Comparison of efficacy between dipeptidyl peptidase-4 inhibitor and sodium-glucose cotransporter 2 inhibitor on metabolic risk factors in Japanese patients with type 2 diabetes mellitus: Results from the CANTABILE study.

AIMS: The aim of this study was to compare the effectiveness of teneligliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, and canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, at reducing a composite outcome of three metabolic risk factors (obesity, hypertension, and dyslipidemia) in Japanese patients with type 2 diabetes mellitus (T2DM) and metabolic risks. METHODS: In this prospective, multicenter, open-label, randomized, parallel-group comparison study, 162 patients with T2DM and one or more metabolic risk factors were randomized into a teneligliptin or canagliflozin group and treated for 24 weeks. The primary endpoint was the composite percentage of subjects who experienced an improvement in at least one metabolic risk after 24 weeks of treatment. RESULTS: The primary endpoint was achieved significantly by more patients in the canagliflozin group than in the teneligliptin group (62.2% vs. 31.3%, p = 0.0004). A ≥ 3% body weight loss was also achieved by significantly more participants in the canagliflozin group than in the teneligliptin group (55.9% vs. 10.5%, p < 0.0001). CONCLUSIONS: This study showed canagliflozin to be more effective at reducing metabolic risks than teneligliptin. In Japanese patients with T2DM and metabolic risk factors, SGLT2 inhibitors may be superior to DPP-4 inhibitors at controlling multiple metabolic risk.

Quantitative enzyme-linked immunosorbent assay (ELISA) for non-enzymatically glycated serum protein.

A competitive ELISA for quantitative determination of glucitollysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine was developed. We applied it to measure non-enzymatically glycated serum proteins. The antiserum obtained by immunizing guinea pigs with reductively glycated human albumin was capable of identifying and quantitating glucitollysine residues of serum proteins in normal and diabetic subjects after reduction of the proteins with sodium borohydride. The ELISA assay developed here had satisfactory reproducibility as judged by the intra-assay precision of 2.3-7.6% and the interassay precision of 6.7-9.8%. Results from this assay procedure correlated well with those from the radioimmunoassay and the boronate affinity chromatography procedure. The data suggested that diabetic serum proteins contained at least three times as much immunochemically detectable glucitollysine residues as normal serum proteins after reduction of the proteins with sodium borohydride. This method allows to quantitate glucitollysine residues on any of the proteins that have been implicated in the pathological sequelae of diabetes.

Characterization of antibodies to advanced glycosylation end products on protein.

Antibodies directed against advanced glycosylation end products (AGEs) formed during a Maillard reaction have been generated and characterized. Since protein-bound AGEs recognized by the antibodies were labile to acid hydrolysis, the antibodies were further characterized by using the AGE-alpha-acetyl-L-lysine methyl ester (AGE-ALME) with a brown and fluorescent property as well as the AGE-proteins. The antibodies reacted with fluorescent compounds, rather than brown pigment compounds, in the AGE-ALME. The fluorescent compounds in the AGE-ALME were separated into four fluorescent compounds by reversed-phase thin layer chromatography (TLC). Of the fluorescent compounds tested, compound 3 (Rf = 0.63), as designated on a TLC plate, showed the highest affinity for the antibodies. In addition, the antibody recognition to the cross-linked oligomers with fluorescence in the AGE-protein was investigated by using bovine pancreatic ribonuclease A (RNase), which is known as a model protein for studying AGE-induced cross-linking. Fluorescence in the AGE-RNase existed in both of the oligomers and the monomer. The cross-linked oligomers exhibited higher affinity to the antibodies than did the monomer, which has a similar degree of fluorescent intensity. These results indicate that our antibodies against cross-linked protein-bound AGEs may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.

[Radioimmunoassay of nonenzymatically glucosylated albumin].

A radioimmunoassay (RIA), to quantitate nonenzymatically glucosylated albumin (GA), was established. Antiserum A was obtained by immunizing guinea pigs with reduced glucosylated guinea pig albumin (RedGlc-GPA), and antiserum B was obtained by immunizing with reduced glucosylated human albumin (RedGlc-HA) and absorbing the antibody against human native albumin. For antiserum A; RedGlc-GPA and NaBH4-reduced guinea pig serum, in which epsilon-N-1-(1-deoxy glucitol) lysine (GL) was present, were effective competitors. But NaBH4-reduced human serum, NaBH4-reduced human hemoglobin, and RedGlc-HA, in which GL were also present, were not so effective competitors. For antiserum B; RedGlc-HA, and RedGlc-GPA, in which GL were present, and GL were equally effective competitors. Thus it seemed that for antiserum A, the antigenic region was not only GL, and for antiserum B, the antigenic region was GL almost specifically. So we used antiserum B for this RIA, and GA in NaBH4-reduced human albumin was quantitated by measuring its ability to compete for antibody binding, compared with that of GL as a standard. Reproducibility of GA-values with this RIA was a enough to use for clinical purpose. With this RIA method, the following results were obtained: 1) These GA values were correlated with GA values, measured by boronate-affinity chromatographic method. 2) Average of GA values in 44 diabetics was significantly higher than that of 24 normal subjects. 3) These GA values were also correlated with HbA1, HbA1c, and fasting blood sugar (FBS). 4) These GA values were more closely correlated with FBS, 2 weeks before, than with FBS, at the same time, and with FBS, 4 weeks before. 5) After insulin treatment to untreated diabetics, these GA values were more rapidly decreased than HbA1c. These result indicated that GA values quantitated in this RIA was available for monitoring blood sugar level for shorter period than that represented by HbA1c.

Renal transplantation between siblings with unrecognized Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of the lysosomal hydrolase, α-galactosidase A (α-Gal A). We report a case of a renal transplant recipient with unrecognized Fabry disease who received the allograft from a sibling donor with unrecognized Fabry disease. The recipient began to show a gradual increase of the serum creatinine with mild proteinuria at 3 years after transplantation. Histopathologic examination revealed finely vacuolated podocytes, demonstrated by ultrastructural examination to contain osmophilic myelin bodies. Furthermore, the recipient showed reduced circulating levels of α-Gal A and elevated urinary levels of globotriaosylceramide. These findings indicated that both the recipient and the donor suffered from Fabry disease of the renal variant phenotype. Enzyme replacement therapy (ERT) was initiated in the recipient, which resulted in a slight decrease of serum creatinine. Although mild proteinuria persisted, initiation of ERT in the recipient led to improvement of the renal function.

Reactivation of antigen-induced arthritis in mice by oral administration of lipopolysaccharide.

We examined whether oral administration of lipopolysaccharide (LPS) from Escherichia coli reactivated antigen-induced arthritis (AIA) in mice that is one of models of human rheumatoid arthritis. To induce AIA, mice were immunized by subcutaneous injection of ovalbumin (OVA) emulsified with complete Freund's adjuvant into the base of the tail (day 0) followed by intraarticular injection of OVA on day 21. To investigate the ability of LPS to reactivate AIA, varying doses of LPS were p.o. administered 48 h after the challenge injection. The results showed that administration of LPS was followed by reactivation of AIA in a dose-related fashion. The reactivation of AIA by LPS was associated with increases in interferon-gamma, interleukin-1beta and tumour necrosis factor-alpha. Polymyxin B sulfate given immediately before administration of LPS suppressed the reactivation of AIA. These findings suggest that LPS from intestinal bacteria may play a role in the reactivation of joint inflammation in which immune responses to pathogenic antigens are involved.

Effect of rolipram, a phosphodiesterase IV inhibitor, on allergic footpad swelling using various adjuvants in mice.

We studied the effect of rolipram, a phosphodiesterase (PDE) IV inhibitor, on allergic footpad swelling in mice. For this study, varying adjuvants including complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and Imject Alum (Alum) were used because the extent of antigen-specifically induced T helper type 1 (Th1) and Th2 responses had been shown to depend on adjuvants used. To induce allergic footpad swelling, we immunized mice with ovalbumin (OVA) emulsified in either CFA or IFA, dissolved in Alum or in phosphate-buffered saline (PBS) as a control (day 0), followed by subcutaneous injection of the antigen into footpads on day 21. Rolipram was given orally to the animals daily from days 0-20. Results showed that treatment with rolipram was followed by an increase in early swelling at 0.5 h and a decrease in late swelling at 6 and 24 h in the CFA group. In the IFA group, rolipram significantly enhanced swelling at, but not after, 30 min. In the Alum and the PBS groups, the PDE inhibitor failed to affect the OVA-specific footpad reaction at all times examined. Treatment of the CFA and IFA groups with rolipram significantly inhibited the production of the Th1 antibody anti-OVA immunoglobulin G2a (IgG2a), and the drug enhanced Th2 cell-dependent anti-OVA IgE production. In both groups, rolipram also enhanced the secretion of Th2 cytokines including interleukin-4 (IL-4) and IL-10. These findings suggest that rolipram may facilitate early allergic footpad swelling mediated by Th2 immune responses, while the late phase of swelling associated with Th1 responses may be attenuated by the PDE IV inhibitor.