Kohji Ohki

Casein Hydrolysate Containing the Antihypertensive Tripeptides Val-Pro-Pro and Ile-Pro-Pro Improves Vascular Endothelial Function Independent of Blood Pressure–Lowering Effects: Contribution of the Inhibitory Action of Angiotensin-Converting Enzyme

Accumulating evidence shows that deterioration of vascular endothelial function underlies the pathophysiology of cardiovascular diseases following lifestyle-related diseases. Both Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which are tripeptides derived from proteolytic hydrolysate of milk casein, inhibit angiotensin-converting enzyme (ACE), suggesting that both VPP and IPP may improve vascular endothelial function, because many ACE inhibitors are known to improve endothelial function. We investigated the effects of ACE-inhibitory food component in humans with mild hypertension, since there has been no report on such effects. The study was conducted by the placebo-controlled, double-blind crossover method in 25 male subjects with mild hypertension. After casein hydrolysate containing both VPP and IPP were administered for 1 week, reactive hyperemia of the left upper forearm was measured using plethysmography as an index of vascular endothelial function. Since one subject dropped out, we analyzed the data of 24 subjects. The reactive hyperemia of the left upper forearm was produced by a 5 min occlusion using inflation of a cuff. The maximum blood flow during reactive hyperemia was 20.8±6.7 mL/min/100 mL tissue in the placebo group, whereas it increased remarkably to 30.0±10.4 mL/min/100 mL tissue in the group administered casein hydrolysate containing both VPP and IPP (p<0.001). There was no change in systemic blood pressure, indicating that the improvement of the vascular endothelial function attributable to VPP and IPP is independent of hemodynamic changes. We conclude that casein hydrolysate containing VPP and IPP improves the vascular endothelial dysfunction in subjects with mild hypertension. The continuous intake of VPP and IPP could help to prevent cardiovascular diseases in hypertensive subjects.

Casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro improves central blood pressure and arterial stiffness in hypertensive subjects: A randomized, double-blind, placebo-controlled trial

OBJECTIVE This trial evaluated the effects of casein hydrolysate containing milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), on central blood pressure and arterial stiffness. METHODS A randomized, double-blind, placebo-controlled trial was conducted in 70 Japanese subjects aged 50-69 years with untreated stage-I hypertension. They were randomly assigned to two groups, which received either placebo tablets or active tablets containing 3.4 mg of VPP and IPP. At the beginning and end of the 8-week intervention, hemodynamic parameters, including central blood pressure and brachial-ankle pulse wave velocity (baPWV), a marker of arterial stiffness, were measured. RESULTS A significant difference in changes in central systolic blood pressure between the groups was observed (active: -11.0±11.0 vs placebo: -4.5±9.6 mmHg, P<0.01). In the active group, reductions in baPWV (-73.9±130.0 vs -8.4±137.1 cm/s, P<0.05), brachial SBP (-10.5±11.5 vs -3.9±9.6 mmHg, P<0.05), and radial mean blood pressure (-7.3±8.9 vs -2.0±7.4 mmHg, P<0.01) were significantly greater as compared with the placebo group. CONCLUSION Casein hydrolysate containing VPP and IPP improves central SBP and baPWV in hypertensive subjects, which suggests VPP and IPP might have beneficial effects on arterial properties.

Milk-Derived Peptides, Val-Pro-Pro and Ile-Pro-Pro, Attenuate Atherosclerosis Development in Apolipoprotein E–Deficient Mice: A Preliminary Study

Milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have angiotensin I-converting enzyme inhibitory activities and blood pressure-lowering effects. We examined the effects of these peptides on the development of atherosclerosis in apolipoprotein E-deficient [apoE(-/-)] mice. For 31 weeks, six-week-old male apoE(-/-) mice received a diet that included one of the following: fermented milk containing both VPP and IPP; casein hydrolysate containing both of these peptides; synthesized VPP; synthesized IPP; enalapril; captopril; or control diet. At the end of feeding, blood biochemistry, aortic atherogenesis, and gene expression by DNA microarray analysis were evaluated. There were no significant changes in the plasma lipid levels and 8-isoprostane, a marker of oxidative stress. The area ratio of intima to media in the aortic arch was significantly lower in the fermented milk, casein hydrolysate, synthesized VPP, enalapril, and captopril groups than in the control group. As is common with diets containing VPP and/or IPP, we observed reductions in mRNA expression of inflammatory cytokines, such as interleukin (IL)-6 and IL-1β, oxidized low-density lipoprotein receptor, and transcription regulators. These results suggest that a continuous intake of VPP and IPP might be beneficial for preventing atherosclerosis caused by hypercholesterolemia.

Lactobacillus helveticus-fermented milk improves learning and memory in mice

Abstract Objectives To investigate the effects of Calpis sour milk whey, a Lactobacillus helveticus–fermented milk product, on learning and memory. Methods We evaluated improvement in scopolamine-induced memory impairment using the spontaneous alternation behaviour test, a measure of short-term memory. We also evaluated learning and working memory in mice using the novel object recognition test, which does not involve primary reinforcement (food or electric shocks). A total of 195 male ddY mice were used in the spontaneous alternation behaviour test and 60 in the novel object recognition test. Results Forced orally administered Calpis sour milk whey powder (200 and 2000 mg/kg) significantly improved scopolamine-induced cognitive impairments (P < 0.05 and P < 0.01, respectively) and object recognition memory (2000 mg/kg; P < 0.05). Discussion These results suggest that Calpis sour milk whey may be useful for the prevention of neurodegenerative disorders, such as Alzheimers disease, and enhancing learning and memory in healthy human subjects; however, human clinical studies are necessary.

Effects of the oriental herbal medicine Bofu-tsusho-san in obesity hypertension: A multicenter, randomized, parallel-group controlled trial (ATH-D-14-01021.R2)

OBJECTIVE There is no clinical evidence that supports the benefit of integrative medicine, defined as combination therapy of oriental and western medicine, on obesity-related hypertension. This study evaluates the efficacy of Bofu-tsusho-san (BOF), an oriental herbal medicine, on the ambulatory blood pressure (BP) profile in hypertensive patients with obesity. METHODS The study design was a multicenter, randomized, open-label, parallel-group controlled trial in 107 hypertensive patients with obesity. Participants were randomly assigned to receive either the conventional control therapy or BOF add-on therapy. In both groups antihypertensive therapy was aimed at achieving the target clinic BP. The primary outcome was change in the ambulatory BP profile from baseline to 24 weeks after randomization. RESULTS Daytime systolic BP variability, an important parameter of ambulatory BP profile, was decreased in the BOF group, and the difference in the changes in daytime systolic BP variability was significant between the BOF and control group (Control vs BOF; the change from baseline in daytime systolic BP variability, 1.0±3.3 vs -1.0±3.3%; p=0.006). CONCLUSION The BOF add-on therapy effectively improved the ambulatory BP variability. This is the first report suggesting that an integrative medicine approach may exert favorable effects on obesity-related hypertension compared with conventional pharmaceutical treatment. CLINICAL TRIAL REGISTRATION UMIN000003878.

Adipocyte‐Specific Enhancement of Angiotensin II Type 1 Receptor‐Associated Protein Ameliorates Diet‐Induced Visceral Obesity and Insulin Resistance

Background The renin–angiotensin system has a pivotal role in the pathophysiology of visceral obesity. Angiotensin II type 1 receptor (AT1R) is a major player in the signal transduction of the renin–angiotensin system, and the overactivation of this signaling contributes to the progression of visceral obesity. We have shown that the AT1R‐associated protein (ATRAP) promotes AT1R internalization from the cell surface into cytoplasm along with the suppression of overactivation of tissue AT1R signaling. In this study, we examined whether the enhancement of adipose ATRAP expression could efficiently prevent diet‐induced visceral obesity and insulin resistance. Methods and Results We generated adipocyte‐specific ATRAP transgenic mice using a 5.4‐kb adiponectin promoter, and transgenic mice and littermate control mice were fed either a low‐ or high‐fat diet for 10 weeks. Although the physiological phenotypes of the transgenic and control mice fed a low‐fat diet were comparable, the transgenic mice exhibited significant protection against high‐fat diet–induced adiposity, adipocyte hypertrophy, and insulin resistance concomitant with an attenuation of adipose inflammation, macrophage infiltration, and adipokine dysregulation. In addition, when mice were fed a high‐fat diet, the adipose expression of glucose transporter type 4 was significantly elevated and the level of adipose phospho‐p38 mitogen‐activated protein kinase was significantly attenuated in the transgenic mice compared with control mice. Conclusions Results presented in this study suggested that the enhancement in adipose ATRAP plays a protective role against the development of diet‐induced visceral obesity and insulin resistance through improvement of adipose inflammation and function via the suppression of overactivation of adipose AT1R signaling. Consequently, adipose tissue ATRAP is suggested to be an effective therapeutic target for the treatment of visceral obesity.

The milk-derived peptides Val-Pro-Pro and Ile-Pro-Pro attenuate arterial dysfunction in L -NAME-treated rats

Both endothelial dysfunction and arterial stiffness are surrogate markers of atherosclerosis and thus cardiovascular (CV) events. The milk-derived peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) inhibit angiotensin-converting enzyme, dilate blood vessels ex vivo and stimulate nitric oxide (NO) production in cells. In this study, we investigated the effects of either VPP or IPP on arterial function and on target organ damage in vivo. Male Wistar rats were treated with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 g l−1), L-NAME+VPP (0.3 g l−1) or L-NAME+IPP (0.3 g l−1) in their drinking water for 8 weeks. Plasma nitrite and nitrate (NOx) levels were significantly increased in normal Wistar rats after supplementation with either VPP or IPP but not in rats that were chronically treated with L-NAME. Acetylcholine-induced vasorelaxation in the thoracic aorta ring was impaired by L-NAME, whereas vasorelaxation was significantly greater in mice treated with L-NAME+VPP for 1 or 4 weeks or L-NAME+IPP for 4 weeks than in mice treated with L-NAME alone. Four weeks of treatment with either VPP or IPP attenuated the increase in pulse wave velocity (PWV) that was induced by L-NAME. Cardiac and renal damage were observed after 8 weeks of treatment with L-NAME, and either VPP or IPP attenuated this damage. These results show that VPP or IPP attenuates arterial dysfunction and suggest that milk-derived peptides might prevent CV damage.

Effects of the Angiotensin Receptor Blocker Olmesartan on Adipocyte Hypertrophy and Function in Mice with Metabolic Disorders

In the present study, we examined the therapeutic effects of olmesartan, an angiotensin II (Ang II) type 1 receptor (AT1R)-specific blocker, in genetically obese diabetic KKAy mice, a model of human metabolic disorders with visceral obesity, with a focus on an olmesartan effect on the adipose tissue. Olmesartan treatment (3 mg/kg per day) for 4 weeks significantly lowered systolic blood pressure but did not affect body weight during the study period in KKAy mice. However, there were three interesting findings possibly related to the pleiotropic effects of olmesartan on adipose tissue in KKAy mice: (1) an inhibitory effect on adipocyte hypertrophy, (2) a suppressive effect on IL-6 gene expression, and (3) an ameliorating effect on oxidative stress. On the other hand, olmesartan exerted no evident influence on the adipose tissue expression of AT1R-associated protein (ATRAP), which is a molecule interacting with AT1R so as to inhibit pathological AT1R activation and is suggested to be an emerging molecular target in metabolic disorders with visceral obesity. Collectively, these results suggest that the blood pressure lowering effect of olmesartan in KKAy mice is associated with an improvement in adipocyte, including suppression of adipocyte hypertrophy and inhibition of the adipose IL-6-oxidative stress axis. Further study is needed to clarify the functional role of adipose ATRAP in the pleiotropic effects of olmesartan.

Renal Tubule Angiotensin II Type 1 Receptor–Associated Protein Promotes Natriuresis and Inhibits Salt‐Sensitive Blood Pressure Elevation

Background Angiotensin II type 1 receptor (AT1R)–associated protein (ATRAP; Agtrap gene) promotes AT1R internalization along with suppression of pathological AT1R activation. In this study, we examined whether enhancement of ATRAP in the renal distal tubules affects sodium handling and blood pressure regulation in response to high salt (HS) loading, using ATRAP transgenic mice on a salt‐sensitive C57BL/6J background. Methods and Results Renal ATRAP transgenic (rATRAP‐Tg) mice, which exhibit renal tubule–dominant ATRAP enhancement, and their wild‐type littermate C57BL/6J mice on a normal salt diet (0.3% NaCl) at baseline were subjected to dietary HS loading (4% NaCl) for 7 days. In rATRAP‐Tg mice, the dietary HS loading–mediated blood pressure elevation was suppressed compared with wild‐type mice, despite similar baseline blood pressure. Although renal angiotensin II level was comparable in rATRAP‐Tg and wild‐type mice with and without HS loading, urinary sodium excretion in response to HS loading was significantly enhanced in the rATRAP‐Tg mice. In addition, functional transport activity of the amiloride‐sensitive epithelial Na+ channel was significantly decreased under saline volume–expanded conditions in rATRAP‐Tg mice compared with wild‐type mice, without any evident change in epithelial Na+ channel protein expression. Plasma membrane AT1R expression in the kidney of rATRAP‐Tg mice was decreased compared with wild‐type mice. Conclusions These results demonstrated that distal tubule–dominant enhancement of ATRAP inhibits pathological renal sodium reabsorption and blood pressure elevation in response to HS loading. The findings suggest that ATRAP‐mediated modulation of sodium handling in renal distal tubules could be a target of interest in salt‐sensitive blood pressure regulation.

Angiotensin II Type 1 Receptor‐Associated Protein Regulates Kidney Aging and Lifespan Independent of Angiotensin

Background The kidney is easily affected by aging‐associated changes, including glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Particularly, renal tubulointerstitial fibrosis is a final common pathway in most forms of progressive renal disease. Angiotensin II type 1 receptor (AT1R)‐associated protein (ATRAP), which was originally identified as a molecule that binds to AT1R, is highly expressed in the kidney. Previously, we have shown that ATRAP suppresses hyperactivation of AT1R signaling, but does not affect physiological AT1R signaling. Methods and Results We hypothesized that ATRAP has a novel functional role in the physiological age‐degenerative process, independent of modulation of AT1R signaling. ATRAP‐knockout mice were used to study the functional involvement of ATRAP in the aging. ATRAP‐knockout mice exhibit a normal age‐associated appearance without any evident alterations in physiological parameters, including blood pressure and cardiovascular and metabolic phenotypes. However, in ATRAP‐knockout mice compared with wild‐type mice, the following takes place: (1) age‐associated renal function decline and tubulointerstitial fibrosis are more enhanced; (2) renal tubular mitochondrial abnormalities and subsequent increases in the production of reactive oxygen species are more advanced; and (3) life span is 18.4% shorter (median life span, 100.4 versus 123.1 weeks). As a key mechanism, age‐related pathological changes in the kidney of ATRAP‐knockout mice correlated with decreased expression of the prosurvival gene, Sirtuin1. On the other hand, chronic angiotensin II infusion did not affect renal sirtuin1 expression in wild‐type mice. Conclusions These results indicate that ATRAP plays an important role in inhibiting kidney aging, possibly through sirtuin1‐mediated mechanism independent of blocking AT1R signaling, and further protecting normal life span.