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Aims To examine the effects of a 24-month treatment with ipragliflozin on carotid intima-media thickness (IMT) in type 2 diabetes patients. Methods and results In this multicenter, prospective, randomized, open-label, and blinded-endpoint investigator-initiated clinical trial, adults with type 2 diabetes and haemoglobin A1C (HbA1c) of 6.0–10.0% (42–86 mmol/mol) were randomized equally to ipragliflozin (50 mg daily) and non-sodium-glucose cotransporter-2 (SGLT2) inhibitor use of standard-care (control group) for type 2 diabetes and were followed-up to 24 months. The primary endpoint was the change in mean common carotid artery IMT (CCA-IMT) from baseline to 24 months. A total of 482 patients were equally allocated to the ipragliflozin (N = 241) and control (N = 241) groups, and 464 patients (median age 68 years, female 31.7%, median type 2 diabetes duration 8 years, median HbA1c 7.3%) were included in the analyses. For the primary endpoint, the changes in the mean CCA-IMT from baseline to 24 months were 0.0013 [95% confidence interval (CI), −0.0155–0.0182] mm and 0.0015 (95% CI, −0.0155–0.0184) mm in the ipragliflozin and control groups, respectively, with an estimated group difference (ipragliflozin-control) of −0.0001 mm (95% CI, −0.0191–0.0189; P = 0.989). A group difference in HbA1c change at 24 months was also non-significant between the treatment groups [−0.1% (95% CI, −0.2–0.1); P = 0.359]. Conclusion Twenty-four months of ipragliflozin treatment did not affect carotid IMT status in patients with type 2 diabetes recruited in the PROTECT study, relative to the non-SGLT2 inhibitor-use standard care for type 2 diabetes.

Background 14-3-3η is an intracellular protein also detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA). It is closely related to disease activity and anti-cyclic citrullinated peptide antibody levels. However, the main source of 14-3-3η and the mechanism of its release into the extracellular space remain unclear. Addressing these two points was the main goal of the current study. Methods The source of 14-3-3η was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4+ cells, and macrophages were selected as candidates among the various cell types in the synovial tissue. Phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL) and cell death of macrophages were studied by phalloidin staining and electron microscopy after stimulation with an oxidative stress inducer (diamide) or tumour necrosis factor (TNF)-α. Extracellular 14-3-3η protein levels were examined by western blotting. Results Macrophages from the synovial tissue from RA, but not osteoarthritis, showed dense and widespread cytoplasmic staining for the 14-3-3η protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-α, but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-α-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with diamide and TNF-α, but not IL-6/sIL-6R. Conclusions Macrophages that highly express 14-3-3η undergo TNF-α-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3η into the extracellular space. The current study provides a novel mechanism for 14-3-3η level increase in the RA synovial fluid.

The purpose of this study was to determine the glycemic profiles of drug-naive type 2 diabetes patients according to hemoglobin A1c (HbA1c) level using continuous glucose monitoring. We aimed to clarify factors associated with HbA1c and average blood glucose level. Patients were divided into three groups according to their HbA1c level (< 7.0% n=23, 7.0% ≤ HbA1c < 8.0% n=17 and ≥ 8.0% n=31), and the factors associated with HbA1c and average glucose of each group were evaluated. Pre-meal glucose levels were the highest before lunch, and the 2 hour postprandial blood glucose level was the lowest after lunch. The pre-meal and postprandial blood glucose levels increased after each meal with increases in HbA1c. Average glucose level was the most significant determinant of HbA1c, whereas pre-meal glucose level at dinner was the most significant determinant of average glucose level, and the range of increase in glucose from pre-meal at dinner was the most significant determinant of standard deviation (SD) of 24 hour glucose levels. HbA1c subgroup analysis indicated that pre-meal glucose level at lunch significantly correlated with average glucose level in the HbA1c < 8.0% group, while pre-meal glucose level at dinner significantly correlated with average glucose level in the HbA1c ≥ 8.0% group. The range of increase in glucose from pre-meal in the morning significantly correlated with SD of 24 hour glucose levels in the HbA1c < 8.0% group, and the postprandial peak glucose level at lunch significantly correlated with SD of 24 hour glucose levels in the HbA1c ≥ 8.0% group. The results suggest that improvement of the average glucose level is necessary to improve the HbA1c levels. For patients with HbA1c < 7.0%, it is important to improve blood glucose level after breakfast and before lunch to decrease the average glucose level. For patients with 7.0% ≤ HbA1c < 8.0%, it is important to improve blood glucose level before lunch and after dinner to decrease the average glucose level. For patients with HbA1c ≥ 8.0%, it is important to improve blood glucose levels after lunch and before dinner to decrease the average glucose level.

It is difficult to detect glycemic excursions using CGM in daily clinical practice. We retrospectively analyzed CGM data in type T2DM to define the correlations between HbA1c and GA levels at admission and the parameters representing glycemic excursions measured by CGM, including the mean amplitude of glycemic excursions (MAGE) and standard deviation (SD). The MAGE correlated significantly with GA and HbA1c, but not with the GA/HbA1c ratio. The SD correlated significantly with GA, HbA1c, and GA/HbA1c. Multivariate analysis identified the GA value to be the most reflective of MAGE. Patients were divided into 2 groups using a MAGE cutoff value of 75 mg/dl, which reflects stable diabetes. There was a significant difference in GA, but not HbA1c, between the groups with low and high mean amplitudes of glycemic excursions. Receiver operating characteristic curve analysis indicated that the cutoff for GA for identifying patients with MAGE of ≤75 mg/dl was 18.1%. Our study identified GA to be the most reflective of glycemic excursions in patients with T2DM. GA can be a useful index of glycemic excursions and treatment optimization to prevent arteriosclerosis.

To determine the factors associated with hypoglycemia in patients with type 2 diabetes mellitus (T2DM) on insulin therapy.This retrospective study included 62 inpatients with T2DM on insulin therapy who underwent 5-day continuous glucose monitoring (CGM). We analyzed the relation between hypoglycemia (defined as blood glucose below 70 mg/dL, as determined by the CGM) and time spent in hypoglycemia with fasting blood glucose, mean blood glucose (MBG), standard deviation (SD), coefficient of variation, minimum blood glucose level, maximum blood glucose level, and the percent time spent with blood glucose levels of180 mg/dL.Twelve patients (19.4%) developed hypoglycemia, and most were maintained on mix insulin therapy alone. In the hypoglycemic group, MBG was lower and SD was higher, than in the non-hypoglycemic group, although HbA1c was not different. Multivariate logistic regression analysis identified MBG and SD as factors related to hypoglycemia. Receiver operating characteristic curve analysis showed that the optimal MBG and SD cutoff values for prediction of hypoglycemia were 150.9 and 41.1 mg/dL, respectively. When subjects were divided into four groups according to these values, time at blood glucose70 mg/dL was longest, and total insulin dosage highest, in the MBG-low/SD-high group.MBG and SD of glucose levels were identified as significant and independent determinants of hypoglycemia in T2DM on insulin therapy. It is important to use the least insulin dose, with the target of minimizing glycemic variability, to achieve good glycemic control without hypoglycemia.

The aim of this retrospective study was to elucidate the association between glucose profile using the continuous glucose monitoring system (CGMS) and microvascular complications in patients with type 2 diabetes mellitus (T2DM). The subjects were 160 inpatients with T2DM. The mean blood glucose (MBG) level, percentage of time in a 24-hour period spent with blood glucose level higher than 180 mg/dl (time at >180 mg/dl), standard deviation (SD), and mean amplitude of glycemic excursions (MAGE) were measured continuously over 48 hours using the CGMS. The primary outcome was the association between microvascular complications and glycemic variability. The secondary outcome was the association between microangiopathies and MBG. The SD and MAGE were not associated with presence of microangiopathies or number of complications. There were also no associations between abnormal vibratory sensation in the bilateral lower extremities, coefficient of variation of the R-R interval (CVRR), retinopathy stage, nephropathy stage, or microalbuminuria. MBG was associated, however, with retinopathy, retinopathy stage, and number of complications. Time at >180 mg/dl correlated with abnormal vibratory sensation in the bilateral lower extremities and presence or stage of retinopathy. MBG and time at >180 mg/dl were not associated with presence or stage of nephropathy. Our findings suggest that broad glycemic variability was not associated with microvascular complications, the number of which increased in patients with a high mean glucose level and long time spent with hyperglycemia. It is important, therefore, to reduce the mean glucose level and time spent with hyperglycemia to prevent future microangiopathies.

Background 14-3-3η is an intracellular protein detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA) [1]. While presence of 14-3-3η is both diagnostic for early and established RA [2] and prognostic for radiographic progression [3], the mechanism of 14-3-3η externalization in RA remains unclear. Objectives To clarify the mechanism of externalization of 14-3-3η into the extracellular space using human PBMC-derived macrophages (Mϕ). Methods Distribution of 14-3-3η in synovial tissue of patients with RA or osteoarthritis (OA) was examined by immunohistochemistry; cellular morphology was studied by confocal microscopy and electron microscopy (EM). Mϕ were stimulated with TNF-α, Diamide (induces ligand-independent TNFR signaling) or IL-6/sIL-6R. Western blotting was used to detect S-358-phosphorylated MLKL (a mediator of necroptosis) and presence of 14-3-3η in Mϕ culture supernatants. Results Dense and widespread staining of 14-3-3η was detected in Mϕ of RA, but not OA, synovial tissues. 14-3-3η and peptidylarginine deiminase 4 (PAD4) co-localized in CD68+ cells (Mϕ) from RA synovial tissue; 14-3-3η was not detected in CD68+ cells from RA lung tissue or CD4+ cells (T cells) from RA synovium. The outer space around the nucleus of healthy control Mϕ treated with TNF-α or Diamide, but not IL-6/sIL-6R, demonstrated abnormal actin distribution by phalloidin staining and presence of cellular and organelle swelling by EM. Further, magnified images showed partial destruction of the cell membrane in TNF-α and Diamide-treated cells. Phosphorylation of MLKL was observed between 20 min and 24 h after stimulation of healthy control Mϕ with TNF-α, with maximum signal at 8 h post-stimulation, but was not observed upon IL-6/sIL-6R stimulation at any time point. After 8 h of stimulation no 14-3-3η was detected in the culture supernatant of healthy control Mϕ endogenously expressing 14-3-3η or Mϕ stimulated with IL-6/sIL-6R, while a high concentration of 14-3-3η was detected in culture supernatants in Mϕ treated with TNF-α or Diamide. Conclusion 14-3-3η protein was abundant in RA, but not OA, synovial tissues and co-localized with PAD4 in CD68+ synovial Mϕ; this close proximity to PAD4 may promote citrullination of 14-3-3η in vivo. Treatment of healthy control Mϕ with TNF-α induced phosphorylation of MLKL and cell swelling and disintegration of the plasma membrane characteristic of necroptosis, correlated with the release of intracellular 14-3-3η protein into cellular supernatants. Our results shed light on mechanism of externalization of 14-3-3η and how it achieves elevated levels in RA synovial fluid. References [1] Kilani RT, et al. J Rheumatol 2007; 34:1650–7. [2] Maksymowych W, et al. J Rheumatol 2014; 41:2104-13. [3] Carrier N, et al. Arthritis Res Ther 2016; 18:50. Competing Interests M. Zaharik and N. Biln are employees of Augurex. Y. Tanaka, has received speaking fees and/or honoraria from Daiichi-Sankyo, Astellas, Eli Lilly, Chugai, Sanofi, Abbvie, Pfizer, YL Biologics, Bristol-Myers, Glaxo-Smithkline, UCB, Mitsubishi-Tanabe, Novartis, Eisai, Takeda, Janssen, Asahi-kasei and has received research grants from Mitsubishi-Tanabe, Bristol-Myers, Eisai, Chugai, Takeda, Abbvie, Astellas, Daiichi-Sankyo, Ono, MSD, Taisho-Toyama. All other authors have nothing to disclose. Disclosure of Interests Gulzhan Trimova: None declared, Kaoru Yamagata: None declared, Shigeru Iwata: None declared, Tong Zhang: None declared, Fumi Uemura: None declared, Minoru Satoh: None declared, Michelle Zaharik: None declared, Norma Biln Shareholder of: Amgen, Employee of: Pfizer, Amgen and Abbott, Shintaro Hirata Grant/research support from: Eli Lilly, UCB, Consultant for: Bristol-Myers Squibb, Jansen, UCB, Paid instructor for: AbbVie, Eisai, Tanabe-Mitsubishi, Speakers bureau: AbbVie, Astellas, Ayumi, Bristol-Myers Squibb, Chugai, Eisai, Eli Lilly, Jansen, Kissei, Pfizer, Sanofi, Takeda, Tanabe-Mitsubishi, UCB, Shingo Nakayamada Grant/research support from: Mitsubishi-Tanabe, Takeda, Novartis and MSD, Speakers bureau: Bristol-Myers, Sanofi, Abbvie, Eisai, Eli Lilly, Chugai, Asahi-kasei and Pfizer, Yoshiya Tanaka Grant/research support from: Abbvie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama, Takeda, Speakers bureau: Abbvie, Asahi-kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Eisai, Glaxo-Smithkline, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer Japan Inc, Sanofi, Takeda, UCB, YL Biologics

Background Hypoglycemia is associated with cardiovascular diseases, increased risk of death. Therefore, it is important to avoid hypoglycemia. The aim of this study was to characterize hypoglycemia according to glycated hemoglobin (HbA1c) level and determine the contributing factors in type 2 diabetes mellitus (T2DM), using continuous glucose monitoring (CGM). Methods T2DM patients (n = 293) receiving inpatient care were divided into five groups according to HbA1c level on admission (Group 1: ≥ 6 to 180 mg/dL. In any of the HbA1c groups, variations in blood glucose level were significantly larger in patients with hypoglycemia than without. Conclusions Hypoglycemia occurred in patients with a wide range of HbA1c on admission (range 6–9%), suggesting that prediction of hypoglycemia based on HbA1c alone is inappropriate. Among patients with low HbA1c, strict control sometimes induce hypoglycemia. Among patients with high HbA1c, the possibility of hypoglycemia should be considered if there is a marked discrepancy between HbA1c and randomly measured blood glucose level. Larger variations in blood glucose level induce hypoglycemia in any of the HbA1c groups. The treatment to reduce variations in blood glucose level is important to prevent hypoglycemia.

A 54-year-old woman was admitted to our hospital for detailed examination of acromegaly because she noticed bilateral hand and finger swelling at the age of 43 and plantar thickening, facial changes and unclear articulation at the age of 49. She had prominent brow ridges, mandibular protrusion, and enlargement of the hands, feet, nasal wings, lips and tongue. Her growth hormone (GH) level was 39.8 ng/ml, insulin-like growth factor-1 (IGF-1) level was 717 ng/ml, GH level was not suppressed (22.9 ng/ml) during a 75-g oral glucose tolerance test (OGTT). Radiography showed cauliflower-like enlargement of the distal phalanx of the fingers, thickening/enlargement of the plantar soft tissues, and increased antero-posterior diameter of the sella turcica. Magnetic resonance imaging showed a mass (21×17 mm) growing towards the right suprasellar region and invading the cavernous sinus. She was diagnosed with acromegaly based on the characteristic physical findings, GH excess, high IGF-1, lack of GH suppression during the 75-g OGTT, and the presence of a pituitary tumor. She was started on octreotide long acting release (Oct-LAR) 20 mg/4w for tumor shrinkage. After three doses, her GH and IGF-1 levels decreased to 2.19 ng/ml (1.69 during the 75-g OGTT) and 205 ng/ml, respectively, meeting cure criteria for acromegaly. In this case, a decrease in GH and IGF-1 levels, tumor shrinkage, and resolution of cavernous sinus invasion allowed the patient to undergo surgery with curative intent (the first-line treatment for acromegaly) without postoperative complications. Thus, preoperative Oct-LAR administration has the potential to improve treatment outcomes of acromegaly.

Bioactive O-β- d -galactopyranosyl-(1 → 4)-O-β- d -galactopyranosyl-(1 → 4)- d -glucopyranose (4′-galactosyl lactose) was site-selectively modified at a reducing end with thiosemicarbazide (TSC). As-synthesized 4′-galactosyl lactose-TSC was immobilized on a gold substrate with cellobiose-TSC as a spacer through spontaneous self-assembly chemisorption via S Au bonding. Quartz crystal microbalance analysis suggested the successful formation of self-assembled monolayers (SAMs) of 4′-galactosyl lactose-TSC and/or cellobiose-TSC. Galactose-binding lectin exhibited the highest affinity for hybrid SAMs with an equimolar ratio of the two oligosaccharide-TSCs, while glucose-binding lectin showed decreasing adsorption with a decrease in cellobiose-TSC ratios. Human hepatocellular carcinoma cells, which recognize galactose residues, efficiently adhered to the hybrid SAMs. Higher enzymatic deethoxylation of ethoxyresorufin via cytochrome P450 appeared on hybrid SAMs. These results suggested that clustering of the bioactive sugars was involved in the cellular responses, possibly via biological carbohydrate–protein interactions. This approach to designing carbohydrate-based scaffolds should provide a basis for the functional development of glyco-decorated biointerfaces for cell culture applications.