Direct Reprogramming Improves Cardiac Function and Reverses Fibrosis in Chronic Myocardial Infarction.

BACKGROUND: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. METHODS: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. RESULTS: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. CONCLUSIONS: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.

Cardiac reprogramming reduces inflammatory macrophages and improves cardiac function in chronic myocardial infarction.

Cardiomyocytes (CMs) have little regenerative capacity. After myocardial infarction (MI), scar formation and myocardial remodeling proceed in the infarct and non-infarct areas, respectively, leading to heart failure (HF). Prolonged activation of cardiac fibroblasts (CFs) and inflammatory cells may contribute to this process; however, therapies targeting these cell types remain lacking. Cardiac reprogramming converts CFs into induced CMs, reduces fibrosis, and improves cardiac function in chronic MI through the overexpression of Mef2c/Gata4/Tbx5/Hand2 (MGTH). However, whether cardiac reprogramming reduces inflammation in infarcted hearts remains unclear. Moreover, the mechanism through which MGTH overexpression in CFs affects inflammatory cells remains unknown. Here, we showed that inflammation persists in the myocardium until three months after MI, which can be reversed with cardiac reprogramming. Single-cell RNA sequencing demonstrated that CFs expressed pro-inflammatory genes and exhibited strong intercellular communication with inflammatory cells, including macrophages, in chronic MI. Cardiac reprogramming suppressed the inflammatory profiles of CFs and reduced the relative ratios and pro-inflammatory signatures of cardiac macrophages. Moreover, fluorescence-activated cell sorting analysis (FACS) revealed that cardiac reprogramming reduced the number of chemokine receptor type 2 (CCR2)-positive inflammatory macrophages in the non-infarct areas in chronic MI, thereby restoring myocardial remodeling. Thus, cardiac reprogramming reduced the number of inflammatory macrophages to exacerbate cardiac function after MI.

Flk1 Deficiency and Hypoxia Synergistically Promote Endothelial Dysfunction, Vascular Remodeling, and Pulmonary Hypertension.

BACKGROUND: The mechanisms underlying pulmonary hypertension (PH) remain largely unknown; further, why advanced vascular remodeling preferentially occurs in arterioles is yet to be answered. VEGF (vascular endothelial growth factor) regulates angiogenesis through Flk1 (fetal liver kinase 1) and Flt1 (fms-like tyrosine kinase 1) on endothelial cells (ECs), which may be related to PH pathogenesis. However, spatiotemporal expression patterns of Flk1 and Flt1 in the pulmonary vascular system and the role of endothelial Flk1 in PH development remain poorly understood. METHODS: We analyzed multiple reporter mice, including Flk1-GFP (green fluorescent protein) bacterial artificial chromosome transgenic (Tg), Flt1-DsRed bacterial artificial chromosome Tg, and Flk1-GFP/Flt1-DsRed double Tg mice, to determine the spatiotemporal expression of Flk1 and Flt1 in hypoxia-induced PH. We also used Cdh5CreERT2/Flk1f/f/Tomato (Flk1-KO [knockout]) mice to induce EC-specific Flk1 deletion and lineage tracing in chronic hypoxia. RESULTS: Flk1 was specifically expressed in the ECs of small pulmonary vessels, including arterioles. Conversely, Flt1 was more broadly expressed in the ECs of large- to small-sized vessels in adult mouse lungs. Intriguingly, Flk1+ ECs were transiently increased in hypoxia with proliferation, whereas Flt1 expression was unchanged. Flk1-KO mice did not exhibit pulmonary vascular remodeling nor PH in normoxia; however, the arteriolar ECs changed to a cuboidal shape with protrusion. In hypoxia, Flk1 deletion exacerbated EC dysfunction and reduced their number via apoptosis. Additionally, Flk1 deletion promoted medial thickening and neointimal formation in arterioles and worsened PH. Mechanistically, lineage tracing revealed that neointimal cells were derived from Flk1-KO ECs. Moreover, RNA sequencing in pulmonary ECs demonstrated that Flk1 deletion and hypoxia synergistically activated multiple pathways, including cell cycle, senescence/apoptosis, and cytokine/growth factor, concomitant with suppression of cell adhesion and angiogenesis, to promote vascular remodeling. CONCLUSIONS: Flk1 and Flt1 were differentially expressed in pulmonary ECs. Flk1 deficiency and hypoxia jointly dysregulated arteriolar ECs to promote vascular remodeling. Thus, dysfunction of Flk1+ ECs may contribute to the pathogenesis of advanced vascular remodeling in pulmonary arterioles.

The Synthesis and Properties of Ladder-Type π-Conjugated Compounds with Pyrrole and Phosphole Rings.

The phosphole ring is known as a useful building block for constructing π-conjugated organic materials. Here, we report ladder-type benzophospholo[3,2-b]indole (BPI) derivatives where the phosphole and the pyrrole rings are directly fused. Compounds 8a-8d with different aryl groups on the phosphorous center were successfully synthesized, and the solid-state structure of 8a was confirmed using X-ray crystallographic analysis. The BPIs exhibit relatively high fluorescence quantum yield (Φ 0.50-0.72) and demonstrate a larger Stokes shift compared with a series of benzophospholo[3,2-b]benzoheteroles. The benzophospholo[3,2-b]carbazole derivative 9, which possesses a benzene ring between the phosphole and the pyrrole rings of the BPI, was also synthesized, and its solid-state structure was confirmed using X-ray crystallographic analysis. Compound 9 was found to show a smaller Stokes shift compared with the BPI.

Prognostic significance of intrahepatic lymphatic invasion in colorectal liver metastases.

BACKGROUND: Intrahepatic lymphatic invasion is an adverse prognostic factor after hepatectomy for colorectal liver metastases (CLMs). However, most patients in previous reports had liver resection before the era of FOLFOX/FIRI-based chemotherapy. METHODS: Forty-six patients who underwent hepatectomy for CLMs from 2004 to 2020 were evaluated. We histologically evaluated portal invasion, intrahepatic lymphatic invasion, and biliary invasion on hematoxylin-eosin slides. We also collected the following clinicopathologic factors: gender, age, timing, the number and maximum size of CLMs, preoperative tumor markers, neutrophil/lymphocyte ratio, location, and lymph node metastases of primary cancer, and chemotherapy after hepatectomy. A multivariate Cox proportional hazard model was used to define the relationship between overall (OS) or disease-free survival (DFS) and clinicopathologic factors. RESULTS: Histological invasions were portal invasion in 8 (17.4 %), intrahepatic lymphatic invasion in 6 (13.0 %), and biliary invasion in 5 (10.9 %). Chemotherapy for recurrence after hepatectomy (n = 29) was performed in 22 and 14 of those who received FOLFOX/FIRI-based chemotherapy. By multivariate analysis, the number of CLMs (p < 0. 01) and presence of intrahepatic lymphatic invasion (p = 0.02) were independent predictors of recurrence. The number of CLMs (p = 0.02) and prehepatectomy carcinoembryonic antigen level (p = 0.02), but not intrahepatic lymphatic invasion (p = 0.18), were independent predictors of survival using multivariate analysis. CONCLUSIONS: The presence of intrahepatic lymphatic invasion adversely affected patient's DFS, but not OS in patients with CLMs in the era of FOLFOX/FIRI chemotherapy. FOLFOX/FIRI-based chemotherapy might improve OS, even in patients with positive intrahepatic lymphatic invasion.

Furan-Containing Chiral Spiro-Fused Polycyclic Aromatic Compounds: Synthesis and Photophysical Properties.

Spiro-fused polycyclic aromatic compounds (PACs) have received growing interest as rigid chiral scaffolds. However, furan-containing spiro-fused PACs have been quite limited. Here, we design spiro[indeno[1,2-b][1]benzofuran-10,10'-indeno[1,2-b][1]benzothiophene] as a new family of spiro-fused PACs that contains a furan unit. The compound was successfully synthesized in enantiopure form and also transformed to its S,S-dioxide derivative and the pyrrole-containing analog via aromatic metamorphosis. The absorption and emission properties of the obtained furan-containing chiral spiro-fused PACs are apparently different from those of their thiophene analogs that have been reported, owing to the increased electron-richness of furan compared to thiophene. All of the furan-containing chiral spiro-fused PACs were found to be circularly polarized luminescent materials.

Transformation of Thia[7]helicene to Aza[7]helicenes and [7]Helicene-like Compounds via Aromatic Metamorphosis.

[n]Helicenes with helically twisted structures have attracted increasing interest owing to their unique properties. Therefore, it has been an important issue to develop facile synthetic methodologies which allow access to a variety of [n]helicenes. Here we report the synthesis of [7]helicenes and [7]helicene-like compounds from the thia[7]helicene as a common starting material. Desulfurative dilithiation of the thia[7]helicene and the subsequent reaction with silicon and phosphorus electrophiles afforded the silole- and phosphole-fused [7]helicene-like compounds, respectively. The cyclopentadiene-fused [7]helicene-like compound and the pyrrole-fused aza[7]helicenes were also successfully synthesized via twofold SNAr reactions of the thia[7]helicene S,S-dioxide with the carbon and nitrogen nucleophiles, respectively. The thia[7]helicene S,S-dioxide showed a slightly red-shifted absorption spectrum than the parent thia[7]helicene, which was well demonstrated by the theoretical calculations. The substituents on the silicon atom of silole-fused [7]helicene-like compounds have little impact on the longest absorption maximum. Such little effect of the substituents on absorption properties was also observed for cyclopentadiene-fused [7]helicene-like compounds and aza[7]helicenes and was well demonstrated by the theoretical calculations. The thia[7]helicene S,S-dioxide and the silole-fused [7]helicene-like compound exhibited bright blue emission, and the cyclopentadiene-fused [7]helicene-like compound and the aza[7]helicenes showed strong violet emission. Each single enantiomer of the aza[7]helicenes showed circularly-polarized luminescence with the dissymmetry factors of 4.2~4.4 × 10-3.

Aromatic Metamorphosis of Thiophenes by Means of Desulfurative Dilithiation.

A new mode of aromatic metamorphosis has been developed, which allows thiophenes and their benzo-fused derivatives to be converted to a variety of exotic heteroles. This transformation involves 1) the efficient generation of key 1,4-dianions by means of desulfurative dilithiation with lithium powder and 2) the subsequent trapping of the dianions with heteroatom electrophiles in a one-pot manner. Via the desulfurative dilithiation, the sulfur atoms of thiophenes are replaced also with a carbon-carbon double bond or a 1,2-phenylene for the construction of benzene rings.

Synthesis and Properties of Spiro-double Sila[7]helicene: The LUMO Spiro-conjugation.

A double helicene with a spiro-Si linker (4) was synthesized by four successive nucleophilic substitutions on SiCl4 . Its (P,P), (M,M) and (P,M) isomers were isolated and characterized by single crystal X-ray analysis. Due to the central spirosilabi[fluorene] moiety, the two helicene units in 4 are symmetrically and nearly perpendicularly arranged. (P,P)-4 and (M,M)-4 exhibit unique optical properties attributable to the LUMO spiro-conjugation between the two sila[7]helicene units.

Covalent Hyperbranched Polymer Self-Assemblies of Three-Way Junction DNA for Single-Molecule Devices.

A hyperbranched polymer (HBP) made of three-way junction (TWJ) DNAs is reported. Three types of 26-mer DNAs with 5'-ends modified with psoralen (PSN) were synthesized. All had self-complementary sequences starting from the 5'-end to the sixth base (AAGCTT), allowing intermolecular hybridization. The base sequences of the remaining 20-mer sites were designed so that upon hybridization, three strands had a TWJ structure with a mass of 25,000 that could be further grown by forming HBPs. PSN photochemically reacts to form interstrand cross-links that increase the polymer stability. Aggregates [(380 ± 44) nm and (65 ± 6) nm] detected with dynamic light scattering for TWJ-DNA solutions were also imaged by electron microscopy and atomic force microscopy, providing evidence of hyperbranched polymerization. The TWJ unit also polymerized on solid substrates such as Au and glass and formed self-assembled monolayers (SAMs). The HBP SAMs were integrated into commercial Pt-interdigitated electrode arrays. The DNA devices had current-voltage curves typical of metal-insulator-metal Schottky diodes; the effective barrier heights and the ideality factors were 0.52 ± 0.002 eV and 21 ± 3.2, respectively. The series resistances were (26 ± 3.3) × 106 Ω, which may provide insights into DNA electron transport. The DNA HBP enables stable electrical connections with probe electrodes and will be an important single-molecule platform.

Solvent-sensitive circularly polarized luminescent compounds bearing a 9,9'-spirobi[fluorene] skeleton.

A series of chiral 9,9'-spirobi[fluorene] (SBF) derivatives with diphenylamino donor and cyano acceptor units were designed as a new family of circularly polarized luminescent materials. The designed SBF derivatives were successfully synthesized from 7,7'-dibromo-9,9'-spirobi[fluorene]-2,2'-diol. No racemization occurred in the synthetic sequence. Therefore, each enantiomer of the SBF derivatives can be prepared from the enantiomerically pure starting material. Absorption and fluorescence spectroscopy and theoretical calculations revealed that the phenylene linker between the donor/acceptor units and the SBF core has a great impact on their photophysical properties. In particular, the phenylene linker was found to induce a red shift in their emission bands. The obtained chiral SBF derivatives exhibited solvent-dependent circularly polarized luminescence owing to their donor-π-acceptor structures.

Efficient Electrogenerated Chemiluminescence of Pyrrolopyrrole Aza-BODIPYs in the Near-Infrared Region with Tripropylamine: Involving Formation of S2 and T2 States.

Efficient electrogenerated chemiluminescences (ECLs) of three pyrrolopyrrole aza-BODIPYs in the near-infrared region by using tripropylamine as a coreactant are reported. Kinetic analysis based on Marcus theory indicates the direct formation of S2 and T2 states through the electron transfer reaction, which affects the ECL efficiencies.

Folding and Assembly of Vanilloid Receptor Secondary-Structure Peptide with Hexahistidine Linker at Nickel-Nitrilotriacetic Acid Monolayer for Capsaicin Recognition.

Herein, we report the self-assembly of a synthetic vanilloid receptor (VR) peptide that selectively binds capsaicin. We synthesized a 26-mer peptide-YSEILFFVQS-HHHHHH-LAMGWTNMLY (S3HS4)-comprising two chemoreceptor domains of transient receptor potential channel (TRPV1) linked by a hexahistidine sequence. High-speed atomic force microscopy (AFM) imaging in water revealed that the peptide structures alternated rapidly between wedge shape and linear forms. Circular dichroism spectroscopy showed that 65% of the amide units in the peptide chain adopted an α-helix structure, which was ascribed to the chemoreceptor domains. S3HS4 developed well-packed monolayers at the Ni-treated thiolated nitrilotriacetic acid self-assembled monolayers by chelation of the hexahistidine segment, as characterized by infrared spectroscopy and AFM, which exhibited statistically constant specific height. Therefore, S3HS4 was expected to fold spontaneously upon chelation, and the resulting helix-turn-helix conformers developed films while uniformly oriented: the tilt angle was 69° from the surface normal to the substrate. According to microgravimetric analysis using a quartz crystal microbalance (QCM), the adsorption was 84 ± 47 pmol cm-2 ( n = 3), which was almost consistent with the saturation adsorption of an α-helix unit. We also used a QCM to investigate the host-guest reactions of S3HS4 and found that the S3HS4-attached QCM-chip-bound capsaicin with an apparent binding constant of (4.2 ± 3.6) × 104 M-1 ( n = 4), whereas there was no evidence of binding to vanillin or acetophenone. Two controls-a blank chip without S3HS4 and a chip modified with a single helical peptide (LAMGWTNMLY-HHHHHH)-produced no capsaicin response. To the best of our knowledge, S3HS4 is the first example of a synthetic VR mimic peptide. We believe that the present surface-directed structure-based design can be used to exploit the α-helix bundle in hexahistidine-linked bishelical peptides.

Association between Body Weight Gain from 20 Years of Age and Diabetic Nephropathy in Japanese Patients with Type 2 Diabetes.

BACKGROUND/AIMS: It has been reported that the body mass index shows a U-shaped association with death from cardiovascular disease (CVD) in the Asian population. The relationship between body weight (BW) gain from early adulthood and diabetic nephropathy remains to be elucidated in Japanese patients with type 2 diabetes. Our aim was to investigate the association between BW gain from early adulthood and diabetic nephropathy in Japanese patients with type 2 diabetes. METHODS: We assessed the BW of 471 consecutive patients with type 2 diabetes and calculated the change in BW from the age of 20 years to the lifetime maximum (ΔBWmax-20y). We then evaluated the relationship of ΔBWmax-20y with the degree of urinary albumin excretion (UAE), which is a useful marker for CVD. RESULTS: ΔBWmax-20y negatively correlated with the logarithm of UAE (r = -0.160, p = 0.002). Multiple regression analysis demonstrated that ΔBWmax-20y was independently correlated with the logarithm of UAE (ß =-0.112, p =0.034). CONCLUSIONS: BW gain from the age of 20 years is correlated with diabetic nephropathy in Japanese patients with type 2 diabetes.

Soritesidine, a Novel Proteinous Toxin from the Okinawan Marine Sponge Spongosorites sp.

A novel protein, soritesidine (SOR) with potent toxicity was isolated from the marine sponge Spongosorites sp. SOR exhibited wide range of toxicities over various organisms and cells including brine shrimp (Artemia salina) larvae, sea hare (Aplysia kurodai) eggs, mice, and cultured mammalian cells. Toxicities of SOR were extraordinary potent. It killed mice at 5 ng/mouse after intracerebroventricular (i.c.v.) injection, and brine shrimp and at 0.34 µg/mL. Cytotoxicity for cultured mammalian cancer cell lines against HeLa and L1210 cells were determined to be 0.062 and 12.11 ng/mL, respectively. The SOR-containing fraction cleaved plasmid DNA in a metal ion dependent manner showing genotoxicity of SOR. Purified SOR exhibited molecular weight of 108.7 kDa in MALDI-TOF MS data and isoelectric point of approximately 4.5. N-terminal amino acid sequence up to the 25th residue was determined by Edman degradation. Internal amino acid sequences for fifteen peptides isolated from the enzyme digest of SOR were also determined. None of those amino acid sequences showed similarity to existing proteins, suggesting that SOR is a new proteinous toxin.

[1]Benzothiophene-Fused Chiral Spiro Polycyclic Aromatic Compounds: Optical Resolution, Functionalization, and Optical Properties.

Spiro polycyclic aromatic compounds have been known as rigid chiral scaffolds. In order to extend their applications, an efficient preparation route to enantiopure derivatives is highly required. Here, we design 10,10'-spirobi[indeno[1,2- b][1]benzothiophene]-7,7'-diol to achieve efficient optical resolution. The compound was successfully synthesized and resolved by chiral HPLC on a semipreparative scale. The absolute configuration of its enantiopure isomer was determined through single-crystal X-ray structure analysis of its derivative. The compound was also transformed into its derivatives with donor-acceptor (D-A) type systems. The obtained chiral D-A type molecules exhibited remarkable solvent-dependence fluorescence and were found to be solvent-sensitive circularly polarized luminescent materials. These results clearly demonstrated the utility of 10,10'-spirobi[indeno[1,2- b][1]benzothiophene]-7,7'-diol as a versatile building block for chiral spiro polycyclic aromatic compounds.

Copolymerization of epoxides with cyclic anhydrides catalyzed by dinuclear cobalt complexes.

The alternating copolymerization of epoxides with cyclic anhydrides (CAs) is a highly diverse synthetic method for polyesters as the polymers' architectures and properties can be easily controlled depending on the combination of two monomers. Thus, a variety of catalyst designs has been reported to prepare the desired copolymers efficiently. We herein report dinuclear cobalt-salen complexes with a benzene ring as a linker and their activities in copolymerization reactions. The dinuclear cobalt complexes showed a higher catalytic activity for the copolymerization of propylene oxide with phthalic anhydride than the corresponding mononuclear cobalt-salen complex and achieved one of the highest turnover frequencies ever reported. A variety of epoxides and CAs were also found to be copolymerized successfully by the dinuclear cobalt complex with a high catalytic activity.

Monolithic Peptide-Nucleic Acid Hybrid Functioning as an Artificial Microperoxidase.

A new peptide nucleic acid (PNA) with an installed peroxidase function has been developed. Fmoc solid phase peptide synthesis prepared a PNA hybrid (VQKCAQCHTVE-(C2H4O)2CH2-[PNA(T)]6-G) that renders the microperoxidase backbone, followed by reconstitution with hemin. The resulting holocompound catalyzed the oxidation of 3,3',5,5'-tetramthylbenzidine by H2O2 to 50% that of natural microperoxidase-11, whereas the apo-form and hemin gave no responses. The peroxidase domain was found to be active toward direct electrochemistry and the PNA hybrid served for gene sensor; in the presence of the target DNA (5'-CATGTATAAAAAA-3'), an electrode-attached DNA probe (5'-TsTsTsTsTsTCTCATACATG-3') showed the ferric-to-ferrous quasi-reversible wave (-276 mV vs Ag/AgCl) through sandwich hybridization. Moreover, the hybridization product could accept H2O2 as an oxidant to enhance the reduction current, which occurred likely based on the iron(II)-center-recycling with specific rate constant of 0.19 s-1.

Ion-Conductive Properties of a Polymer Electrolyte Based on Ethylene Carbonate/Ethylene Oxide Random Copolymer.

A random copolymer of ethylene oxide with CO2 , namely, poly(ethylene carbonate/ethylene oxide) (P(EC/EO)), has been synthesized as a novel candidate for polymer electrolytes. Electrolyte composed of P(EC/EO) and lithium bis(fluorosulfonyl)imide has an ionic conductivity of 0.48 mS cm-1 and a Li transference number (t+ ) of 0.66 at 60 °C. To study ion-conductive behavior of P(EC/EO)-based electrolytes, the Fourier transform infrared (FT-IR) technique is used to analyze the interactions between Li+ and functional groups of the copolymer. The carbonate groups may interact preferentially with Li+ rather than the ether groups in P(EC/EO). This study suggests that copolymerization of carbonate and flexible ether units can realize both high conductivity and t+ for polymer electrolytes. High-performance P(EC/EO) electrolyte is expected to be a candidate material for use in all-solid-state batteries.

Caffeine intake enhances the benefits of sodium glucose transporter 2 inhibitor.

BACKGROUND: The effect of sodium glucose transporter 2 (SGLT-2) inhibitors is dependent on the glomerular filtration rate. It has been reported that caffeine intake increases glomerular filtration rate. However, the effect of caffeine intake on urinary glucose excretion in patients who take SGLT-2 inhibitors is unclear. METHODS: Six patients with type 2 diabetes took part in a randomized, open-label, crossover pilot study. The patients took SGLT-2 inhibitors (ipragliflozin) for 9 days. On day 3, 6 and 9, the patients were assigned to one of three studies: Water 500, patients drank 500 mL of water in 3 h; Water 1500, patients drank 1500 mL of water in 3 h; and Caffeine 500, patients drank 500 mL of water with 400 mg of caffeine in 3 h. In all of the studies, the patients' urine was collected over a 6-h period. In addition, we enrolled 60 patients with type 2 diabetes who newly took SGLT-2 inhibitors in a 3-month follow-up cohort study to investigate the effect of caffeine intake on glucose control. Caffeine intake was evaluated using questionnaires. RESULTS: The 6-h median (interquartile range) urinary glucose excretion was 9.5 (8.5-9.7) g in Water 500, 12.2 (10.3-27.2) g in Water 1500 and 15.7 (11.4-21.4) g in Caffeine 500 (p = 0.005 vs Water 500). In the cohort study, multiple regression analysis demonstrated that log (caffeine intake) was associated with a change in HbA1c (ß = -0.299, p = 0.043) after adjusting for covariates. CONCLUSIONS: Caffeine intake enhanced the effect of SGLT-2 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.