Purification and biochemical characterization of a novel secretory dipeptidyl peptidase IV from porcine serum.

Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC50 value of 8.473 µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25 µg.

Potential Hypoglycemic Effect of Pomegranate Juice and Its Mechanism of Action: A Systematic Review.

Pomegranate juice (PJ) has gained popularity attributed to its phenolic compounds and their medicinal properties. Its potential hypoglycemic effect has been related to enzymatic inhibition, insulin release, and the protection of pancreatic tissue. These effects depend on several aspects, such as the content and composition of phenols in pomegranate and characteristics of the organism that consumes the juice. The objective of this study was to systematically review scientific evidence investigating the hypoglycemic effect of PJ; the factors that affect bioactive compounds; and the mechanisms of action attributed to this effect. Human and rodent in vivo and in vitro studies were retrieved from PubMed, Scopus, and ScienceDirect databases. After reviewing the articles, it was identified that the methodologies and results among the scientific evidence were quite heterogeneous. Despite these limitations, many of the in vivo and in vitro studies found important hypoglycemic effects from PJ, as well as an increase in the function of ß-cell, insulin secretion, a significantly lower activity of α-amylase enzyme, an inhibition of the enzyme α-glucosidase and dipeptidyl peptidase-4 (DPP-4), and the protection against DNA damage. Determining the potential health benefits of polyphenols contained in the pomegranate is limited for multiple factors that could affect the efficacy of PJ. Overall, the results of this review suggest the need for further experimentation, using controlled variable factors and testing the effect of PJ under similar experimental conditions.

Dipeptidyl peptidase-4 inhibitors can inhibit angiotensin converting enzyme.

Angiotensin-1 converting enzyme inhibitors (ACEIs) improve insulin sensitivity. Inhibitors of dipeptidyl peptidase-4 (DPP-4) are anti-diabetic drugs with several cardio-renal effects. Both ACE and DPP-4 share common features. Thus, we tested if they could be inhibited by one inhibitor. First, in silico screening was used to investigate the ability of different DPP-4 inhibitors or ACEIs to interact with DPP-4 and ACE. The results of screening were then extrapolated into animal study. Fifty Sprague Dawley rats were randomly assigned into 5 groups treated with vehicle, captopril, enalapril, linagliptin or sitagliptin. Both low and high doses of each drug were tested. Baseline blood samples and samples at days 1, 8, 10, 14 were used to measure plasma DPP-4 and ACE activities and angiotensin II levels. Active glucagon-like peptide-1 (GLP-1) levels were measured after oral glucose challenge. All tested DPP-4 inhibitors could interact with ACE at a relatively reasonable binding energy while most of the ACEIs only interacted with DPP-4 at a predicted high inhibition constant. In rats, high dose of sitagliptin was able to inhibit ACE activity and reduce angiotensin II levels while linagliptin had only a mild effect. ACEIs did not significantly affect DPP-4 activity or prevent GLP-1 degradation. It seems that some DPP-4 inhibitors could inhibit ACE and this could partially explain the cardio-renal effects of these drugs. Further studies are required to determine if such inhibition could take place in clinical settings.

Study on regulation of adipokines on body fat distribution and its correlation with metabolic syndrome in type 2 diabetes mellitus.

BACKGROUND: This paper aimed to investigate the regulating role of adipokine expression level in body fat distribution of patients with type 2 diabetes mellitus (T2MD) as well as its correlation with metabolic syndrome (MS). METHODS: One hundred forty patients with T2MD admitted in the Endocrinology Department of the First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine from January 2017 to July 2017 were selected; their body height and weight were measured to calculate Body Mass Index (BMI); patients with a BMI ≤23.9 kg/m2 were included into control group (N.=49), and those with a BMI ≥24 kg/m2 into observation group (N.=91). Based on whether the patients were complicated with MS, they were divided into non-metabolic syndrome (N-MS) group (N.=79) and MS group (N.=61); the levels of serum dipeptidyl peptidase-4 (DPP-4), adiponectin (ADPN) and leptin as well as the contents of body fat and lean tissue of the two groups of patients were measured. RESULTS: The serum DPP-4 level in observation group was remarkably elevated compared with that in control group (P<0.05), but there were no significant differences in the leptin and ADPN levels between the two groups (P>0.05). The serum DPP-4 and leptin levels were positively correlated with the total body fat mass (FAT) of the patients in observation group (r=0.461, P=0.004; r=0.433, P=0.007); DPP-4 level had a positive correlation with trunk fat mass (TRUNK F) (r=0.545, P=0.001) and limb fat mass (LIMB F) (r=0.412, P=0.005); the leptin level was positively correlated with LIMB F (r=0.513, P=0.001); the leptin and ADPN levels were negatively correlated with the content of lean tissue (r=-0.476, P=0.001; r=-0.344, P=0.021). Compared with those in N-MS group, the levels of serum DPP-4 and leptin were increased significantly, while the ADPN level was decreased notably (P<0.05) in MS group. CONCLUSIONS: The adipokines DPP-4 and leptin in the serum can influence the body fat distribution of patients with T2MD; there is an important association of DPP-4, leptin and ADPN levels with MS, which may be used as therapeutic targets for multiple metabolism disorders of T2MD.

Correction of vitamin D deficiency facilitated suppression of IP-10 and DPP IV levels in patients with chronic hepatitis C: A randomised double-blinded, placebo-control trial.

Vitamin D deficiency was common among patients with chronic hepatitis C (CHC) and had negative influence on treatment outcome. Correction of vitamin D deficiency improved treatment response. Interferon gamma-induced protein 10 (IP-10) and enzyme dipeptidyl peptidase-4 (DPP IV) involved in inflammatory responses in CHC. Their higher levels at pretreatment of CHC could predict poorer responses. Vitamin D suppressed expression of IP-10 from monocytes in vitro. In CHC patients, DPP IV involved in IP-10 regulation. We hypothesized that correction of vitamin D insufficiency or deficiency in CHC patients might restore immune dysregulation through a pathway linked to the TH1/Th2 cytokines, IP-10 or DPP IV. We conducted a double-blind, placebo-controlled trial. 80 CHC patients with vitamin D levels less than 30 ng/mL were assigned to receive vitamin D (40) or placebo (40) supplements for 6 weeks. The levels of 25-hydroxyvitamin D [25(OH)D], Th1/Th2 cytokines, IP-10 and DPP IV were measured at baseline and at the 6th week. At the end of study, the mean 25(OH)D level in vitamin D group was significantly increased and normalised. There were no changes in the level of Th1/Th2 cytokines. Our important finding revealed that upon correction of vitamin D insufficiency or deficiency, the serum IP-10 and DPP IV levels were decreased significantly as compare to the placebo group (delta changes; 83.27 vs -133.80; 95% CI [-326.910, -40.758], p = 0.0125, and 271.04 vs -518.69; 95% CI [-1179,15, -59.781], p = 0.0305, respectively. As previous evidences suggested that each factor individually influenced and predicted outcome of CHC treatment. Our results offer a new insight and help to piece the puzzle of vitamin D deficiency, IP-10 and DPP IV together in CHC. TRIAL REGISTRATION: Thai Clinical Trials Registry TCTR20160429001.

Oral Administration of Collagen Hydrolysates Improves Glucose Tolerance in Normal Mice Through GLP-1-Dependent and GLP-1-Independent Mechanisms.

The aim of this study was to evaluate the antidiabetic properties of collagen hydrolysates (CHs). CHs exhibited dipeptidyl peptidase-IV inhibitory activity and stimulated glucagon-like-peptide-1 (GLP-1) secretion in vitro. We also determined whether CHs improve glucose tolerance in normal mice. Oral administration of CHs suppressed the glycemic response during the oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT), but the effects were weaker in IPGTT than in OGTT. CHs had no effect on the gastric emptying rate. A pretreatment with the GLP-1 receptor antagonist, exendin 9-39 (Ex9), partially reversed the glucose-lowering effects of CHs, but only when coadministered with glucose. CHs administered 45 min before the glucose load potentiated the glucose-stimulated insulin secretion. This potentiating effect on insulin secretion was not reversed by the pretreatment with Ex9, it appeared to be enhanced. These results suggest that CHs improve glucose tolerance by inhibiting intestinal glucose uptake and enhancing insulin secretion, and also demonstrated that GLP-1 was partially involved in the inhibition of glucose uptake, but not essential for the enhancement of insulin secretion.

Improvement of glycemic control in streptozotocin-induced diabetic rats by Atlantic salmon skin gelatin hydrolysate as the dipeptidyl-peptidase IV inhibitor.

In our previous study, Atlantic salmon skin gelatin hydrolysed with flavourzyme possessed 42.5% dipeptidyl-peptidase (DPP)-IV inhibitory activity at a concentration of 5 mg mL(-1). The oral administration of the hydrolysate (FSGH) at a single dose of 300 mg per day in streptozotocin (STZ)-induced diabetic rats for 5 weeks was evaluated for its antidiabetic effect. During the 5-week experiment, body weight increased, and the food and water intake was reduced by FSGH in diabetic rats. The daily administration of FSGH for 5 weeks was effective for lowering the blood glucose levels of diabetic rats during an oral glucose tolerance test (OGTT). After the 5-week treatment, plasma DPP-IV activity was inhibited; the plasma activity of glucagon-like peptide-1 (GLP-1), insulin, and the insulin-to-glucagon ratio were increased by FSGH in diabetic rats. The results indicate that FSGH has the function of inhibiting GLP-1 degradation by DPP-IV, resulting in the enhancement of insulin secretion and improvement of glycemic control in STZ-induced diabetic rats.

Noopept normalizes parameters of the incretin system in rats with experimental diabetes.

Experiments on adult Wistar rats with streptozotocin-induced diabetes showed that antihyperglycemic activity of an original nootropic and neuroprotective drug Noopept (N-phenylacetyl-L-prolylglycine ethyl ester) is more pronounced under conditions of oral application than after intraperitoneal injection. These data provided a basis for studying the effect of Noopept on major indexes of the incretin system. Streptozotocin was shown to decrease the concentrations of incretin GLP-1 and insulin in the blood. Noopept had a normalizing effect on these parameters. This influence of Noopept was not related to the inhibition of a major enzyme metabolizing incretins (dipeptidyl peptidase IV). A reference drug sitagliptin also increased the contents of incretins and insulin, which was associated with the inhibition of dipeptidyl peptidase IV. It is known that GLP-1 increases NGF expression in the insular system. Our results suggest that the increase in incretin activity contributes to the antiapoptotic effect of Noopept on pancreatic ß cells. The mechanism for an increase in blood GLP-1 level after oral application of Noopept requires further investigations.

Attenuation of renovascular damage in Zucker diabetic fatty rat by NWT-03, an egg protein hydrolysate with ACE- and DPP4-inhibitory Activity.

BACKGROUND: Dipeptidyl peptidase 4 (DPP4) and angiotensin-converting enzyme (ACE) are important target enzymes in glycemic control and renovascular protection. Here, we studied the effect of NWT-03, an egg protein hydrolysate with DPP4- and ACE-inhibitory activity, on renovascular damage in Zucker diabetic fatty (ZDF) rats. Comparisons were made to rats treated with vildagliptin (VIL), included as a positive control for the effect of DPP4 inhibition. METHODS: ZDF rats received NWT-03 (1 g/kg/day) or VIL (3 mg/kg/day) from 10 to 25 weeks of age. Metabolic and renal functions were assessed; the kidney was removed for histological analysis of glomerulosclerosis and expression of pro-inflammatory/fibrotic markers (RT-PCR and Western blotting); and the aorta was removed for studies of endothelium-dependent relaxation (EDR). FINDINGS: Hyperinsulinemic ZDF rats typically developed signs of type-2 diabetes and renovascular damage, as evidenced by albuminuria, glomerulosclerosis, and impaired EDR. Neither NWT-03 nor VIL improved metabolic parameters; for VIL, this was despite a 5-fold increase in glucagon-like peptide (GLP)-1 levels. NWT-03 and VIL both reduced renal interleukin (Il)-1ß/Il-13 mRNA expression and glomerulosclerosis. However, only NWT-03 additionally decreased renal tumor necrosis factor (TNF)-α mRNA and P22(phox) protein expression, reduced albuminuria, and restored aortic EDR. Indomethacin added to the organ bath instantly improved aortic EDR, indicating a role for cyclooxygenase (COX)-derived contractile prostanoids in opposing relaxation in ZDF rats. This indomethacin effect was reduced by NWT-03, but not by VIL, and coincided with decreased renal COX-1/2 protein expression. CONCLUSION AND INTERPRETATION: Long-term supplementation with the egg protein hydrolysate NWT-03 attenuated renovascular damage in this preclinical rat model of type 2 diabetes. A comparison to the DPP4-inhibitor VIL suggests that the effects of NWT-03 were related to both ACE- and DPP4-inhibitory properties. The development of protein hydrolysates with a multiple-targeting strategy may be of benefit to functional food formulations.

CD26/dipeptidyl-peptidase IV and adenosine deaminase serum levels in psoriatic patients treated with cyclosporine, etanercept, and psoralen plus ultraviolet A phototherapy.

OBJECTIVE: The aim of this study is to determine serum levels of soluble forms of CD26/dipeptidyl-peptidase IV (DPP-IV) and adenosine deaminase (ADA), thought to be markers of T-cell activation, and changes in their levels in response to cyclosporine, etanercept, and psoralen plus ultraviolet A (PUVA) treatments with respect to disease activity. METHODS: This study is designed as a prospective clinical study with a control group and three months of follow-up. The study included 41 patients with psoriasis and 41 healthy controls that were older than 18years of age. There were three different treatment groups: PUVA (n=15), cyclosporine (n=15), and etanercept (n=11). To determine disease severity of patients with psoriasis, psoriasis area and severity index (PASI) scores were calculated. RESULTS: Only mean serum ADA levels were different between patients with psoriasis [mean1±standard deviation (SD)=13.9±3.3U/ml] and control group (mean±SD=12±3.5U/ml). Mean serum ADA levels were significantly higher before treatment than after treatment (mean±SD=12.4±3.4U/ml). Contrarily, following three months of therapy, mean serum CD26 levels increased significantly from 777.7±214.6 to 835.3±203ng/ml (P<0.05) and mean serum DPP-IV activity increased significantly from 12.1±4 to 15.9±4.2nmol/min (P<0.05). There was no correlation between ADA and CD 26/DPP-IV with PASI values. CONCLUSIONS: The results show that ADA might be a useful marker indicating disease activity and T-cell activation. As significant changes were observed in serum CD26/DPP-IV before and after treatment, we think CD26/DPP-IV might play a role in psoriasis pathogenesis, which should be clarified by further studies.

Evaluation of the antidiabetic effects of dipeptidyl peptidase-IV inhibitor ASP8497 in streptozotocin-nicotinamide-induced mildly diabetic mice.

Gastrointestinal hormone glucagon-like peptide-1 (GLP-1) has a potent glucose-dependent insulinotropic effect and is rapidly degraded by dipeptidyl peptidase (DPP)-IV. Therefore, the use of DPP-IV inhibitors is being actively explored as a novel approach to the treatment of type 2 diabetes. The present study investigated the antidiabetic effects of the DPP-IV inhibitor ASP8497 in streptozotocin-nicotinamide-induced mildly diabetic mice which possess aggravation of glucose tolerance due to loss of early-phase insulin secretion. ASP8497 exhibited good oral bioavailability with potent inhibition of plasma DPP-IV activity. This inhibitory activity lasted up to 24 h when administered at 5 mg/kg twice a day or 10 mg/kg once a day. A single oral administration of ASP8497 (0.3-3 mg/kg) significantly improved glucose tolerance by increasing plasma insulin and GLP-1 levels during the oral glucose or liquid meal tolerance tests. These effects were seen not only immediately, but also 8 h after administration. In contrast, ASP8497 (0.3-10 mg/kg) had no significant effect on blood glucose and plasma insulin levels under fasting conditions. Furthermore, repeated administration of ASP8497 (5 mg/kg twice a day or 10 mg/kg once a day) for 25 days significantly decreased nonfasting blood glucose and HbA(1c) levels. These results suggest that ASP8497 is a potent and long-acting DPP-IV inhibitor that improves glucose tolerance through glucose-dependent insulinotropic action via the elevation of the GLP-1 level in streptozotocin-nicotinamide-induced mildly diabetic mice. It is expected to be useful as a therapeutic agent for impaired glucose tolerance and type 2 diabetes.

Dipeptide boronic acid inhibitors of dipeptidyl peptidase IV: determinants of potency and in vivo efficacy and safety.

Dipeptidyl peptidase IV (DPP-IV; E.C. 3.4.14.5), a serine protease that degrades the incretin hormones GLP-1 and GIP, is now a validated target for the treatment of type 2 diabetes. Dipeptide boronic acids, among the first, and still among the most potent DPP-IV inhibitors known, suffer from a concern over their safety. Here we evaluate the potency, in vivo efficacy, and safety of a selected set of these inhibitors. The adverse effects induced by boronic acid-based DPP-IV inhibitors are essentially limited to what has been observed previously for non-boronic acid inhibitors and attributed to cross-reactivity with DPP8/9. While consistent with the DPP8/9 hypothesis, they are also consistent with cross-reactivity with some other intracellular target. The results further show that the potency of simple dipeptide boronic acid-based inhibitors can be combined with selectivity against DPP8/9 in vivo to produce agents with a relatively wide therapeutic index (>500) in rodents.

[Levels of adenosine deaminase and dipeptidyl peptidase IV in patients with panic disorder]. / Panik bozuklugunda adenozin deaminaz ve dipeptidil peptidaz IV enzim düzeyleri.

OBJECTIVE: Adenosine deaminase and dipeptidyl peptidase IV are enzymes connected to T cells that play an important role in immune system functioning. In this study, in order to understand the immune processes in panic disorder, we determined the serum levels of adenosine deaminase and dipeptidyl peptidase IV in medication-free panic disorder patients and compared them to those of healthy controls. METHOD: Enzymes levels were determined in blood samples of 24 healthy controls and 33 panic disorder patients diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-IV that were medication free during the previous month and medically healthy. RESULTS: Levels of both enzymes were significantly higher in panic disorder patients than in the controls (P<0.001 for adenosine deaminase and P<0.05 for dipeptidyl peptidase IV). The levels of the enzymes did not correlate with sociodemographic variables, duration of the disorder, presence of agoraphobia, presence of stressors, number of panic attack symptoms, and Hamilton depression and anxiety rating scale scores. In addition, the 2 enzymes? levels did not correlate with each other. There was a correlation between Hamilton anxiety rating scale score and the number of panic attack symptoms (P<0.001); however, Hamilton anxiety rating scale scores were not correlated with the other variables. CONCLUSION: Our results suggest that there may be a primary or secondary impaired immune state in the course of panic disorder, as there is in many other psychiatric disorders, such as major depression. Future studies with larger samples are needed to clarify the relationship between the immune system and panic disorder.

Discovery of non-covalent dipeptidyl peptidase IV inhibitors which induce a conformational change in the active site.

A series of non-covalent inhibitors of the serine protease dipeptidyl peptidase IV (DPP-IV) were found to adopt a U-shaped binding conformation in X-ray co-crystallization studies. Remarkably, Tyr547 undergoes a 70 degrees side-chain rotation to accommodate the inhibitor and allows access to a previously unexposed area of the protein backbone for hydrogen bonding.

2-[3-[2-[(2S)-2-Cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl]- 1,2,3,4-tetrahydroisoquinoline: a potent, selective, and orally bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a new type of antidiabetic drugs. Most known DPP-IV inhibitors often resemble the dipeptide cleavage products, with a proline mimic at the P1 site. As off-target inhibitions of DPP8 and/or DPP9 have shown profound toxicities in the in vivo studies, it is important to develop selective DPP-IV inhibitors for clinical usage. To achieve this, a new class of 2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors was synthesized. SAR studies resulted in a number of DPP-IV inhibitors, having IC(50) values of <50 nM with excellent selectivity over both DPP8 (IC(50) > 100 microM) and DPP-II (IC(50) > 30 microM). Compound 21a suppressed the blood glucose elevation after an oral glucose challenge in Wistar rats and also inhibited plasma DPP-IV activity for up to 4 h in BALB/c mice. The results show that compound 21a possesses in vitro and in vivo activities comparable to those of NVP-LAF237 (4), which is in clinical development.

Dipeptidyl peptidase IV and adenosine deaminase activity. Decrease in depression.

Dipeptidyl peptidase IV (DPPIV) and adenosine deaminase (ADA), two T cell associated enzymes, are known to have a possible interaction and play essential roles in immune system functioning. On the other hand, depression has been shown to be accompanied with some immune-inflammatory alterations. In this regard, in order to make a contribution to the understanding of the ongoing immune disturbances in depression, serum DPPIV and ADA activities were determined in minor and major depressives and compared with healthy controls. Both enzyme activities were found to be decreased in major depressives compared to controls while only DPPIV activity was significantly lower in major depressives than the minor depressives. There were significant inverse relationships between enzyme activities and the severity of depression. Moreover, a positive intracorrelation was found between decreased DPPIV and ADA levels. The correlated decrease in DPPIV and ADA, might be a further support for their possible association. Results also suggest that decreased enzyme activities might reflect the impaired immune state in depression while major depressed patients might have a greater tendency to immune dysfunction than the minor depressed ones.