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Astaxanthin Lowers Blood Pressure and Increases Insulin Sensitivity in Rats: Are These Effects Interdependent?

Harry G. Preuss,1,✉ Bobby Echard,1 Eiji Yamashita,2 and  Nicholas V. Perricone3

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The present investigation in Sprague-Dawley rats (SD) was designed to examine effects of astaxanthin (Asta) at different doses on elevated blood pressure (BP) and glucose-insulin perturbations produced by heavy sucrose ingestion. We also examined effects of Asta on BP during restraint stress. SD were divided into six groups each containing eight rats. All SD ate a basic diet of ground regular rat chow with sucrose added at 30% w/w. The Control group received only the basic diet containing added sucrose, while the other five groups each received the same diet with added test material: captopril, (30 mg/Kg), pioglitazone (15.0 mg/Kg), low Asta (25 mg/Kg), medium Asta (50 mg/kg) or high Asta (100 mg/Kg). Many tests were carried out to examine the mechanisms behind the effects of Asta on BP (serum ACE activity, losartan challenge, and LNAME challenge) and the glucose-insulin system (glucose tolerance, HOMA measurement, and insulin challenge). In SD, a relatively low dose of Asta decreased SBP, but produced no major changes in the glucose-insulin system simulating results from a previous study using Zucker Fatty Rats. Increasing the dose of Asta resulted in both a lowering of elevated systolic BP and enhanced insulin sensitivity determined by many different estimations. BP lowering was consistent with changes in the renin-angiotensin (RAS) and nitric oxide (NO) systems. At the examined doses of each, captopril lowered BP in SD without influencing glucose-insulin metabolism, whereas pioglitazone favorably affected glucose-insulin metabolism while showing essentially no effects on BP. Accordingly, Asta beneficially affects both sucrose-induced elevations of BP and insulin resistance at relatively high doses in SD. Also, Asta at higher doses lessens restraint stress, whereas, captopril and pioglitazone did not at the doses examined, even though they influenced the BP and glucose-insulin systems respectively.

Keywords: Astaxanthin, BP effect, Insulin resistance, Restraint stress, renin-angiotensin system

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In a recent study examining effects of astaxanthin (Asta) on elevated blood pressure (BP) and glucose-insulin perturbations in Zucker Fatty Rats (ZFR), we found under conditions examined that this carotenoid at the doses under investigation lowered systolic BP (SBP) consistently, but had no significant influences on glucose-insulin metabolism 1. Our findings in the ZFR, considered by many to be an excellent model of the Metabolic Syndrome 2-4, raised two difficulties in interpreting the data: (1) studies from other laboratories had found that Asta ameliorates both BP elevations and glucose-insulin perturbations 5 and (2) previous reports had strongly suggested that hypertension and other chronic disturbances making up the Metabolic Syndrome are driven by the development of insulin resistance 5-7. Plausible explanations for the first disparity are the differences in dosing and/or rat species used among the various studies. Concerning the second need for clarification, further corroboration of the lack of direct dependence between insulin resistance and hypertension is necessary to substantiate this possibility.

Accordingly, the purpose of the present investigation was to compare with our original results the effects of Asta at different doses and in a different species of rat, Sprague-Dawley (SD), on BP and glucose-insulin perturbations. As a secondary gain, we wished to determine whether Asta ameliorates other forms of stress similar to previous findings with heat stress 1.

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The Animal Welfare Board at Georgetown University Medical Center approved the protocol for this investigation. Forty-eight male Sprague-Dawley rats (SD), obtained from Taconic Farms, Germantown, NY, were used. The SD, weighing between 252-324 g at the beginning of the studies, were housed in a constant temperature room with a light-dark phase of 12 hours each. The SD were divided into six groups, each containing eight rats. The Control group received only the basic diet containing the added sucrose, while the other five groups each received the basic diet with added sucrose containing on a weight of chow basis: captopril, (30 mg/Kg), pioglitazone (15.0 mg/Kg), low Asta (25 mg/Kg), medium Asta (50 mg/kg) or high Asta (100 mg/Kg). The amount of Asta added was based on 2% w/w content of astaxanthin to Haematococcus pluvialis powder1.

The SD were followed for approximately eight months while consuming their respective diets. Body weights and SBP were routinely measured. At the two month and eight month periods of study, specialized tests [intraperitoneal glucose-tolerance (ipGTT), insulin-challenge (ICT), losartan challenge, homeostatic model assessment (HOMA-IR), and Nw-nitro-L arginine-methyl ester hydrochloride (LNAME) challenge] along with blood chemistry assessments were carried out. Details of the procedures are given below.

Body Weight (BW)

BW was estimated by routine scale measurements. Two reading taken at least 10 minutes apart on the given day had to be within two grams of each other or the procedure was repeated until the weights were consistently within the two gram range.

Food and Water Intake

Food and water intakes were estimated close to the midpoint of the study (Day 121) by subtracting the volume or weight of the remaining fluid and food from the amounts premeasured 24 hours earlier.

Blood Pressure (BP)

SBP was measured by tail plethysmography 8 using two different instruments. As in many of our previous studies and for the majority of our measurements, we used an instrument from Narco Biosciences (Houston, TX) 9,10. This allowed us to rapidly measure SBP with a beeper sound system. The second reading was performed on an instrument obtained from Kent Scientific Corporation (Torrington, CT). This is a computerized, non-invasive tail cuff acquisition system that utilizes a specially designed differential pressure transducer to non-invasively measure the blood volume in the tail. The latter instrument not only allowed us to record SBP, but also diastolic BP (DBP), mean BP (MBP), and cardiac rate. A previous report showed that the SBP readings were essentially similar between the two instruments 11. Rats were allowed free access to their diet and water until SBP and the other cardiovascular readings were obtained after a slight warming between 13.00 h and 17.00 h. Multiple readings on individual rats at each session were taken. To be accepted, SBP measurements on a given rat had to be virtually stable. Over the course of study, readings were recorded at multiple time points.

In most cases, the following tests were carried out at two (weeks 7-9) and eight (weeks 31-33) months to give an acute and sub chronic reading.

Blood Chemistries

Blood for chemical analysis was obtained at two and eight months following removal of food the night before. Chemistry data were obtained via dry chemistry procedures using a Johnson and Johnson Vitros 250 instrument. The glucose and insulin levels from the fasting bloods were used to derive a homeostatic model assessment (HOMA-IR) value to assess insulin sensitivity 12. Immunoreactive rat insulin was determined on fasting blood specimens by radioimmunoassay (Diagnostic Products Corporation, Los Angeles CA).

Intraperitoneal glucose tolerance test (ipGTT)

During the ipGTT, performed at two and eight months, glucose (2.5 g/Kg BW) was injected intraperitoneally (i.p.) to challenge tolerance. Drops of blood were obtained from the rat tail at 0, 15, 30, 60, and 120 minutes post injection. Statistical comparisons of serum glucose were made by differences from initial baseline in area under the curve (AUC) over the two hours of study. Glucose was estimated using commercial glucose strips (Lifescan, One Touch Ultra, Melitas, CA).

Insulin Challenge Testing (ICT)

Testing was commenced after 17-19 hours of food deprivation at two and eight months. For ICT, 0.6 unit of regular insulin/kg BW (Eli Lilly Co., Indianapolis, IN) was administered intraperitoneally (i.p.) to determine the decrease in glucose levels from the initial baseline. Blood for glucose determinations was obtained from the tail vein at 7.5 minutes after injection. Glucose was estimated using commercial glucose strips (Lifescan, One Touch Ultra, Melitas, CA). At the two months level, a variation of this procedure was carried out where both glucose 250 mg/Kg and 0.6 units of regular insulin/kg were injected simultaneously, and the glucose rise above baseline was measured. In the first test, a greater decrease in glucose levels soon after insulin injection before various checks and balances can come into significant play suggests greater sensitivity; and in the second procedure, a lesser early elevation in circulating glucose after glucose challenge suggests greater sensitivity to insulin.

Losartan Challenge

This test using the angiotensin receptor blocker, losartan, was also performed at two and eight months. After performing baseline SBP readings, SD from all dietary groups were given 20 mg/kg losartan orally via gastric lavage 13. Three and six hours after lavage, SBP was remeasured. The decreased SBP after losartan was used to estimate activity of the RAS with a greater decrease in SBP connoting augmented system activity 14. Previous studies established that the lowest values of BP reached a plateau for a couple of hours after the 6h mark.

Serum angiotensin converting enzyme (ACE) Activity

ACE refers to the angiotensin converting enzyme. ACE, necessary for the production of angiotensin 2 which is chiefly responsible for the presser effect in the RAS system, was measured by a commercial kit (Sigma Co. Ltd, St. Louis, MO) 15. This spectrophotometric method utilizes the synthetic tripeptide substrate N-[3-(2-furyl)acryloyl]--phenylalanylglcylglcine (FAPGG). FAPGG is hydrolyzed by ACE to furylacryloylphenylalanine (FAP) and glycylglycine. Hydrolysis of FAPGG results in a decreased absorbency at 340 nm. Serum ACE activity was determined by comparing the sample reaction rate to that obtained with an appropriate ACE calibrator. 

Nw-nitro-L arginine-methyl ester hydrochloride (LNAME) Challenge

Effects of nitric oxide synthase (NOS) inhibition on SBP were measured 13. After baseline measurements of SBP, the NOS inhibitor Nw-nitro-L arginine-methyl ester hydrochloride (LNAME) was given intraperitoneally (i.p.) at a dose of 10 mg/kg. Each rat received a single dose of LNAME. Measurements of SBP were taken at four, seven, 10, 15, and 20 minutes post injection. The area under the curve relative to baseline was used to estimate activity of the NO system with a greater increase in SBP connoting augmented system activity.

Statistical Analyses

Results are presented as mean ± SEM. SBP and BW were examined by repeated measures, 2-way analyses of variance (one factor being group and the second factor being time of examination). Where a significant effect of regimen was detected by repeated measures ANOVA (p<0.05), the Dunnett t test was used to establish which differences between means reached statistical significance 16. When the data from two columns were analyzed at a single time point, Student's t test was used. Statistical significance was set at a p < 0.05.

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