Yusuke Kashiwagi

Expression of SGLT1 in Human Hearts and Impairment of Cardiac Glucose Uptake by Phlorizin during Ischemia-Reperfusion Injury in Mice

Objective Sodium-glucose cotransporter 1 (SGLT1) is thought to be expressed in the heart as the dominant isoform of cardiac SGLT, although more information is required to delineate the subtypes of SGLTs in human hearts. Moreover, the functional role of SGLTs in the heart remains to be fully elucidated. We herein investigated whether SGLT1 is expressed in human hearts and whether SGLTs significantly contribute to cardiac energy metabolism during ischemia-reperfusion injury (IRI) via enhanced glucose utilization in mice. Methods and Results We determined that SGLT1 was highly expressed in both human autopsied hearts and murine perfused hearts, as assessed by immunostaining and immunoblotting with membrane fractionation. To test the functional significance of the substantial expression of SGLTs in the heart, we studied the effects of a non-selective SGLT inhibitor, phlorizin, on the baseline cardiac function and its response to ischemia-reperfusion using the murine Langendorff model. Although phlorizin perfusion did not affect baseline cardiac function, its administration during IRI significantly impaired the recovery in left ventricular contractions and rate pressure product, associated with an increased infarct size, as demonstrated by triphenyltetrazolium chloride staining and creatine phosphokinase activity released into the perfusate. The onset of ischemic contracture, which indicates the initiation of ATP depletion in myocardium, was earlier with phlorizin. Consistent with this finding, there was a significant decrease in the tissue ATP content associated with reductions in glucose uptake, as well as lactate output (indicating glycolytic flux), during ischemia-reperfusion in the phlorizin-perfused hearts. Conclusions Cardiac SGLTs, possibly SGLT1 in particular, appear to provide an important protective mechanism against IRI by replenishing ATP stores in ischemic cardiac tissues via enhancing availability of glucose. The present findings provide new insight into the significant role of SGLTs in optimizing cardiac energy metabolism, at least during the acute phase of IRI.

Aerobic interval exercise training in the afternoon reduces attacks of coronary spastic angina in conjunction with improvement in endothelial function, oxidative stress, and inflammation.

BackgroundCoronary spasm plays an important role in the pathogenesis of ischemic heart disease. Endothelial function is impaired in patients with coronary spasm. Exercise training has been shown to improve endothelial function. ObjectiveWe examined the effects of aerobic interval exercise training (AIT) on attacks in conjunction with endothelial function in patients with coronary spastic angina. Participants and methodsThe study participants were 26 patients with rest angina (19 men and 7 women, mean age 61.7±11.7 years) in whom coronary spasm was documented and no severe organic lesions were found. The numbers of attacks and of individuals with attacks were examined in conjunction with endothelial function, oxidative stress, inflammation, and insulin resistance before and after 3 successive days of AIT. ResultsThe number of attacks/patient and the ratio of patients with attacks/5 days decreased [from 2 (1, 7) to 0 (0, 2), P<0.001, and from 23/26 (88.5%) to 10/26 (38.5%), P<0.001] in conjunction with the improvement in endothelial function assessed by improved flow-mediated dilatation (4.8±2.7 vs. 6.9±2.8%, P<0.001), plasma levels of diacron-reactive oxygen metabolites (363±58 vs. 349±61 U.CARR, P=0.001), interleukin-6[1.63 (1.33, 2.22) vs. 1.39 (1.09, 2.02) pg/ml, P=0.012], high-sensitivity C-reactive protein [0.087 (0.041, 0.136) vs. 0.063 (0.028, 0.085) mg/dl, P=0.028], and homeostasis model assessment-insulin resistance [1.79 (1.41, 2.39) vs. 1.54 (1.17, 1.79) mg/dl µU/ml, P=0.005] after AIT. ConclusionAIT in the afternoon suppressed the attacks in conjunction with improvement in endothelial function, oxidative stress, inflammation, and insulin resistance in patients with coronary spastic angina.

Possible increase in insulin resistance and concealed glucose-coupled potassium-lowering mechanisms during acute coronary syndrome documented by covariance structure analysis

Objective Although glucose-insulin-potassium (GIK) therapy ought to be beneficial for ischemic heart disease in general, variable outcomes in many clinical trials of GIK in acute coronary syndrome (ACS) had a controversial impact. This study was designed to examine whether “insulin resistance” is involved in ACS and to clarify other potential intrinsic compensatory mechanisms for GIK tolerance through highly statistical procedure. Methods and results We compared the degree of insulin resistance during ACS attack and remission phase after treatment in individual patients (n = 104). During ACS, homeostasis model assessment of insulin resistance (HOMA-IR) values were significantly increased (P<0.001), while serum potassium levels were transiently decreased (degree of which was indicated by ΔK) (P<0.001). This finding provides a renewed paradox, as ΔK, a surrogate marker of intrinsic GIK cascade activation, probably reflects the validated glucose metabolism during ischemic attack. Indeed, multiple regression analysis revealed that plasma glucose level during ACS was positively correlated with ΔK (P = 0.026), whereas HOMA-IR had no impact on ΔK. This positive correlation between ΔK and glucose was confirmed by covariance structure analysis with a strong impact (β: 0.398, P = 0.015). Intriguingly, a higher incidence of myocardial infarction relative to unstable angina pectoris, as well as a longer hospitalization period were observed in patients with larger ΔK, indicating that ΔK also reflects disease severity of ACS. Conclusions Insulin resistance most likely increases during ACS; however, ΔK was positively correlated with plasma glucose level, which overwhelmed insulin resistance condition. The present study with covariance structure analysis suggests that there are potential endogenous glucose-coupled potassium lowering mechanisms, other than insulin, regulating glucose metabolism during ACS.

Preconditioning actions of aldosterone through p38 signaling modulation in isolated rat hearts

Although persistent excessive actions of aldosterone have unfavorable effects on the cardiovascular system, primarily via mineralocorticoid receptor (MR)-dependent pathways, the pathophysiological significance of aldosterone cascade activation in heart diseases has not yet been fully clarified. We herein examined the effects of short-term aldosterone stimulation at a physiological dose on cardiac function during ischemia-reperfusion injury (IRI). In order to study the effects of aldosterone preconditioning, male Wistar rat Langendorff hearts were perfused with 10(-9) mol/l of aldosterone for 10 min before ischemia, and the response to IRI was assessed. Although aldosterone did not affect the baseline hemodynamic parameters, preconditioning actions of aldosterone significantly improved the recovery in left ventricular contractility and left ventricular end-diastolic pressure associated with a reduced activity of creatine phosphokinase released into the perfusate after ischemia-reperfusion. Notably, the MR inhibitor eplerenone did not abrogate these beneficial effects. Biochemical analyses revealed that p38MAPK phosphorylation was significantly increased during aldosterone preconditioning before ischemia, whereas its phosphorylation was substantially attenuated during sustained ischemia-reperfusion, compared with the results for in the non-preconditioned control hearts. This dual regulation of p38MAPK was not affected by eplerenone. The phosphorylation levels of other MAPKs were not altered by aldosterone preconditioning. In conclusion, the temporal induction of the aldosterone cascade, at a physiological dose, has favorable effects on cardiac functional recovery and injury following ischemia-reperfusion in a MR-independent manner. Phasic dynamism of p38MAPK activation may play a key role in the physiological compensatory pathway of aldosterone under severe cardiac pathological conditions.

Coronary spastic angina is associated with insulin resistance - possible involvement of endothelial dysfunction.

ObjectiveIn this study, we examined whether coronary spastic angina (CSA) is associated with insulin resistance. BackgroundThere is increasing evidence that insulin resistance is associated with endothelial dysfunction. Patients with CSA show endothelial dysfunction. MethodsThe study participants include 111 CSA patients (81 men and 30 women, mean age 62±12 years) and 53 participants without CSA (24 men and 29 women, mean age 63±10 years), serving as the controls. The oral glucose tolerance test was performed, and anthropometric parameters, plasma glucose and insulin levels, lipid profiles, and other laboratory parameters were evaluated. ResultsHomeostasis model assessment of insulin resistance (HOMA-IR), Log HOMA-IR, the quantitative insulin sensitivity check index, the insulin sensitivity index, and insulin resistance 60–120 min after glucose load (log post-glucose-IR) were calculated as surrogate markers of insulin resistance. The number of men, the number of smokers, log post-glucose-IR, the insulin sensitivity index, and fasting plasma glucose levels were higher in CSA patients compared with controls (P=0.001, 0.001, 0.004, 0.012, and 0.013, respectively), whereas plasma high-density lipoprotein cholesterol levels were lower (P<0.001). ConclusionInsulin resistance on glucose load (log post-glucose-IR), plasma high-density lipoprotein cholesterol levels, and smoking were significantly associated with CSA (r=0.225, P=0.004; r=−0.313, P<0.001; and r=0.258, P=0.001, respectively).

Increased urinary aldosterone excretion is associated with subcutaneous not visceral, adipose tissue area in obese individuals: a possible manifestation of dysfunctional subcutaneous adipose tissue

Aldosterone is reported to be associated with obesity and is a risk factor for metabolic syndrome. Metabolic abnormalities are more strongly associated with visceral adipose tissue (VAT) than with subcutaneous adipose tissue (SAT).

Pioglitazone, a peroxisome proliferator-activated receptor γ activator, suppresses coronary spasm.

ObjectiveWe examined whether a peroxisome proliferator-activated receptor &ggr; (PPAR-&ggr;) agonist, pioglitazone, suppresses coronary spasm. BackgroundPatients with coronary spastic angina (CSA) also have endothelial dysfunction and inflammation. Activation of PPAR-&ggr; improves endothelial dysfunction and inflammation. Participants and methodsThe study participants included 73 consecutive CSA patients (47 men and 26 women, mean age 63.6±10.4 years) who were admitted to our institution with a suspicion of CSA because of episodes of chest discomfort occurring mostly at rest in whom coronary spasm was induced by an intracoronary acetylcholine injection and a repeat acetylcholine provocation injection was administered after 6 months of follow-up. Thirty-six of the patients were administered pioglitazone15–30 mg/day added on calcium channel blockers (CCBs) (pioglitazone group) and 37 were administered CCBs alone (control group). Clinical and laboratory data were also examined before and after 6 months of follow-up and the results between the two groups were compared. ResultsCoronary spasm was suppressed in 18/36 patients (50.0%) in the pioglitazone group (P<0.001) and 8/37 patients (21.6%) in the control group (P=0.008) after 6 months of treatment. Coronary spasm was thus significantly reduced in the pioglitazone group compared with the control group (P=0.011). The levels of total white blood cell count and high-sensitivity C-reactive protein decreased significantly (P<0.001 and P<0.001, respectively) in the pioglitazone group, whereas these levels did not differ in the control group (P=0.15 and 0.39, respectively) after the treatment. ConclusionPioglitazone added on CCBs significantly reduced coronary spasm compared with CCBs alone after 6 months of treatment. Pioglitazone may thus prove to be a novel therapy for coronary spasm.

Suppression of Primary Aldosteronism and Resistant Hypertension by the Peroxisome Proliferator-activated Receptor Gamma Agonist Pioglitazone

Abstract:The peroxisome proliferator-activated receptor gamma (PPAR &ggr;) agonists have been reported to have antiproliferative and tumor-suppressive effects. We report a case of 55-year-old man with primary aldosteronism (PA) whose hyperaldosteronism was suppressed with the PPAR &ggr; agonist pioglitazone. He had drug-resistant hypertension, hypokalemia, and diabetes mellitus. The diagnosis of PA was confirmed by the oral sodium loading test (20.5 &mgr;g/d of urinary aldosterone) and Captopril challenge test (19.5 ng/dL of plasma aldosterone). Computed tomography imaging revealed no apparent adrenal mass. The result of the posture stimulation test was consistent with the diagnosis of idiopathic adrenal hyperplasia. On administration of pioglitazone (30 mg/d) and nifedipine (40 mg/d), hypertension and hypokalemia improved and plasma aldosterone decreased for more than 6 months. The sodium loading test done after 6 months of the administration revealed the near normal results (11.2 ng/dL of plasma aldosterone and 13.1 &mgr;g/d of urinary aldosterone). The findings indicated that pioglitazone suppressed PA.

The thermogenic actions of natriuretic peptide in brown adipocytes: The direct measurement of the intracellular temperature using a fluorescent thermoprobe

In addition to the various effects of natriuretic peptides (NPs) on cardiovascular systems, increasing attention is being paid to the possibility that NPs induce adipose tissue browning and activate thermogenic program. We herein established a direct intracellular temperature measurement system using a fluorescent thermoprobe and investigated the thermogenic effects of A-type NP (ANP) on brown adipocytes. The thermoprobe was successfully introduced into rat brown adipocytes, and the temperature dependent change in fluorescence intensity ratio was measured using a fluorescence microscope. After one-hour incubation with ANP, the degree of the change in fluorescence intensity ratio was significantly higher in ANP-treated (P < 0.01) adipocytes compared to untreated controls. The ANP treatment increased uncoupling protein-1 (UCP1) mRNA levels, which is one of the markers of thermogenesis in adipocytes, while the intracellular ATP content was not changed, indicating mitochondrial uncoupled respiration. Intriguingly, these thermogenic actions of ANP were more prominent when brown adipocytes were incubated at 35 °C than at 37 °C. Moreover, the increase in the intracellular temperature and the expression of UCP1 induced by ANP were cancelled by p38MAPK inhibition. Taken together, this study directly demonstrated the thermogenic actions of ANP in brown adipocytes through the use of a novel method of intracellular temperature measurement.

Tissue thrombin is associated with the pathogenesis of dilated cardiomyopathy

BACKGROUND Thrombin is a serine protease known to be the final product of the coagulation cascade. However, thrombin plays other physiological roles in processes such as gastric contractions and vessel wound healing, and a state of coagulability is increased in patients with dilated cardiomyopathy (DCM). In this study, we investigate the role of thrombin in the pathogenesis of DCM. The purpose of this study is to clarify the role of thrombin in the pathogenesis of DCM and investigate the possibility of treatment against DCM by thrombin inhibition. METHODS We investigated the expression of thrombin in the left ventricles of five patients with DCM who underwent the Batista operation and four patients without heart disease. Furthermore, we investigated the involvement of thrombin in the development of DCM using knock-in mice with a deletion mutation of cardiac troponin T that causes human DCM (∆K210 knock-in mouse) (B6;129-Tnnt2tm2Mmto) and assessed the effects of a direct thrombin inhibitor, dabigatran on ∆K210 knock-in mice using echocardiographic examinations, the Kaplan-Meier method and Western blotting. RESULTS The immunohistochemical analysis showed a strong thrombin expression in the DCM patients compared to the patients without heart disease. In immunohistochemical analysis, a strong thrombin expression was observed in the heart tissues analysis in the ∆K210 knock-in mice. Dabigatran administration significantly improved fractional shortening according to the echocardiographic examination and the survival outcomes in ∆K210 knock-in mice. CONCLUSION Tissue thrombin is involved in the pathogenesis of DCM and thrombin inhibition can be beneficial for the treatment of DCM.