adipose tissue-specific Sirt7 knockout mice have higher body temperature and energy expenditure. SIRT7 deficiency increases the protein level of UCP1, a key regulator of brown adipose tissue thermogenesis. Mechanistically, we found that SIRT7 deacetylates insulin-like growth factor 2 mRNA-binding protein 2, an RNA-binding protein that inhibits the translation of Ucp1 mRNA, thereby enhancing its inhibitory action on Ucp1. Furthermore, SIRT7 attenuates the expression of batokine genes, such as fibroblast growth factor 21. In conclusion, we propose that SIRT7 serves as an energy-saving factor by suppressing brown adipose tissue functions.

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 ß-cell line.

KCNQ1, located on 11p15.5, encodes a voltage-gated K(+) channel with six transmembrane regions, and loss-of-function mutations in the KCNQ1 gene cause hereditary long QT syndrome. Recent genetic studies have identified that single nucleotide polymorphisms located in intron 15 of the KCNQ1 gene are strongly associated with type 2 diabetes and impaired insulin secretion. In order to understand the role of KCNQ1 in insulin secretion, we introduced KCNQ1 into the MIN6 mouse ß-cell line using a retrovirus-mediated gene transfer system. In KCNQ1 transferred MIN6 cells, both the density of the KCNQ1 current and the density of the total K(+) current were significantly increased. In addition, insulin secretion by glucose, pyruvate, or tolbutamide was significantly impaired by KCNQ1-overexpressing MIN6 cells. These results suggest that increased KCNQ1 protein expression limits insulin secretion from pancreatic ß-cells by regulating the potassium channel current.