Insulin receptor‐insulin interaction kinetics using multiplex surface plasmon resonance

Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor‐insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real‐time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR‐A). We used a scaffold approach in which anti‐insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR‐A capture. The multiplex SPR system (ProteOn XPR36?, Bio‐Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two‐site binding pattern, consistent with the literature, with a high‐affinity site (dissociation constant K_D1 = 38.1 ± 0.9 nM) and a low‐affinity site (K_D2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K_D1 = 73.2 ± 1.8 nM and K_D2 = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high‐affinity site was converted to the low‐affinity site. The new SPR assay enables insulin‐eIR‐A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling. Copyright © 2013 John Wiley & Sons, Ltd.     

Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells

We have produced a series of hybrid IgG₁-IgG_2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differnces between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse γ¹ and γ^2a gene segments. Crossovers occured through out these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H₂L₂ tetramers and properly glycosylated. In addition, all 11 immunoglbulins have identical antigen combining sites specific for the fluorescent hapten ε-dansyll-L-lysine. Protein A binding was used as probe of the structural integrity of the Fc portion of the variant antibodies. Differeneces in protein A binding between IgG₁ and IgG_2a appear to be due to amino acid differances at postions 252 (Thr→Met) and 254 (Thr→Ser) of the heavy chain (EU numbering).     

Hepatic Insulin Resistance in Obese Non-Diabetic Subjects and in Type 2 Diabetic Patients

Objective: Obese non-diabetic patients are characterized by an extra-hepatic insulin resistance. Whether obese patients also have decreased hepatic insulin sensitivity remains controversial. Research Methods and Procedures: To estimate their hepatic insulin sensitivity, we measured the rate of exogenous insulin infusion required to maintain mildly elevated glycemia in obese patients with type 2 diabetes, obese non-diabetic patients, and lean control subjects during constant infusions of somatostatin and physiological low-glucagon replacement infusions. To account for differences in insulin concentrations among the three groups of subjects, an additional protocol was also performed in healthy lean subjects with higher insulin infusion rates and exogenous dextrose infusion. Results: The insulin infusion rate required to maintain glycemia at 8.5 mM was increased 4-fold in obese patients with type 2 diabetes and 1.5-fold in obese non-diabetic patients. The net endogenous glucose production (measured with 6,6-²H₂-glucose) and total glucose output (measured with 2-²H₁-glucose) were ∼30% lower in the patients than in the lean subjects. Net endogenous glucose production and total glucose output were both markedly increased in both groups of obese patients compared with lean control subjects during hyperinsulinemia. Discussion: Our data indicate that both obese non-diabetic and obese type 2 diabetic patients have a blunted suppressive action of insulin on glucose production, indicating hepatic and renal insulin resistance.     

Cellular subpopulations in the expression of IgG1.

Consistent with the surface display of IgG₁, antigen-primed B cells are sensitive to functional elimination by anti-Ig and C. This depletion of IgG₁ responses, assayed in adoptive transfer, is accompanied by increased responses of other isotypes, a phenomenon termed compensation. An analysis of the tertiary response reveals that cells sensitive to functional elimination are precursors to memory cells as well as plaque-forming cells. To investigate control mechanisms governing preferential expression of IgG₁, and the compensation active after elimination of IgG₁, increased numbers of carrier-primed cells were transferred and antigen dose was increased to adoptive recipients. Doses of carrier-primed cells or antigen were found which allowed maximal expression of both IgG₁ and non-IgG₁ isotypes. In recipients of anti-Ig and C-treated B cells, increased numbers of carrier-primed cells did not further increase the compensatory expression of non-IgG₁ isotypes. A discussion is presented of compensation as a result of limiting inductive signals to B cells.     

Insulin binding and glucose transport in the R3230AC mammary adenocarcinoma

Cells dissociated from the R3230AC mammary adenocarcinoma from intact and diabetic rats were examined for insulin binding and glucose transport. The K_d for insulin binding, ～ 10^?10 M, was similar in all tumors studied. However, the apparent number of receptor sites per cell increased in cells from diabetic rats. Kinetic analysis of 3-0-methyl glucose (3-OMG) entry showed both diffusional and passive carrier characteristics. Insulin (4 × 10^?9 M) in vitro did not affect diffusional entry, whereas the hormone altered the passive carrier system, as reflected by an increase in K_m and V_max. Insulin decreased initial velocity of glucose transport at 4–6 mM glucose levels but increased initial velocity of glucose transport at 20 mM glucose. An explanation of the role of insulin on tumor growth in vivo from effects on glucose transport in vitro is proposed.     

ARA290 Improves Insulin Release and Glucose Tolerance in Type 2 Diabetic Goto-Kakizaki Rats

Effects of ARA290 on glucose homeostasis were studied in type 2 diabetic Goto-Kakizaki (GK) rats. In GK rats receiving ARA290 daily for up to 4 wks, plasma glucose concentrations were lower after 3 and 4 wks, and hemoglobin A_1c (Hb A_1c) was reduced by ~20% without changes in whole body and hepatic insulin sensitivity. Glucose-stimulated insulin secretion was increased in islets from ARA290-treated rats. Additionally, in response to glucose, carbachol and KCl, islet cytoplasmic free Ca²⁺ concentrations, [Ca²⁺]_i, were higher and the frequency of [Ca²⁺]_i oscillations enhanced compared with placebo. ARA290 also improved stimulus–secretion coupling for glucose in GK rat islets, as shown by an improved glucose oxidation rate, ATP production and acutely enhanced glucose-stimulated insulin secretion. ARA290 also exerted an effect distal to the ATP-sensitive potassium (K_ATP) channel on the insulin exocytotic pathway, since the insulin response was improved following islet depolarization by KCl when K_ATP channels were kept open by diazoxide. Finally, inhibition of protein kinase A completely abolished effects of ARA290 on insulin secretion. In conclusion, ARA290 improved glucose tolerance without affecting hematocrit in diabetic GK rats. This effect appears to be due to improved β-cell glucose metabolism and [Ca²⁺]_i handling, and thereby enhanced glucose-induced insulin release.     

Effect of Insulin Analogues on Insulin/IGF1 Hybrid Receptors: Increased Activation by Glargine but Not by Its Metabolites M1 and M2

Background

In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids.

Methodology

To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP₃) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP₃ production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells.

Results

Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP₃ production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP₃ production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin.

Conclusion

Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.     

Production of auto-anti-idiotypic antibody during the normal immune response: IX. Characteristics of the auto-anti-idiotype antibody and its production

Using hapten-reversible inhibition of plaque formation as an assay for auto-anti-idiotype antibody (anti-Id) and as a means for following idiotype (Id) expression, we have obtained evidence that following immunization with trinitrophenyl (TNP) conjugates (a) there are differences in Id expression in the anti-TNP antibody response to different TNP conjugates although there is some overlap; (b) different strains, although showing some differences in Id expression, tend to produce cross-reactive Ids, thus no obvious allotype linked inheritance of Id expression is observed in this heterogeneous immune response; (c) the auto-anti-Id produced following immunization with TNP-Brucella abortus or TNP-Ficoll tends to be of the IgG_2a and IgG_2b isotypes.     

Investigations on the specificity of thiophilic interaction for monoclonal antibodies of different subclasses

A comparative study was carried out to investigate the influence of different mouse antibody subclasses on the chromatographic behaviour on thiophilic supports. Cell-free supernatants from different mouse-mouse hybridoma cultures in a standard medium were purified on thiophilic agarose and Fractogel EMD TA. The adsorption capacities and purification factors were monitored under optimised adsorption conditions. The different isotypes did not differ significantly regarding capacity of the thiophilic matrix, but the purity of the eluted antibody fractions was significantly lower for the IgG_2a subclass compared to all other murine antibodies. A significant copurification of proteins from cell culture supernatant with antibodies of the IgG_2a subclass indicated a restriction in the universal nature of thiophilic interaction.     

Age-related prevalence of antibodies to infective larvae ofWuchereria bancrofti in normal individuals from a filaria-endemic region

Antibody isotypic levels (IgM, IgE and IgG subclasses) to infective larvae (L₃) ofWuchereria bancrofti were measured in 104 normal individuals from a filaria-endemic region in Orissa. The titres of antibodies were considerably higher in adults (n = 25, 25.1± 3.8 year) than in children (n = 52, 7.1 ± 2.1 year). Young children (n = 14) less than four years were seronegative to all isotypes other than IgM, the sero-conversion to which was achieved in the children (n=15) by the age of 7.5±1.2 years. The prevalence of other isotypes increased with age and reached a maximum in early adulthood (18.6 ±1.6 years), which persisted in older adults (> 30 years). However, the increase in IgG₃ prevalence with age was less marked. IgG₂ was detected only after 10 years of age. Compared to the high prevalence (100%) of IgM, IgE, IgG₁, and IgG₂, in adults. IgG₃ and IgG₄ prevalences were low, 35% and 28% respectively. IgA level to L₃ antigen was found to be extremely low even in adults. These data indicate that the prevalence of L₃ antibodies was different for different isotypes and the acquisition of antibody response essentially followed an age dependent pattern.     

Immunoglobulin isotype distribution of malaria-specific antibodies produced during infection with Plasmodium chabaudi adami and Plasmodium yoelii

The antibody response of mice to Plasmodium chabaudi adami and Plasmodium yoelii has been compared using a solid phase isotype-specific radioimmunoassay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Serological cross-reactivity between these parasites was substantial. Studies using a radioimmunoassay detecting all classes of malaria-specific antibody demonstrated that during the early part of infection it was not possible to distinguish between homologous and heterologous reactions. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 50% or more of the protein antigens detected were apparently shared by both parasites although the intensity of bands was always greater with homologous reactions. However, the distribution of isotypes in the antibody (Ab) response differed in the two infections. P. chabaudi infections were characterized by a predominant and persistent IgM response, moderate IgG₂ and IgG₃ and little significant IgG₁ response during a primary infection. By contrast, IgM antibodies were transient in P. yoelii infection, IgG₂ was the predominant isotype, and both IgG₁ and IgG₃ antibodies were present during a primary infection. These differences in isotypes were also detected when sera were tested on the heterologous antigen extracts suggesting that antigens shared by P. chabaudi and P. yoelii do not necessarily induce similar antibody responses in the two infections.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Basal immunoglobulin serum concentration and isotype distribution in relation to the polygenic control of antibody responsiveness in mice

Serum Ig concentration and isotype distribution were determined in the high (H) and low (L) responder lines selected for antibody response to complex immunogens. Data were recorded in normal and postimmunization sera from the H and L lines produced by five independent selective breedings (selections I, 11, III, IV, and V). Ig levels were much higher in H than in L mice of all the selections. In four selections this interline difference increased further after immunization with the selection antigens. This is in agreement with the general effect of the polygenic control of antibody responses operating in H and L lines. The Ig isotype profiles of normal sera were different in each line; however, similitudes were noticed between H and L lines in selections I and If. In contrast, in selections III, IV, and V a similar interline difference was observed: the lack of IgG2a isotype in L lines only. After immunization there were minor alterations of the isotype profiles except in the H lines of selections III and IV, in which a clear inverse modification of IgG₁ and IgG_2a proportions occurred. The characteristic pattern of each selection may be partially dependent on isotype-restricted regulatory effects in relation to the immunization procedure used for selective breeding.     

Somatic cell hybridization of mouse myeloma cells

Somatic cell hybrids between different mouse myeloma cell lines have been readily isolated using modifications of existing techniques. The hybrid nature of these cells was established by HAT or HAT-ouabain selective procedures, their chromosome number, and, in one case, H-2 surface antigen expression. Three hybrid cell lines are described here in detail: an IgG_2b, ? X IgG_2a, ?; an IgG₁, ? X IgG_2b, ?; and an IgG₁, ? X IgM, λ. In all cases, both parental types of H and L chains are expressed in the hybrid cells and no new chains are observed. However, molecules possessing disulfide-bonded mixtures of parental H and/or L chains are seen. Analysis of subclones of these hybrids indicates considerable stability in the expression of the immunoglobulins for up to 13 months. However, segregant clones no longer synthesizing one or more of the parental H or L chains arise frequently.     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

A defucosylated anti-PD-L1 monoclonal antibody 13-mG2a-f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. A strong correlation has been reported between PD-L1 expression in tumor cells and negative prognosis in cancer patients. Previously, we established an anti-PD-L1 monoclonal antibody (mAb), L₁Mab-13 (IgG₁, kappa), by immunizing mice with PD-L1-overexpressing CHO-K1 cells. L₁Mab-13 specifically reacts with endogenous PD-L1 in lung cancer cell lines in flow cytometry and Western blot applications, and stains a plasma membrane-like pattern in lung cancer tissues via immunohistochemical analysis. In this study, we investigated whether L₁Mab-13 reacts with oral cancer cell lines and exerts antitumor activities. Because L₁Mab-13 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), we first converted the subclass of L₁Mab-13 from IgG₁ into IgG_2a (13-mG_2a), and further produced a defucosylated version (13-mG_2a-f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 13-mG_2a-f was confirmed using fucose-binding lectins, such as Aleuria aurantia and Pholiota squarrosa lectins. The dissociation constants (K_D) for 13-mG_2a-f in SAS and HSC-2 oral cancer cells were determined via flow cytometry to be 2.8 × 10^?9 M and 4.8 × 10^?9 M, respectively, indicating that 13-mG_2a-f possesses extremely high binding affinity. In vitro analysis demonstrated that 13-mG_2a-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 13-mG_2a-f significantly reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. Taken together, these data demonstrate that treatment with 13-mG_2a-f may represent a useful therapy for patients with PD-L1-expressing oral cancers.     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.     

IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses,Fc-Glycans and a Fab-Peptide Sequence

The Fc-glycan profile of IgG₁ anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG₁, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG₄ peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG₄, the ACPA-IgG₄ Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG₁ compared to FT-IgG₁ were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG₁ relative to FT-IgG₁ as well as lower content of IgG₂ (p = 0.0000001) and elevated content of IgG₄ (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG₁ and ACPA-IgG₄ are distinct. Given that IgG₁ and IgG₄ have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.     

Dipeptidyl peptidase-IV inhibition prevents blood–retinal barrier breakdown,inflammation and neuronal cell death in the retina of type 1 diabetic rats

Diabetic retinopathy, a leading cause of vision loss in working-age population, is often associated with inflammation and apoptosis. We have previously reported that sitagliptin, a DPP-IV inhibitor, exerts beneficial effects in the retina of type 2 diabetic animals. The present study aimed to evaluate whether sitagliptin can exert protective effects in the retina of type 1 diabetic animals by a mechanism independent of insulin secretion and glycemia normalization.Streptozotocin-induced diabetic rats were treated orally with sitagliptin (5 mg/kg/day) for the last two weeks of 4 weeks of diabetes. Sitagliptin treatment did not change the weight and glucose, HbA_1c or insulin levels. However, it prevented the diabetes-induced increase in DPP-IV/CD26 activity and levels in serum and retina. Sitagliptin also prevented the increase in blood–retinal barrier (BRB) permeability and inhibited the changes in immunoreactivity and endothelial subcellular distribution of occludin, claudin-5 and ZO-1 proteins induced by diabetes. Furthermore, sitagliptin decreased the retinal inflammatory state and neuronal apoptosis.Sitagliptin inhibited the BRB breakdown in a type 1 diabetic animal model, by a mechanism independent of normalization of glycemia, by preventing changes in tight junctions (TJs) organization. Sitagliptin also exerted protective effects against inflammation and pro-apoptotic state in the retina of diabetic rats. Altogether, these results suggest that sitagliptin might be envisaged to be used to prevent or delay some of the alterations associated with the development of diabetic retinopathy.     

Critical Role of Gap Junction Coupled KATP Channel Activity for Regulated Insulin Secretion

Pancreatic β-cells secrete insulin in response to closure of ATP-sensitive K⁺ (K_ATP) channels, which causes membrane depolarization and a concomitant rise in intracellular Ca²⁺ (Ca_i). In intact islets, β-cells are coupled by gap junctions, which are proposed to synchronize electrical activity and Ca_i oscillations after exposure to stimulatory glucose (>7 mM). To determine the significance of this coupling in regulating insulin secretion, we examined islets and β-cells from transgenic mice that express zero functional K_ATP channels in approximately 70% of their β-cells, but normal K_ATP channel density in the remainder. We found that K_ATP channel activity from approximately 30% of the β-cells is sufficient to maintain strong glucose dependence of metabolism, Ca_i, membrane potential, and insulin secretion from intact islets, but that glucose dependence is lost in isolated transgenic cells. Further, inhibition of gap junctions caused loss of glucose sensitivity specifically in transgenic islets. These data demonstrate a critical role of gap junctional coupling of K_ATP channel activity in control of membrane potential across the islet. Control via coupling lessens the effects of cell–cell variation and provides resistance to defects in excitability that would otherwise lead to a profound diabetic state, such as occurs in persistent neonatal diabetes mellitus.