DPP4 Deficiency Preserved Cardiac Function in Abdominal Aortic Banding Rats

Dipeptidyl peptidase-4 (DPP4) enzyme inhibition has been reported to increase plasma glucagon-like peptide-1 (GLP-1) level for controlling postprandial glucose concentration. A prominent GLP-1 level in DPP4-deficient rats contributed to the resistance of endotoxemia and myocardial infarction. DPP4 deficiency also increased the capability against H₂O₂-induced stress in cardiomyocyte. However, long term effect of loss DPP4 activity on cardiac performance remained unclear. We used abdominal aortic banding (AAB) to induce pressure overload in wild-type and DPP4-deficient rats, and investigated the progression of heart failure. Cardiac histology and function were determined. Blood sample was collected for the plasma biochemical marker measurement. Heart weight to body weight ratio increased 1.2-fold after 6 weeks of AAB surgery. Cardiac function was compensated against pressure overload after 6 weeks of AAB surgery, but progressed to deterioration after 10 weeks of AAB surgery. AAB induced cardiac dysfunction was alleviated in DPP4-deficient rats. DPP4 activity increased significantly in wild-type rats after 10 weeks of AAB surgery, but remained unchanged in DPP4-deficient rats. In contrast, GLP-1 concentration was elevated by AAB after 6 weeks of surgery in DPP4-deficient rats, and remained high after 10 weeks of surgery. Ang II level markedly increased after 6 weeks of AAB surgery, but were less in DPP4-deficient rats. Massive collagen deposits in wild-type rat hearts appeared after 10 weeks of AAB surgery, which were alleviated in DPP4-deficient rats. Long term deficiency of DPP4 activity improved cardiac performance against pressure overload in rat, which may be attributed to a great quantity of GLP-1 accumulation during AAB.     

StAR protein and steroidogenic enzyme expressions in the rat Harderian gland

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5α-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology.     

Association of DPP4 Gene Polymorphisms with Type 2 Diabetes Mellitus in Malaysian Subjects

BackgroundGenetic polymorphisms of the Dipeptidyl Peptidase 4 (DPP4) gene may play a role in the etiology of type 2 diabetes mellitus (T2DM). This study aimed to investigate the possible association of single nucleotide polymorphisms (SNPs) of the DPP4 gene in Malaysian subjects with T2DM and evaluated whether they had an effect on the serum levels of soluble dipeptidyl peptidase 4 (sDPP-IV).MethodTen DPP4 SNPs were genotyped by TaqMan genotyping assays in 314 subjects with T2DM and 235 controls. Of these, 71 metabolic syndrome (MetS) subjects were excluded from subsequent analysis. The odds ratios (ORs) and their 95% confidence interval (CIs) were calculated using multiple logistic regression for the association between the SNPs of DPP4 and T2DM. In addition, the serum levels of sDPP-IV were investigated to evaluate the association of the SNPs of DPP4 with the sDPP-IV levels.ResultsDominant, recessive, and additive genetic models were employed to test the association of DPP4 polymorphisms with T2DM, after adjusting for age, race, gender and BMI. The rs12617656 was associated with T2DM in Malaysian subjects in the recessive genetic model (OR = 1.98, p = 0.006), dominant model (OR = 1.95, p = 0.008), and additive model (OR = 1.63, p = 0.001). This association was more pronounced among Malaysian Indians, recessive (OR = 3.21, p = 0.019), dominant OR = 3.72, p = 0.003) and additive model (OR = 2.29, p = 0.0009). The additive genetic model showed that DPP4 rs4664443 and rs7633162 polymorphisms were associated with T2DM (OR = 1.53, p = 0.039), and (OR = 1.42, p = 0.020), respectively. In addition, the rs4664443 G>A polymorphism was associated with increased sDPP-IV levels (p = 0.042) in T2DM subjects.ConclusionsDPP4 polymorphisms were associated with T2DM in Malaysian subjects, and linked to variations in sDPP-IV levels. In addition, these associations were more pronounced among Malaysian Indian subjects.     

DPP4 Gene DNA Methylation in the Omentum is Associated With Its Gene Expression and Plasma Lipid Profile in Severe Obesity

Severely obese subjects with the metabolic syndrome (MS) have higher dipeptidyl peptidase‐4 (DPP4) expression in their visceral adipose tissue (VAT) compared to obese individuals without MS. We tested the hypothesis that methylation level of CpG sites in the DPP4 promoter CpG island in VAT was genotype‐dependent and associated with DPP4 mRNA abundance and MS‐related phenotypes. The VAT DNA was extracted in 92 severely obese premenopausal women undergoing biliopancreatic derivation for the treatment of obesity. Women were nondiabetic and none of them used medication to treat MS features. Cytosine methylation rates (%) of 102 CpG sites in the DPP4 CpG island were assessed by pyrosequencing of sodium bisulfite‐treated DNA. Methylation rates were >10% for CpG sites 94–102. Their mean methylation rate (%Meth_94–102) was different between genotypes for DPP4 polymorphisms rs13015258 (P = 0.001), rs17848915 (P = 0.0004), and c.1926 G>A (P = 0.001). The %Meth_94–102 correlated negatively with DPP4 mRNA abundance (r = ?0.25, P < 0.05) and positively with plasma high‐density lipoprotein (HDL) cholesterol concentrations (r = 0.22, P < 0.05), whereas DPP4 mRNA abundance correlated positively with plasma total‐/HDL‐cholesterol ratio (r = 0.25; P < 0.05). In the VAT of nondiabetic severely obese women, genotype‐dependent methylation levels of specific CpG sites in the DPP4 promoter CpG island were associated with DPP4 gene expression and variability in the plasma lipid profile. Higher DPP4 gene expression in VAT and its relationship with the plasma lipid profile may be explained by actually unknown DPP4 biological effect or, to another extent, may also be a marker of VAT inflammation known to be associated with metabolic disturbances.     

Hidden Disease Susceptibility and Sexual Dimorphism in the Heterozygous Knockout of Cyp51 from Cholesterol Synthesis

We examined the genotype-phenotype interactions of Cyp51^+/− mice carrying one functional allele of lanosterol 14α-demethylase from cholesterol biosynthesis. No distinct developmental or morphological abnormalities were observed by routine visual inspection of Cyp51^+/− and Cyp51^+/+ mice and fertility was similar. We further collected a large data-set from female and male Cyp51^+/− mice and controls fed for 16 weeks with three diets and applied linear regression modeling. We used 3 predictor variables (genotype, sex, diet), and 39 response variables corresponding to the organ characteristics (7), plasma parameters (7), and hepatic gene expression (25). We observed significant differences between Cyp51^+/− and wild-type mice in organ characteristics and blood lipid profile. Hepatomegaly was observed in Cyp51^+/− males, together with elevated total and low-density lipoprotein cholesterol. Cyp51^+/− females fed high-fat, high-cholesterol diet were leaner and had elevated plasma corticosterone compared to controls. We observed elevated hepatocyte apoptosis, mitosis and lipid infiltration in heterozygous knockouts of both sexes. The Cyp51^+/− females had a modified lipid storage homeostasis protecting them from weight-gain when fed high-fat high-cholesterol diet. Malfunction of one Cyp51 allele therefore initiates disease pathways towards cholesterol-linked liver pathologies and sex-dependent response to dietary challenge.     

Hsd3b2 associated in modulating steroid hormone synthesis pathway regulates the differentiation of chicken embryonic stem cells into spermatogonial stem cells

Steroid hormones regulate differentiation of various types of cell during embryogenesis. Testosterone is one of the androgens that bind to receptors to regulate gene expression and promote spermatogenesis. Our results showed that testosterone, as a product of steroid hormones synthesis pathway, could facilitate the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The analysis of the steroid hormones synthesis pathway demonstrated that 3beta‐hydroxysteroid dehydrogenase2 (Hsd3b2) plays a major role in the synthesis of testosterone. In the absence of Hsd3b2, the expression of downstream genes such as Cyp1a1, Ugt1a1, and Hsd17b7 was not maintained. This reduction is probably due to the down‐regulation of the steroid hormones synthesis pathway. Furthermore, qRT‐PCR, immunofluorescence, and flow cytometry analysis confirmed that the steroid hormones synthesis pathway could facilitate the differentiation of ESCs. Altogether, these results lead to a model in which Hsd3b2 regulates ESCs differentiation via modulating the activity of steroid hormones synthesis pathway.     

CREM modulates the circadian expression of CYP51, HMGCR and cholesterogenesis in the liver

We show for the first time that isoforms of the cAMP response element modulator Crem, regulate the circadian expression of Cyp51 and other cholesterogenic genes in the mouse liver. In the wild type mice the expression of Cyp51, Hmgs, Fpps, and Sqs is minimal between CT12 and CT16 and peaks between CT20 and CT24. Cyp51, Fpps, and Sqs lost the circadian behavior in Crem−/− livers while Hmgcr is phase advanced from CT20 to CT12. This coincides with a phase advance of lathosterol/cholesterol ratio, as detected by GC-MS. Overexpression of CREMτ and ICER has little effect on the Hmgcr proximal promoter while they influence expression from the CYP51 promoter. Our data indicate that Crem-dependent regulation of Cyp51 in the liver results in circadian expression of this gene. We propose that cAMP signaling might generally be involved in the circadian regulation of cholesterol synthesis on the periphery.     

Characterization of the hypothalamic-pituitary-gonadal axis in estrogen receptor (ER) Null mice reveals hypergonadism and endocrine sex reversal in females lacking ERalpha but not ERbeta

To determine the role of each estrogen receptor (ER) form (ERalpha, ERbeta) in mediating the estrogen actions necessary to maintain proper function of the hypothalamic-pituitary-gonadal axis, we have characterized the hypothalamic-pituitary-gonadal axis in female ER knockout (ERKO) mice. Evaluation of pituitary function included gene expression assays for Gnrhr, Cga, Lhb, Fshb, and Prl. Evaluation of ovarian steroidogenic capacity included gene expression assays for the components necessary for estradiol synthesis: i.e. Star, Cyp11a, Cyp17, Cyp19, Hsd3b1, and Hsd17b1. These data were corroborated by assessing plasma levels of the respective peptide and steroid hormones. alphaERKO and alphabetaERKO females exhibited increased pituitary Cga and Lhb expression and increased plasma LH levels, whereas both were normal in betaERKO. Pituitary Fshb expression and plasma FSH were normal in all three ERKOs. In the ovary, all three ERKOs exhibited normal expression of Star, Cyp11a, and Hsd3b1. In contrast, Cyp17 and Cyp19 expression were elevated in alphaERKO but normal in betaERKO and alphabetaERKO. Plasma steroid levels in each ERKO mirrored the steroidogenic enzyme expression, with only the alphaERKO exhibiting elevated androstenedione and estradiol. Elevated plasma testosterone in alphaERKO and alphabetaERKO females was attributable to aberrant expression of Hsd17b3 in the ovary, representing a form of endocrine sex reversal, as this enzyme is unique to the testes. Enhanced steroidogenic capacity in alphaERKO ovaries was erased by treatment with a GnRH antagonist, indicating these phenotypes to be the indirect result of excess LH stimulation that follows the loss of ERalpha in the hypothalamic-pituitary axis. Overall, these findings indicate that ERalpha, but not ERbeta, is indispensable to the negative-feedback effects of estradiol that maintain proper LH secretion from the pituitary. The subsequent hypergonadism is illustrated as increased Cyp17, Cyp19, Hsd17b1, and ectopic Hsd17b3 expression in the ovary.     

Cholesterol-lowering effects of dietary pomegranate extract and inulin in mice fed an obesogenic diet

BackgroundIt has been demonstrated in animal studies that both polyphenol-rich pomegranate extract (PomX) and the polysaccharide inulin, ameliorate metabolic changes induced by a high-fat diet, but little is known about the specific mechanisms.ObjectiveThis study evaluated the effect of PomX (0.25%) and inulin (9%) alone or in combination on cholesterol and lipid metabolism in mice.MethodsMale C57BL/6 J mice were fed high-fat/high-sucrose [HF/HS (32% energy from fat, 25% energy from sucrose)] diets supplemented with PomX (0.25%) and inulin (9%) alone or in combination for 4 weeks. At the end of intervention, serum and hepatic cholesterol, triglyceride levels, hepatic gene expression of key regulators of cholesterol and lipid metabolism as well as fecal cholesterol and bile acid excretion were determined.ResultsDietary supplementation of the HF/HS diet with PomX and inulin decreased hepatic and serum total cholesterol. Supplementation with PomX and inulin together resulted in lower hepatic and serum total cholesterol compared to individual treatments. Compared to HF/HS control, PomX increased gene expression of Cyp7a1 and Cyp7b1, key regulators of bile acid synthesis pathways. Inulin decreased gene expression of key regulators of cholesterol de novo synthesis Srebf2 and Hmgcr and significantly increased fecal elimination of total bile acids and neutral sterols. Only PomX in combination with inulin reduced liver and lipid weight significantly compared to the HF/HS control group. PomX showed a trend to decrease liver triglyceride (TG) levels, while inulin or PomX-inulin combination had no effect on either serum or liver TG levels.ConclusionDietary PomX and inulin supplementation decreased hepatic and serum total cholesterol by different mechanisms and the combination leading to a significant enhancement of the cholesterol-lowering effect.     

DA-1229, a novel and potent DPP4 inhibitor, improves insulin resistance and delays the onset of diabetes

AimTo characterize the pharmacodynamic profile of DA-1229, a novel dipeptidyl peptidase (DPP) 4 inhibitor.Main methodsEnzyme inhibition assays against DPP4, DPP8 and DPP9. Antidiabetic effects of DA-1229 in HF-DIO mice and young db/db mice.Key findingsDA-1229 was shown to potently inhibit the DPP4 enzyme in human and murine soluble forms and the human membrane-bound form with IC₅₀ values of 0.98, 3.59 and 1.26 nM, respectively. As a reversible and competitive inhibitor, DA-1229 was more selective to human DPP4 (6000-fold) than to human DPP8 and DPP9. DA-1229 (0.1–3 mg/kg) dose-dependently inhibited plasma DPP4 activity, leading to increased levels of plasma GLP-1 and insulin, and thereby lowering blood glucose levels in mice. In high fat diet-fed (HF) mice, a single oral dose of 100 mg/kg of DA-1229 reduced plasma DPP4 activity by over 80% during a 24 h period. Long-term treatment with DA-1229 for 8 weeks revealed significant improvements in glucose intolerance and insulin resistance, accompanied by significant body weight reduction. However, it remains unclear whether there is a direct causal relationship between DPP4 inhibition and body weight reduction. In young db/db mice, the DA-1229 treatment significantly reduced blood glucose excursions for the first 2 weeks, resulting in significantly lower levels of HbA1c at the end of the study. Furthermore, the pancreatic insulin content of the treatment group was significantly higher than that of the db/db control.SignificanceDA-1229 as a novel and selective DPP4 inhibitor improves the insulin sensitivity in HF mice and delays the onset of diabetes in young db/db mice.     

Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.     

Association between 12α-hydroxylated bile acids and hepatic steatosis in rats fed a high-fat diet

High-fat (HF) diet induces hepatic steatosis that is a risk factor for noncommunicable diseases such as obesity, type 2 diabetes and cardiovascular disease. Previously, we found that HF feeding in rats increases the excretion of fecal bile acids (BAs), specifically 12α-hydroxylated (12αOH) BAs. Although the liver is the metabolic center in our body, the association between hepatic steatosis and 12αOH BAs in HF-fed rats is unclear. Thus, we investigated extensively BA composition in HF-fed rats and evaluated the association between hepatic steatosis and 12αOH BAs. Acclimated male inbred WKAH/HkmSlc rats were divided into two groups and fed either control or HF diet for 8 weeks. Feeding HF diet increased hepatic triglyceride and total cholesterol concentrations, which correlated positively with 12αOH BAs concentrations but not with non-12αOH BAs in the feces, portal plasma and liver. Accompanied by the increase in 12αOH BAs, the rats fed HF diet showed increased fat absorption and higher mRNA expression of liver Cidea. The enhancement of 12αOH BA secretion may contribute to hepatic steatosis by the promotion of dietary fat absorption and hepatic Cidea mRNA expression. The increase in 12αOH BAs was associated with enhanced liver cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) mRNA expression. There was a significant increase in 7α-hydroxycholesterol, a precursor of BAs, in the liver of HF-fed rats. Altogether, these data suggest that the HF diet increases preferentially 12αOH BAs synthesis by utilizing the accumulated hepatic cholesterol and enhancing mRNA expression of Cyp7a1 and Cyp8b1 in the liver.     

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells

Background

Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established.

Methodology

Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity.

Results and Conclusions

In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC₅₀) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.     

Advanced paternal age increased metabolic risks in mice offspring

ObjectivesThe Developmental Origins of Health and Disease Science indicate that chronic diseases in adulthood are associated with prenatal and early-life traits. Our study aimed to explore the metabolic phenotype of offspring from advanced paternal age (APA) and the inherited alterations in sperm.Methods3-month-old (Young father, YF-F0) and 21-month-old male (Old Father, OF-F0) C57BL/6J mice were used to study paternal aging's effect on offspring. Blood glucose testing, lipid analysis, indirect calorimetry and RNA sequencing were performed.ResultsThe characterized metabolic changes in OF-F1 male mice offspring were glucose intolerance, hepatic lipid accumulation, increased adipocytes and impaired energy balance that lasted until they were elderly. Gene expression in both 8-week-old and 52-week-old offspring livers significantly altered in lipid metabolism- and thermogenesis-related pathways. PPAR signaling pathway was activated in both young and elderly offspring livers as indicated by significant upregulation of Cyp7a1, Cyp8b1, Cyp4a10, Cyp4a31, Fabp2, and Scd1. These targeted genes were also confirmed to be increased in offspring adipocytes. Furthermore, when examined the differentially expressed genes in F0 and F1 sperm, upregulated pathways including cholesterol metabolism, type II diabetes mellitus and endocrine resistance were strongly related to the APA offspring phenotype. Importantly, approximately 46.7% of enriched pathways in the sperm of APA offspring were consistent with those of APA fathers.ConclusionsThese findings added evidence of the connection between paternal gametes and alterations in progeny genome and raised the possibility that inherited alterations in sperm contribute to the intergenerational effects of paternal aging offspring's chronic metabolic risks.     

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)-1β/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.     

Kv4.2 and Accessory Dipeptidyl Peptidase-like Protein 10 (DPP10) Subunit Preferentially Form a 4:2 (Kv4.2:DPP10) Channel Complex

Kv4 is a member of the voltage-gated K⁺ channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2-DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of the DPP10 formed dimers in the plasma membrane, and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2-DPP10 complex; Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2-DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2-DPP10 complex was variable depending on the relative expression level of each subunit, with a preference for 4:2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10.