Epac2a-null mice exhibit obesity-prone nature more susceptible to leptin resistance.

BACKGROUND: The exchange protein directly activated by cAMP (Epac), which is primarily involved in cAMP signaling, has been known to be essential for controlling body energy metabolism. Epac has two isoforms: Epac1 and Epac2. The function of Epac1 on obesity was unveiled using Epac1 knockout (KO) mice. However, the role of Epac2 in obesity remains unclear. METHODS: To evaluate the role of Epac2 in obesity, we used Epac2a KO mice, which is dominantly expressed in neurons and endocrine tissues. Physiological factors related to obesity were analyzed: body weight, fat mass, food intake, plasma leptin and adiponectin levels, energy expenditure, glucose tolerance, and insulin and leptin resistance. To determine the mechanism of Epac2a, mice received exogenous leptin and then hypothalamic leptin signaling was analyzed. RESULTS: Epac2a KO mice appeared to have normal glucose tolerance and insulin sensitivity until 12 weeks of age, but an early onset increase of plasma leptin levels and decrease of plasma adiponectin levels compared with wild-type mice. Acute leptin injection revealed impaired hypothalamic leptin signaling in KO mice. Consistently, KO mice fed a high-fat diet (HFD) were significantly obese, presenting greater food intake and lower energy expenditure. HFD-fed KO mice were also characterized by greater impairment of hypothalamic leptin signaling and by weaker leptin-induced decrease in food consumption compared with HFD-fed wild-type mice. In wild-type mice, acute exogenous leptin injection or chronic HFD feeding tended to induce hypothalamic Epac2a expression. CONCLUSIONS: Considering that HFD is an inducer of hypothalamic leptin resistance and that Epac2a functions in pancreatic beta cells during demands of greater work load, hypothalamic Epac2a may have a role in facilitating leptin signaling, at least in response to higher metabolic demands. Thus, our data indicate that Epac2a is critical for preventing obesity and thus Epac2a activators may be used to manage obesity and obesity-mediated metabolic disorders.

Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

Although glucose is physiologically the most important regulator of insulin secretion, glucose-induced insulin secretion is modulated by hormonal and neural inputs to pancreatic ß-cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca²âº , and phospholipid-derived molecules by activating G protein-coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration-dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)-dependent and Epac2A-dependent mechanisms. Many of the proteins expressed in ß-cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras-like small G protein Rap in a cAMP-dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic ß-cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin-related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes.

Cross-Sectional and Longitudinal Associations of Creatinine-to-Cystatin C Ratio with Sarcopenia Parameters in Older Adults.

OBJECTIVES: Accumulating evidence from cross-sectional studies suggests that the serum creatinine-to-cystatin C ratio (CCR) may be a useful biomarker for sarcopenia. This study aimed to assess the cross-sectional and longitudinal associations of CCR with sarcopenia and its parameters in community-dwelling older adults. DESIGN: Cross-sectional and longitudinal study. SETTING AND PARTICIPANTS: This 6-year prospective cohort study included the repeated measurement data from 1,253 Japanese residents (662 males and 591 females) aged ≥65 years who underwent medical checkups in Kusatsu and Hatoyama, Japan. A total of 4,421 observations were collected. MEASUREMENTS: The CCR was grouped into quartiles by sex (Q1-Q4) using Q4 as the reference category. Sarcopenia was defined according to the Asian Working Group for Sarcopenia 2019 algorithm. Skeletal muscle mass index (SMI) measured using segmental multifrequency bioelectrical impedance analysis, handgrip strength (HGS), usual gait speed (UGS), and maximal gait speed (MGS) were measured repeatedly as sarcopenia parameters. The association of the CCR with changes in sarcopenia, SMI, HGS, UGS, and MGS during the 6-year period were analyzed using a generalized linear mixed-effects model. RESULTS: The prevalence of sarcopenia at baseline was 13.1% (11.9% in males and 14.5% in females). In a cross-sectional analysis, the CCR quartile was inversely associated with sarcopenia and was positively associated with SMI, HGS, and MGS (P for trend < 0.001). In a longitudinal analysis during the 6 years, a significant increase in sarcopenia in Q2 (B = 1.1% point/year; P = 0.026 for group-by-time interaction) and significant declines in SMI (B = -0.01 kg/m2/year; P = 0.044 for group-by-time interaction) and MGS (B = -0.008 m/sec/year; P = 0.041 for group-by-time interaction) in Q1 were observed compared with Q4. However, the dose-response relationship was significant only for MGS (P = 0.033 for trend). No significant group-by-time interaction was observed for HGS. CCR was not significantly associated with UGS either cross-sectionally or longitudinally. CONCLUSIONS: CCR is a useful biomarker regarding the status of sarcopenia. It may be used for sarcopenia screening even in older adults whose physical function is difficult to assess. However, further longitudinal studies are needed to determine whether CCR can be a predictor of future sarcopenia.

Role of major histocompatibility complex class II in the development of autoimmune type 1 diabetes and thyroiditis in rats.

Although the MHC class II 'u' haplotype is strongly associated with type 1 diabetes (T1D) in rats, the role of MHC class II in the development of tissue-specific autoimmune diseases including T1D and autoimmune thyroiditis remains unclear. To clarify this, we produced a congenic strain carrying MHC class II 'a' and 'u' haplotypes on the Komeda diabetes-prone (KDP) genetic background. The u/u homozygous animals developed T1D similar to the original KDP rat; a/u heterozygous animals did develop T1D but with delayed onset and low frequency. In contrast, none of the a/a homozygous animals developed T1D; about half of the animals with a/u heterozygous or a/a homozygous genotypes showed autoimmune thyroiditis. To investigate the role of genetic background in the development of thyroiditis, we also produced a congenic strain carrying Cblb mutation of the KDP rat on the PVG.R23 genetic background (MHC class II 'a' haplotype). The congenic rats with homozygous Cblb mutation showed autoimmune thyroiditis without T1D and slight to severe alopecia, a clinical symptom of hypothyroidism such as Hashimoto's thyroiditis. These data indicate that MHC class II is involved in the tissue-specific development of autoimmune diseases, including T1D and thyroiditis.

Cell signalling in insulin secretion: the molecular targets of ATP, cAMP and sulfonylurea.

Clarification of the molecular mechanisms of insulin secretion is crucial for understanding the pathogenesis and pathophysiology of diabetes and for development of novel therapeutic strategies for the disease. Insulin secretion is regulated by various intracellular signals generated by nutrients and hormonal and neural inputs. In addition, a variety of glucose-lowering drugs including sulfonylureas, glinide-derivatives, and incretin-related drugs such as dipeptidyl peptidase IV (DPP-4) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists are used for glycaemic control by targeting beta cell signalling for improved insulin secretion. There has been a remarkable increase in our understanding of the basis of beta cell signalling over the past two decades following the application of molecular biology, gene technology, electrophysiology and bioimaging to beta cell research. This review discusses cell signalling in insulin secretion, focusing on the molecular targets of ATP, cAMP and sulfonylurea, an essential metabolic signal in glucose-induced insulin secretion (GIIS), a critical signal in the potentiation of GIIS, and the commonly used glucose-lowering drug, respectively.

Treating diabetes today: a matter of selectivity of sulphonylureas.

It is well known that sulphonylureas (SUs), commonly used in the treatment of type 2 diabetes mellitus, stimulate insulin secretion by closing ATP-sensitive K(+) (K(ATP) ) channels in pancreatic ß-cells by binding to the SU receptor SUR1. SUs are now known also to activate cAMP sensor Epac2 (cAMP-GEFII) to Rap1 signalling, which promotes insulin granule exocytosis. For SUs to exert their full effects in insulin secretion, they are required to activate Epac2 as well as to inhibit the ß-cell K(ATP) channels. As Epac2 is also necessary for potentiation of glucose-induced insulin secretion by cAMP-increasing agents, such as incretin, Epac2 is a target of both cAMP and SUs. The distinct effects of various SUs appear to be because of their different actions on Epac2/Rap1 signalling as well as K(ATP) channels. Differently from other SUs, gliclazide is unique in that it is specific for ß-cell K(ATP) channel and does not activate Epac2.

Difference between "Physical Fitness Age" Based on Physical Function and Chronological Age Is Associated with Obesity, Hyperglycemia, Depressive Symptoms, and Low Serum Albumin.

OBJECTIVES: This study aimed to (1) develop the physical fitness age, which is the biological age based on physical function, (2) evaluate the validity of the physical fitness age for the assessment of sarcopenia, and (3) examine the factors associated with the difference between physical fitness age and chronological age. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: Community-dwelling older adults and outpatients. MEASUREMENTS: A formula for calculating the physical fitness age was created based on the usual walking speed, handgrip strength, one-leg standing time, and chronological age of 4,076 older adults from the pooled data of community-dwelling and outpatients using the principal component analysis. For the validation of the physical fitness age, we also used pooled data from community-dwelling older adults (n = 1929) and outpatients (n = 473). Sarcopenia was diagnosed according to the Asian Working Group for Sarcopenia 2019 consensus. The association of D-age (the difference between physical and chronological ages) with cardiovascular risk factors, renal function, and cardiac function was examined. RESULTS: The receiver operating characteristic analysis, with sarcopenia as the outcome, showed that the area under the curve (AUC) of physical fitness age was greater than that of chronological age (AUC 0.87 and 0.77, respectively, p < 0.001). Binomial logistic regression analysis revealed that the D-age was significantly associated with sarcopenia after adjustment for covariates (odds ratio 1.22, 95% confidence interval 1.19-1.26; p <0.001). In multivariate linear regression analysis with D-age as the dependent variable, D-age was independently associated with a history of diabetes mellitus (or hemoglobin A1c as a continuous variable), obesity, depression, and low serum albumin level. D-age was also correlated with estimated glomerular filtration rate derived from serum cystatin C, brain natriuretic peptide, and ankle-brachial index, reflecting some organ function and arteriosclerosis. CONCLUSIONS: Compared to chronological age, physical fitness age calculated from handgrip strength, one-leg standing time, and usual walking speed was a better scale for sarcopenia. D-age, which could be a simple indicator of physical function, was associated with modifiable factors, such as poor glycemic control, obesity, depressive symptoms, and malnutrition.

Interaction of Eating Status and Dietary Variety on Incident Functional Disability among Older Japanese Adults.

OBJECTIVES: To examine whether eating status and dietary variety were associated with functional disability during a 5-year follow-up analysis of older adults living in a Japanese metropolitan area. DESIGN: A 5-year follow-up study. SETTING: Ota City, Tokyo, Japan. PARTICIPANTS: A total of 10,308 community-dwelling non-disabled adults aged 65-84 years. MEASUREMENTS: Eating status was assessed using a self-reported questionnaire. Dietary variety was assessed using the dietary variety score (DVS). Based on the responses, participants were classified according to eating alone or together and DVS categories (low: 0-3; high: 4-10). Functional disability incidence was prospectively identified using the long-term care insurance system's nationally unified database. Multilevel survival analyses calculated the adjusted hazard ratio (HR) and 95% confidence interval (CI) for incident functional disability. RESULTS: During a 5-year follow-up, 1,991 (19.3%) individuals had functional disabilities. Eating status or DVS were not independently associated with incident functional disability. However, interaction terms between eating status and DVS were associated with functional disability; HR (95% CI) for eating together and low DVS was 1.00 (0.90-1.11), eating alone and high DVS was 0.95 (0.77-1.17), and eating alone and low DVS was 1.20 (1.02-1.42), compared to those with eating together and high DVS. CONCLUSION: Older adults should avoid eating alone or increase dietary variety to prevent functional disability. This can be ensured by providing an environment of eating together or food provision services for eating a variety of foods in the community.

cAMP-GEFII is a direct target of cAMP in regulated exocytosis.

Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.

Menin interacts with ß-catenin in osteoblast differentiation.

Menin promotes the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage by interacting with the BMP-2 signaling molecules Smad1/5, and Runx2. However, the relationship between menin and the Wnt-ß-catenin pathway in bone is unclear. Reduction of menin expression by transfection of a menin antisense construct did not alter the levels of ß-catenin in mouse mesenchymal C2C12 and osteoblastic MC3T3-E1 cells. However, menin co-immunoprecipitated with ß-catenin as well as LEF-1 in C2C12 and MC3T3-E1 cells. Reduction of menin expression by antisense menin transfection antagonized ß-catenin-induced transcriptional activity of the pGL3-OT luciferase reporter construct in C2C12 and MC3T3-E1 cells. Antisense menin transfection antagonized the BMP-2 and ß-catenin-stimulated increases in Runx2 and alkaline phosphatase levels in C2C12 cells. The data show that menin interacts with ß-catenin in mouse mesenchymal and osteoblastic cells, and suggest that the interaction is important for osteoblast differentiation.

Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice.

AIMS/HYPOTHESIS: We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K(+) (K(ATP)) channels (Kir6.2(-/-) mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. METHODS: Pancreas perfusion experiments were performed using wild-type (Kir6.2(+/+)) or Kir6.2(-/-) mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2(-/-) mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K(ATP) channels with glibenclamide. RESULTS: The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2(-/-) pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K(ATP) channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2(+/+), as in Kir6.2(-/-) pancreases. CONCLUSIONS/INTERPRETATION: Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2(-/-) beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.

Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing.

A point mutation in the human insulin receptor gene in a patient with type A insulin resistance alters the amino acid sequence within the tetrabasic processing site of the proreceptor molecule from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser. Epstein-Barr virus-transformed lymphocytes from this patient synthesize an insulin receptor precursor that is normally glycosylated and inserted into the plasma membrane but is not cleaved to mature alpha and beta subunits. Insulin binding to these cells is severely reduced but can be increased about fivefold by gentle treatment with trypsin, accompanied by the appearance of normal alpha subunits. These results indicate that proteolysis of the proreceptor is necessary for its normal full insulin-binding sensitivity and signal-transducing activity and that a cellular protease that is more stringent in its specificity than trypsin is required to process the receptor precursor.

Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor.

A member of the inwardly rectifying potassium channel family was cloned here. The channel, called BIR (Kir6.2), was expressed in large amounts in rat pancreatic islets and glucose-responsive insulin-secreting cell lines. Coexpression with the sulfonylurea receptor SUR reconstituted an inwardly rectifying potassium conductance of 76 picosiemens that was sensitive to adenosine triphosphate (ATP) (IKATP) and was inhibited by sulfonylureas and activated by diazoxide. The data indicate that these pancreatic beta cell potassium channels are a complex composed of at least two subunits--BIR, a member of the inward rectifier potassium channel family, and SUR, a member of the ATP-binding cassette superfamily. Gene mapping data show that these two potassium channel subunit genes are clustered on human chromosome 11 at position 11p15.1.

Protective role of ATP-sensitive potassium channels in hypoxia-induced generalized seizure.

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels are activated by various metabolic stresses, including hypoxia. The substantia nigra pars reticulata (SNr), the area with the highest expression of K(ATP) channels in the brain, plays a pivotal role in the control of seizures. Mutant mice lacking the Kir6.2 subunit of K(ATP) channels [knockout (KO) mice] were susceptible to generalized seizures after brief hypoxia. In normal mice, SNr neuron activity was inactivated during hypoxia by the opening of the postsynaptic K(ATP) channels, whereas in KO mice, the activity of these neurons was enhanced. K(ATP) channels exert a depressant effect on SNr neuronal activity during hypoxia and may be involved in the nigral protection mechanism against generalized seizures.

Roles of cAMP signalling in insulin granule exocytosis.

Insulin secretion is regulated by a series of complex events generated by various intracellular signals including Ca(2+), ATP, cAMP and phospholipid-derived signals. Glucose-stimulated insulin secretion is the principal mode of insulin secretion, and the mechanism potentiating the secretion is critical for physiological responses. Among the various intracellular signals involved, cAMP is particularly important for amplifying insulin secretion. Recently, glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-IV (DPP-IV) inhibitors have been developed as new antidiabetic drugs. These drugs all act through cAMP signalling in pancreatic beta-cells. Until recently, cAMP was generally thought to potentiate insulin secretion through protein kinase A (PKA) phosphorylation of proteins associated with the secretory process. However, it is now known that in addition to PKA, cAMP has other targets such as Epac (also referred to as cAMP-GEF). The variety of the effects mediated by cAMP signalling may be linked to cAMP compartmentation in the pancreatic beta-cells.

Score-Based and Nutrient-Derived Dietary Patterns Are Associated with Depressive Symptoms in Community-Dwelling Older Japanese: A Cross-Sectional Study.

OBJECTIVES: This study evaluated associations of score-based and nutrient-derived dietary patterns with depressive symptoms in community-dwelling older Japanese. DESIGN: Cross-sectional study. SETTING: Community-based. PARTICIPANTS: 982 community-dwelling adults aged 65 years or older. MEASUREMENTS: Score-based pattern was assessed by using dietary variety score (DVS), which covers 10 food group items in Japanese meals. Nutrient-derived dietary patterns were identified by using reduced rank regression (RRR), with folate, vitamin C, magnesium, calcium, iron, and zinc intakes as response variables. Depressive symptoms were assessed with the Geriatric Depression Scale. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for these dietary patterns in multivariate logistic regression analyses with potential confounders. The lowest consumption category was used as the reference group. RESULTS: The prevalence of depressive symptoms was 13.5%. Higher DVS was associated with fewer depressive symptoms (OR=0.52, 95% CI=0.27-1.03 for the highest vs the lowest DVS; P for trend=0.031). The first RRR dietary pattern score was characterized by high intakes of fish, soybean products, potatoes, most vegetables, mushrooms, seaweeds, fruits, and green tea and a low intake of rice and was inversely associated with the prevalence of depressive symptoms (OR=0.53, 95% CI=0.30-0.92; P for trend=0.030). CONCLUSION: Greater dietary variety and a dietary pattern characterized by high intakes of fish, soybean products, potatoes, most vegetables, mushrooms, seaweeds, fruit, and green tea and a low intake of rice were consistently associated with lower prevalence of depressive symptoms in community-dwelling older Japanese. Therefore, both patterns identified the components of dietary habits essential to depression prevention.

A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels.

We have cloned an isoform of the sulfonylurea receptor (SUR), designated SUR2. Coexpression of SUR2 and the inward rectifier K+ channel subunit Kir6.2 in COS1 cells reconstitutes the properties of K(ATP) channels described in cardiac and skeletal muscle. The SUR2/Kir6.2 channel is less sensitive than the SUR/Kir6.2 channel (the pancreatic beta cell KATP channel) to both ATP and the sulfonylurea glibenclamide and is activated by the cardiac K(ATP) channel openers, cromakalim and pinacidil, but not by diazoxide. In addition, SUR2 binds glibenclamide with lower affinity. The present study shows that the ATP sensitivity and pharmacological properties of K(ATP) channels are determined by a family of structurally related but functionally distinct sulfonylurea receptors.