Ghrelin augments the expressions and secretions of proinflammatory adipokines, VEGF120 and MCP-1, in differentiated 3T3-L1 adipocytes.

Ghrelin is a physiological-active peptide with growth hormone-releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin-direct-effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3-L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9-fold increase on vascular endothelial growth factor-120 (VEGF(120)) releases (p < 0.01) and the 1.7-fold on monocyte chemoattractant protein-1 (MCP-1) (p < 0.01) from 3T3-L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c-Jun NH2 -terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4-fold (p < 0.01) and 1.6-fold (p < 0.01) in the ghrelin-stimulated adipocytes, respectively. Furthermore, the treatment with LY294002 (50 µmol/L) and Wortmannin (10nmol/L), inhibitors of phosphatidylinositol 3-kinase (PI3K), significantly decreased the amplified VEGF(120) secretion by 29% (p < 0.01) and 28% (p < 0.01) relative to the cells stimulated by ghrelin alone, respectively, whereas these inhibitors had no effects on increased MCP-1 release. On the other hand, JNK inhibitor SP600125 (10 µmol/L) clearly reduced the increased MCP-1, but not VEGF(120), release by 35% relative to the only ghrelin-stimulated cells (p < 0.01). In conclusion, ghrelin can enhance the secretions of proinflammatory adipokines, VEGF(120) and MCP-1, but fails to affect IL-10 and adiponectin which are considered to be anti-inflammatory adipokines. Moreover, this augmented VEGF(120) release is invited through the activation of PI3K pathways and the MCP-1 is through JNK pathways. Consequently, our results strongly suggest that ghrelin can induce the development of diabetes via its direct-action in peripheral tissues as well as via in CNS.

The association between impaired proinsulin processing and type 2 diabetes mellitus in non-obese Japanese individuals.

We aimed to examine the association between impaired proinsulin processing in pancreatic beta cells and type 2 diabetes mellitus in non-obese Japanese patients. Participants were divided into groups for normal glucose tolerance, prediabetes, and type 2 diabetes based on the oral glucose tolerance test (OGTT). Activities of prohormone convertase (PC) 1/3 and PC2 in fasting states were estimated. Multiple regression analysis was undertaken to ascertain if alteration of the activities of these enzymes contributes to the development of impaired glucose tolerance by comparison with HOMA-ß and the oral disposition index (DI(O)). Overall, 452 subjects were included. PC1/3 activity tended to decrease in type 2 diabetes compared with normal glucose tolerance. PC2 activity showed no difference among the three groups. Decreased estimated PC1/3 activity was significantly associated with type 2 diabetes after adjustment for sex, age, creatinine, triglycerides, HOMA-ß and DI(O). Odds ratios (95% CI) of PC1/3, HOMA-ß, and DI(O) were 2.16 (1.12-4.19), 3.44 (1.82-6.52) and 14.60 (7.87-27.11), respectively. Furthermore, decreased PC1/3(≤1.7) combined with decreased HOMA-ß (≤30) had a sensitivity of 73% and specificity of 62%. Decreased PC1/3 activity may be a useful measurement of beta-cell function alongside decreased HOMA-ß or DI(O). A combined decrease in estimated fasting PC1/3 activity and HOMA-ß measurement led to suspicion of type 2 diabetes in the non-obese Japanese population studied.

Mediobasal hypothalamic PTEN modulates hepatic insulin resistance independently of food intake in rats.

Phosphatase and tensin homolog (PTEN) dephosphorylates phosphatidylinositol (PI) 3,4,5-triphosphate and antagonizes PI 3-kinase. Insulin acts in the mediobasal hypothalamus (MBH) to not only suppress food intake and weight gain but also improve glucose metabolism via PI 3-kinase activation. Thus, the blocking of hypothalamic PTEN is a potential target for treating obesity as well as diabetes. However, genetic modification of PTEN in specific neuronal populations in the MBH yielded complex results, and no postnatal intervention for hypothalamic PTEN has been reported yet. To elucidate how postnatal modification of hypothalamic PTEN influences food intake as well as glucose metabolism, we bidirectionally altered PTEN activity in the MBH of rats by adenoviral gene delivery. Inhibition of MBH PTEN activity reduced food intake and weight gain, whereas constitutive activation of PTEN tended to induce the opposite effects. Interestingly, the effects of MBH PTEN intervention on food intake and body weight were blunted by high-fat feeding. However, MBH PTEN blockade improved hepatic insulin sensitivity even under high-fat-fed conditions. On the other hand, constitutive activation of MBH PTEN induced hepatic insulin resistance. Hepatic Akt phosphorylation and the G6Pase expression level were modulated bidirectionally by MBH PTEN intervention. These results demonstrate that PTEN in the MBH regulates hepatic insulin sensitivity independently of the effects on food intake and weight gain. Therefore, hypothalamic PTEN is a promising target for treating insulin resistance even in states of overnutrition.

Possible involvement of PI3K-dependent pathways in the increased VEGF120 release from osteoblastic cells preloaded with palmitate in vitro.

It have been reported that abnormal bone metabolism often occurs in patients with type 2 diabetes, but the underlying mechanisms remain to be elucidated. In recent years dyslipidemia (hyperlipidemia) has been presumed to have an influence on bone metabolism. In addition, the involvements of VEGF and MCP-1 derived from osteoblasts in bone abnormal metabolism were also observed. Thus, we investigated the pathogenic mechanism of this abnormal bone metabolism, which is included in the regulation of VEGF and MCP-1 secretions from osteoblasts, by using UMR-106 osteosarcoma cells as an osteoblast cell model and treating them with palmitate in order to mimic a state of hyperlipidemia. Palmitate-preloaded cells showed the significant increase of VEGF120 release (1.8-fold vs. control cells, p<0.01). Moreover, the treatment with palmitate significantly increased VEGF-A mRNA with the maximal 2.5-fold upregulation at 12h after the treatment (p<0.01). However, MCP-1 release was not affected by palmitate. Moreover, the amplified VEGF120 secretion with palmitate was significantly decreased by the treatment with TLR4 antagonist or PI3K pathway inhibitors, LY294002 and wortmannin (p<0.01, respectively). On the other hand, the stimulation with TNF-α, which osteoclasts were able to release, significantly enhanced MCP-1 secretion (p<0.01), but had no effect on VEGF120. On the contrary IL-1ß amplified VEGF120 release (p<0.01), but not MCP-1. These results suggest that palmitate can increase VEGF120 release from UMR-106 osteosarcoma cells, which is accelerated at the transcriptional level, and this increase of VEGF120 release may be mediated though, at least partly, TLR4 and the PI3K pathways. In addition, we also verified that TNF-α and IL-1ß, which are considered to be derived from osteoclasts, amplified the secretions of MCP-1 and VEGF120 from UMR-106 cells, respectively.

The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells.

Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

Long-term effects of ipragliflozin and pioglitazone on metabolic dysfunction-associated steatotic liver disease in patients with type 2 diabetes: 5 year observational follow-up of a randomized, 24 week, active-controlled trial: Effect of ipragliflozin in MASLD.

AIMS/INTRODUCTION: We conducted a 5 year post-trial monitoring study of our previous randomized 24 week, open-label, active-controlled trial that showed beneficial effects of ipragliflozin on metabolic dysfunction-associated steatotic liver disease (MASLD), identical to those of pioglitazone. MATERIALS AND METHODS: In our previous trial, 66 patients with MASLD and type 2 diabetes were randomly assigned to receive either ipragliflozin (n = 32) or pioglitazone (n = 34). Upon its conclusion, 61 patients were monitored for 5 years for outcome measures of MASLD, glycemic, and metabolic parameters. Differences between the two groups were analyzed at baseline, 24 weeks, and 5 years; changes in outcome measures from baseline were also evaluated. RESULTS: At 5 years, the mean liver-to-spleen attenuation ratio increased by 0.20 (from 0.78 ± 0.24 to 0.98 ± 0.20) in the ipragliflozin group and by 0.26 (from 0.76 ± 0.26 to 1.02 ± 0.20) in the pioglitazone group (P = 0.363). Similarly, ipragliflozin and pioglitazone significantly improved serum aminotransferase, HbA1c, and fasting plasma glucose levels over 5 years. In the ipragliflozin group, significant reductions in body weight and visceral fat area observed at 24 weeks were sustained throughout the 5 years (-4.0%, P = 0.0075 and -7.6%, P = 0.045, respectively). Moreover, ipragliflozin significantly reduced the values of fibrosis markers (serum ferritin and FIB-4 index), was well tolerated, and had a higher continuation rate for 5 years compared with pioglitazone. CONCLUSIONS: Ipragliflozin and pioglitazone improved MASLD and glycemic parameters over 5 years. In the ipragliflozin group, significant reductions in body weight and visceral fat mass persisted for 5 years.

Hypoglycemic effects of colestimide on type 2 diabetic patients with obesity.

Recent studies have shown colestimide, a bile acid-binding resin, to also exert a glucose-lowering effect via amelioration of insulin resistance. To evaluate the effects of colestimide on glucose metabolism and to elucidate the underlying mechanism, we conducted a 6-month, open-label pilot study on 43 type 2 diabetic patients with obesity (BMI ≥ 25). The subjects were randomized to either treatment with colestimide 4g/day (T group, n=23) or continuation of their current therapy (C group, n=20). In the T group patients, mean HbA1c and fasting glucose improved markedly (from 7.71 ± 0.32% to 6.97 ± 0.20%; from 147.4 ± 7.3mg/dL to 127.0 ± 5.0mg/dL, respectively), while obesity-related parameters, i.e. body weight, waist circumference, and visceral fat and subcutaneous fat as determined by umbilical slice abdominal CT, showed no significant changes. Fractionation analyses of serum bile acids revealed significantly increased cholic acids (CA) and decreased chenodeoxycholic acids (CDCA) in the T group patients. However, no correlation was observed between these changes and ΔHbA1c. According to logistic regression analysis, baseline HbA1c was the only variable predicting the decrease of HbA1c (>0.5%) among sex, age, BMI, total cholesterol, ΔCA and ΔCDCA. The index of insulin resistance, i.e. HOMA-R, did not improve, and the index of ß cell function, i.e. HOMA-ß, actually increased significantly. These results suggests that, in obese patients with type 2 diabetes, the mechanism underlying improved glycemic control with colestimide treatment involves enhanced ß cell activity rather than improved insulin resistance.

Noninvasive evaluation of kidney hypoxia and fibrosis using magnetic resonance imaging.

Interstitial fibrosis and hypoxia accelerate the progression of CKD, but clinical tools to quantitate these factors in patients are lacking. Here, we evaluated the use of two magnetic resonance imaging (MRI) techniques, diffusion-weighted (DW)-MRI and blood oxygen level-dependent (BOLD)-MRI, to assess kidney fibrosis and hypoxia of the cortex in 142 patients with either diabetic nephropathy (n = 43), CKD without diabetes (n = 76), or acute kidney injury (AKI) (n = 23). Apparent diffusion coefficient (ADC) values of DW-MRI correlated with estimated glomerular filtration rates (eGFR) in the diabetic nephropathy and CKD groups (r(2) = 0.56 and r(2) = 0.46, respectively). Although the T2* values of BOLD-MRI and eGFR displayed good correlation in the CKD group (r(2) = 0.38), we did not observe a significant correlation between these values in the diabetic nephropathy group, suggesting that factors other than tubulointerstitial alteration determine the degree of hypoxia in the renal cortex. In the AKI group, neither the T2* nor ADC values correlated with eGFR. Renal biopsies from patients with CKD demonstrated that the T2* and ADC MRI values correlated with renal pathology. Taken together, ADC and T2* values appear to serve as accurate indices for evaluating renal tubulointerstitial alterations and parenchymal hypoxia, respectively, in the cortex. Functional MRI can thus contribute to multilateral, noninvasive, in vivo assessment of kidney function.

Activation of NAD(P)H:quinone oxidoreductase 1 prevents arterial restenosis by suppressing vascular smooth muscle cell proliferation.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (betaL) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. betaL significantly reduced the neointimal formation induced by balloon injury. betaL also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. betaL increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the betaL-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by betaL is mediated by LKB1 in the presence of NQO1. Taken together, these results show that betaL inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs.

Effects of Dapagliflozin Compared with Sitagliptin and Metformin in Drug-Naïve Japanese Patients with Type 2 Diabetes: A 12-Week, Open-Label, Randomized, Active-Controlled Trial.

INTRODUCTION: To compare the efficacy and tolerability of dapagliflozin with those of sitagliptin and metformin in patients with type 2 diabetes who have never received glucose-lowering agents. METHODS: In this randomized, 12-week, open-label, active-controlled trial, 32 patients were randomly assigned to receive dapagliflozin 5 mg, sitagliptin 50 mg, or metformin 1000 mg per day for 12 weeks. At baseline and at week 12, the patients underwent a meal tolerance test (MTT). RESULTS: After 12 weeks of treatment, the changes in fasting and postprandial plasma glucose and plasma glucose area under the curve (AUC)0-120 min levels during the MTT from baseline were significantly improved in the three study groups, and there were no significant differences among the three study groups (P < 0.05). The mean changes in glycated hemoglobin (HbA1c) from baseline to week 12 were - 0.96%, - 1.24%, and - 1.40% in the dapagliflozin, sitagliptin, and metformin groups, respectively. Although there was no significant difference among the three study groups, the lowering effect of HbA1c tended to be greater in the metformin group than in the dapagliflozin group. In contrast, the insulin AUC0-120 min levels at week 12 significantly decreased only in the dapagliflozin group (P = 0.049). Similarly, body weight was significantly reduced only in the dapagliflozin group (- 2.1 kg [- 2.7%], P = 0.047). Moreover, dapagliflozin significantly improved serum adiponectin levels (P = 0.003). However, there were no significant differences in the changes in these glycemic and metabolic parameters among the three study groups. No serious adverse events were documented in any group. CONCLUSIONS: Dapagliflozin exerted beneficial effects similar to sitagliptin and metformin on glycemic parameters. In addition, dapagliflozin significantly reduced body weight and insulin AUC levels and improved serum adiponectin levels. Therefore, we suggest that these three hypoglycemic agents could be viable first-line medications for drug-naïve Japanese patients with type 2 diabetes. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN000024427).

High molecular weight adiponectin activates AMPK and suppresses cytokine-induced NF-kappaB activation in vascular endothelial cells.

Various isoforms of adiponectin circulate in the plasma. We purified high molecular weight (HMW) adiponectin from human plasma. HMW adiponectin was observed to activate AMP-activated protein kinase (AMPK), thereby increasing the phosphorylation of eNOS and NO production in endothelial cells. On the other hand, cells preincubated with HMW adiponectin had reduced TNFalpha-induced NF-kappaB activation. HMW adiponectin by itself was found to modestly activate NF-kappaB, which was significantly enhanced by inhibition of AMPK/eNOS activation. Thus, HMW adiponectin might have dual action, both pro and anti-inflammatory. An initial period of NF-kappaB activation by HMW adiponectin might be proinflammatory, but it could be counteracted by activation of AMPK/eNOS, which lead to a potential reduction in a second activation of NF-kappaB against inflammatory stimuli.

SH3 domain of the phosphatidylinositol 3-kinase regulatory subunit is responsible for the formation of a sequestration complex with insulin receptor substrate-1.

Class IA phosphatidylinositol 3-kinase (PI 3-kinase), which is composed of a 110kDa catalytic subunit and a regulatory subunit, plays a key role in most insulin dependent cellular responses. To date, five mammalian regulatory subunit isoforms have been identified, including two 85kDa proteins (p85alpha and p85beta), two 55kDa proteins (p55gamma and p55alpha), and one 50kDa protein (p50alpha). In the present study, we overexpressed these recombinant proteins, tagged with green fluorescent proteins (GFP), in CHO-IR cells and investigated intracellular localizations in both the presence and the absence of insulin stimulation. Interestingly, in response to insulin, only p85alpha and p85beta redistributed to isolated foci in the cells, while both were present throughout the cytoplasm in quiescent cells. In contrast, p55s accumulated in the perinuclear region irrespective of insulin stimulation, while p50alpha behaved similarly to control GFP. Immunofluorescent antibodies against endogenous IRS-1 revealed IRS-1 to be co-localized in the p85 foci in response to insulin. As both insulin receptors and p110alpha catalytic subunits were absent from these foci on immunofluorescence study, only p85 and IRS-1 were suggested to form a sequestration complex in response to insulin. To determine the domain responsible for IRS-1 complex formation, we prepared and overexpressed the SH3 domain deletion mutant of p85alpha in CHO-IR cells. This mutant failed to form foci, suggesting the SH3 domain of regulatory subunits to be responsible for formation of the p85-IRS-1 sequestration complex. In conclusion, our study revealed the SH3 domain of PI 3-kinase to play a critical role in intracellular localizations, including formation of foci with IRS-1 in response to insulin.

Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues.

AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in multiple cell signaling pathways that has become an attractive therapeutic target for vascular diseases. It is not clear whether rottlerin, an inhibitor of protein kinase Cdelta, activates AMPK in vascular cells and tissues. In the present study, we have examined the effect of rottlerin on AMPK in vascular smooth muscle cells (VSMCs) and isolated rabbit aorta. Rottlerin reduced cellular ATP and activated AMPK in VSMCs and rabbit aorta; however, inhibition of PKCdelta by three different methods did not activate AMPK. Both VSMCs and rabbit aorta expressed the upstream AMPK kinase LKB1 protein, and rottlerin-induced AMPK activation was decreased in VSMCs by overexpression of dominant-negative LKB1, suggesting that LKB1 is involved in the upstream regulation of AMPK stimulated by rottlerin. These data suggest for the first time that LKB1 mediates rottlerin-induced activation of AMPK in vascular cells and tissues.

Long-Term Effects of Ipragliflozin on Diabetic Nephropathy and Blood Pressure in Patients With Type 2 Diabetes: 104-Week Follow-up of an Open-Label Study.

BACKGROUND: In our previous study, we investigated the efficacy of ipragliflozin, a sodium-glucose cotransporter (SGLT) 2 inhibitor on diabetic nephropathy in patients with type 2 diabetes and demonstrated that ipragliflozin significantly improved diabetic nephropathy in addition to reducing HbA1c and body weight. Herein, we conducted post-trial monitoring to determine whether these lowering effects on blood glucose and body weight or the beneficial effects on diabetic nephropathy were maintained long-term (104 weeks) after starting ipragliflozin treatment. METHODS: Initially, during a 24-week interventional trial period, a 50 mg dose of ipragliflozin was administered to 50 patients with type 2 diabetes without changing other treatments. During the post-trial monitoring period, these patients returned to hospital-based diabetes care according to their clinical needs. We continued monitoring their clinical data for 104 weeks in each hospital and analyzed the results on an intention-to-treat basis. RESULTS: The improvements in glycemic control and body weight reduction provided by 24-week ipragliflozin administration were maintained for 104 weeks. Despite a transient decrease during the intervention period, the estimated glomerular filtration rate (eGFR) was restored to near the baseline level at 104 weeks. Notably, in patients with diabetic nephropathy, the median urinary albumin-to-creatinine ratio (UACR) was significantly decreased from 119.2 (98.9 - 201.8) at baseline to 36.9 (19.7 - 204.7) mg/gCr at 104 weeks. In addition, eGFR was stable for 104 weeks, showing no decrease. In contrast, a significant positive correlation between UACR and blood pressure observed at 24 weeks disappeared after discontinuation of the intervention therapy. CONCLUSIONS: The well-controlled HbA1c and body weight reductions were maintained for 104 weeks of post-trial follow-up. Moreover, ipragliflozin significantly reduced urinary albumin excretion in patients with diabetic nephropathy without decreasing eGFR.

Is renoprotection by angiotensin receptor blocker dependent on blood pressure?: the Saitama Medical School, Albuminuria Reduction in Diabetics with Valsartan (STAR) study.

To explore the effects of various antihypertensive regimes on microalbuminuria, an angiotensin II receptor blocker (ARB), valsartan, was substituted for or added to treatment with a calcium channel blocker (CCB). After a 6-month CCB baseline period, 28 Japanese hypertensive patients with incipient diabetic nephropathy (defined as a urinary albumin excretion [UAE] of 30-300 mg/g creatinine), were assigned to two groups according to their blood pressure (BP) levels: in patients with a BP of more than 130/85 mmHg (n=17), valsartan was added to the CCB (Group A), while in patients with a BP <130/85 mmHg, valsartan alone was given (Group B: n=11) for 12 months. UAE was determined before and at 3, 6 and 12 months after the initiation of ARB. Although the initial BP was significantly higher in Group A (150/83 mmHg) than Group B (127/77 mmHg), BP was decreased to 141/78 mmHg in Group A and slightly, but not significantly, increased to 130/82 mmHg in Group B. In both groups, UAE was significantly decreased after ARB treatment (to 89% of the basal value in Group A and to 40.5% of the basal value in Group B) and did not differ each other and the amount of decrease did not differ significantly between the two groups. These results suggest that combination therapy with an ARB and CCB is very effective in lowering BP and UAE in cases in which BP is not well controlled, while, even in patients with a sufficient BP control of <130/85 mmHg, the use of ARB singly resulted in a significant decrease in UAE without a further decrease in BP, implying that the ARB had a renoprotective action independent of changes in BP.

Fluorescent indicators for imaging protein phosphorylation in single living cells.

To visualize signal transduction based on protein phosphorylation in living cells, we have developed genetically encoded fluorescent indicators, named phocuses. Two different color mutants of green fluorescent protein (GFP) were joined by a tandem fusion domain composed of a substrate domain for the protein kinase of interest, a flexible linker sequence, and a phosphorylation recognition domain that binds with the phosphorylated substrate domain. Intramolecular interaction of the substrate domain and the adjacent phosphorylation recognition domain within a phocus was dependent upon phosphorylation of the substrate domain by protein kinase, which influenced the efficiency of fluorescence resonance energy transfer (FRET) between the GFPs within a phocus. In the present study, we employed phocuses composed of insulin signaling proteins to visualize protein phosphorylation by the insulin receptor. This method may provide a general approach for studying the dynamics of protein phosphorylation-based signal transduction in living cells.

[Adiponectin receptor regulation in diabetic states].

Two adiponectin receptors were recently identified, AdipoR1 and R2. AdipoR1 expression was increased approximately 2.5-fold in muscle tissues from insulin-deficient diabetic mice, but normalized with insulin administration. AdipoR1 expression was decreased by 44% in insulin-resistant obese, as compared to lean, mice. These results indicate AdipoR1 expression to correlate inversely with plasma insulin levels. Insulin treatment for 24 h decreased AdipoR1 expression by approximately 60% in C2C12 myocytes. In addition, this effect was mediated by the phosphatidylinositol-3 kinase/Foxo 1 dependent pathway. In contrast, AdipoR2 expression was not significantly altered in diabetic states. Our results indicate that regulation of AdipoR1, but not AdipoR2, may be involved in glucose and lipid metabolism in diabetic states.

Effects of Ipragliflozin on Diabetic Nephropathy and Blood Pressure in Patients With Type 2 Diabetes: An Open-Label Study.

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT2) inhibitors are novel agents used to treat type 2 diabetic patients. We investigated the efficacy of the SGLT2 inhibitor ipragliflozin on diabetic nephropathy in Japanese patients with type 2 diabetes. METHODS: A 50 mg dose of ipragliflozin was administered for 24 weeks to 50 patients with type 2 diabetes who were concomitantly managed with diet and exercise therapy alone or antidiabetic medications other than SGLT2 inhibitors. RESULTS: At the end of the 24-week ipragliflozin treatment, significant decreases in mean glycated hemoglobin (HbA1c) (1.0±1.2%) and body weight (2.7 ± 2.5 kg) were observed; in addition, median urinary albumin-to-creatinine ratio (UACR) significantly decreased from 15.5 (8.0 - 85.7) to 12.9 (7.4 - 36.3) mg/gCr. Sub-analysis by renal function at baseline revealed that median UACR in patients with estimated glomerular filtration rate (eGFR) ≥ 90 mL/min/1.73 m2 decreased significantly from 12.3 (7.5 - 89.6) to 10.6 (5.8 - 27.3) mg/gCr. Furthermore, mean eGFR decreased significantly from 102.4 ± 8.6 to 93.6 ± 10.5 mL/min/1.73 m2 in these patients. In contrast, UACR and eGFR did not change significantly in patients with eGFR < 90. In addition, analysis of the relationship between the amount of change in UACR and blood pressure at 24 weeks revealed a significant positive correlation between UACR and SBP values, independently of the presence of diabetic nephropathy. CONCLUSIONS: Our results indicate that ipragliflozin may facilitate HbA1c control and body weight reduction. Furthermore, our results also raise the possibility that ipragliflozin significantly reduces urinary albumin levels and improves glomerular hyperfiltration in a subset of patients with type 2 diabetes.

Comparison of Ipragliflozin and Pioglitazone Effects on Nonalcoholic Fatty Liver Disease in Patients With Type 2 Diabetes: A Randomized, 24-Week, Open-Label, Active-Controlled Trial.

OBJECTIVE: To compare the efficacy of ipragliflozin versus pioglitazone in patients with type 2 diabetes complicated by nonalcoholic fatty liver disease (NAFLD). RESEARCH DESIGN AND METHODS: In this open-label, randomized, active-controlled trial, we randomly assigned 66 patients with type 2 diabetes and NAFLD to receive ipragliflozin 50 mg (n = 32) or pioglitazone 15-30 mg (n = 34) orally once daily. The primary outcome was a change from baseline in the liver-to-spleen attenuation ratio (L/S ratio) on computed tomography at week 24. RESULTS: At week 24, the mean ± SD L/S ratio had increased by 0.22 (from 0.80 ± 0.24 to 1.00 ± 0.18) in the ipragliflozin group and 0.21 (from 0.78 ± 0.26 to 0.98 ± 0.16) in the pioglitazone group (P = 0.90). Serum aspartate and alanine aminotransferase levels, HbA1c, and fasting plasma glucose were similarly reduced in the two treatment groups. Nevertheless, body weight and visceral fat area showed significant reductions only in the ipragliflozin group compared with the pioglitazone group (P < 0.0001 and P = 0.0013, respectively). There were no serious adverse events in either group. CONCLUSIONS: Compared with pioglitazone, ipragliflozin exerts equally beneficial effects on NAFLD and glycemic control during the treatment of patients with type 2 diabetes complicated by NAFLD. Furthermore, ipragliflozin significantly reduced body weight and abdominal fat area.