Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship With Insulin Resistance

Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship... Abstract Context Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown. Objective To investigate whole body methionine and homocysteine kinetics and the effects of insulin in essential hypertension. Design and Setting Eight hypertensive male subjects and six male normotensive controls were infused with l-[methyl-2H3,1-13C]methionine for 6 hours. In the last 3 hours a euglycemic, hyperinsulinemic clamp was performed. Steady-state methionine and homocysteine kinetics were determined in postabsorptive and hyperinsulinemic conditions. Results Postabsorptive hypertensive subjects had elevated homocysteine concentrations (+30%, P = 0.035) and slightly (by 15% to 20%) but insignificantly lower methionine rates of appearance (Ras) (P = 0.07 to P = 0.05) and utilization for protein synthesis (P = 0.06) than postabsorptive normotensive controls. Hyperinsulinemia suppressed methionine Ra and protein synthesis, whereas it increased homocysteine trans-sulfuration, clearance, and methionine transmethylation (the latter only in the normotensive subjects). However, in the hypertensive subjects trans-sulfuration was significantly lower (P < 0.05) and increased ~50% less [by +1.59 ± 0.34 vs +3.45 ± 0.52 µmol/kg lean body mass (LBM) per hour, P < 0.005] than in normotensive controls. Homocysteine clearance through trans-sulfuration was ~50% lower in hypertensive than in normotensive subjects (P < 0.005). In the hypertensive subjects, insulin-mediated glucose disposal was ~45% lower (460 ± 44 vs 792 ± 67 mg/kg LBM per hour, P < 0.0005) than in normotensive controls and was positively correlated with the increase of trans-sulfuration (P < 0.0015). Conclusions In subjects with essential hypertension, hyperhomocysteinemia is associated with decreased homocysteine trans-sulfuration and probably represents a feature of insulin resistance. Homocysteine is an independent vascular risk factor (1, 2), is often elevated in arterial hypertension (3), and has been proposed as an independent risk factor for hypertension (4). Hyperhomocysteinemia has been associated with tissue and organ toxicity through a variety of possible mechanisms (5). Homocysteine is produced within the methionine cycle from S-adenosyl homocysteine demethylation (6) and is removed through either remethylation to methionine or trans-sulfuration to cystathionine and subsequent oxidation. Homocysteine removal is therefore important to prevent its accumulation in plasma and tissues. The methionine cycle is strictly associated with the provision of methylene tetrahydrofolate via the one-carbon methyl cycle and is a controlling step for numerous methylation reactions (7). The one-carbon methyl cycle is relevant in several physiological and pathological conditions, such as nutrition, growth, aging, and metabolic diseases (8–10). Methionine is an essential amino acid and therefore also important for protein synthesis. It is abundant in meat proteins and a precursor of both homocysteine and glutathione (6). Homocysteine is an amino acid not present in the proteins but is involved in oxidative processes that can modify proteins and mediate a number of adverse reactions, including vascular damage (5, 11). The mechanisms whereby homocysteine concentration is elevated in hypertension are not fully understood. Homocysteine can accumulate in blood because of increased production, decreased removal, or both. Whether any of these metabolic steps is altered in subjects with essential hypertension has not yet been investigated. Hypertensive subjects are often insulin-resistant in respect to glucose utilization (12). Insulin in turn affects some methionine metabolic steps also in vivo. We have previously reported that euglycemic hyperinsulinemia stimulates methionine transmethylation, as well as homocysteine trans-sulfuration and clearance, in healthy subjects (13). This insulin effect was impaired in an insulin-resistant condition such as type 2 diabetes mellitus (14). Therefore, one hypothesis is that in hypertensive subjects insulin resistance is extended to some methionine/homocysteine metabolic steps, possibly representing a cause of hyperhomocysteinemia. The methionine–homocysteine cycle can be investigated in vivo with stable isotope techniques (15). Therefore, the aim of this study was to measure methionine and homocysteine kinetics in human subjects affected by essential hypertension, both in the postabsorptive (p.a) state and after the insulin stimulus. Subjects and Methods Subjects Eight male subjects with essential hypertension and six male normotensive controls were enrolled. In the hypertensive subjects, age, body weight (BW), estimated lean body mass (LBM) (16), and body mass index (BMI) were not different from those of the normotensive controls (Table 1). Subjects had no history of diabetes or impaired glucose tolerance, and their fasting plasma glucose concentration and hemoglobin A1c (HbA1c) (data not reported) were normal. All subjects were adapted to a standard mixed, balanced diet containing ~50%–55% calories as carbohydrates, ~15%–20% as proteins, and ~30% as lipids. No specific dietary assessment was performed. However, no patient reported any major dietary deviation from usual, after a brief inquiry at the enrollment visit in the outpatient unit. All but one of the eight hypertensive subjects were treated with one or more hypotensive medications [five with angiotensin-converting enzyme inhibitors (ACE-Is), of whom two were also treated with diuretics and two with β-blockers; two with angiotensin receptor blockers (ARBs); one with calcium antagonists]. One subjects was not being treated yet because his hypertension was recently discovered. Two hypertensive and three normotensive subjects were also treated for hypercholesterolemia (with statins). All drugs were suspended the night before the study day. Table 1. Subjects’ Clinical and Biochemical Characteristics   Hypertensive Subjects (N = 8)  Normotensive Subjects (N = 6)  P  Age, y  59 ± 5  48 ± 4  0.13  Weight, kg  87.8 ± 5.1  79.7 ± 3.5  0.31  BMI, kg  27.6 ± 1.0  26.2 ± 1.1  0.39  LBM, kg  56.2 ± 2.6  54.8 ± 1.6  0.27  % LBM  68.2 ± 1.3  69.1 ± 1.7  0.54  Fasting glucose, mmol/L  4.9 ± 0.1  4.9 ± 0.1  0.69  Fasting insulin, pmol/L  53 ± 10  33 ± 8  0.16  HOMA index  1.98 ± 0.38  1.20 ± 0.32  0.19    Hypertensive Subjects (N = 8)  Normotensive Subjects (N = 6)  P  Age, y  59 ± 5  48 ± 4  0.13  Weight, kg  87.8 ± 5.1  79.7 ± 3.5  0.31  BMI, kg  27.6 ± 1.0  26.2 ± 1.1  0.39  LBM, kg  56.2 ± 2.6  54.8 ± 1.6  0.27  % LBM  68.2 ± 1.3  69.1 ± 1.7  0.54  Fasting glucose, mmol/L  4.9 ± 0.1  4.9 ± 0.1  0.69  Fasting insulin, pmol/L  53 ± 10  33 ± 8  0.16  HOMA index  1.98 ± 0.38  1.20 ± 0.32  0.19  Values are expressed as means ± standard error. View Large The aims of the protocol were explained in detail to each subject, and each subject signed an informed consent form before the study. The protocol was approved by the Ethics Committee of the Medical Faculty at the University of Padova, Italy, and was performed according to the Helsinki Declaration (as revised in 1983). The studies began in 2010. Experimental design The study was performed as described in detail elsewhere (13). Briefly, subjects were admitted to the Metabolism Unit at 7 am on the study day after an overnight fast. Two 18-gauge polyethylene catheters were placed intravenously, one in an antecubital vein of the right arm, another in a contralateral wrist vein in a retrograde fashion, the hand being maintained in a Plexiglas box heated at 55°C, for arterialized venous blood sampling. After preinfusion blood and expired air sampling, a primed, continuous infusion of l-[methyl-2H3,1-13C]methionine (Isotech, Miamisburg, OH; isotope purity 99% for both the methyl-2H3 and the 13C isotopes) was started. The priming dose was equivalent to 45 times the constant infusion rate per minute, or ~0.03 µmol/kg BW per minute. Blood samples were initially drawn every 30 minutes for 120 minutes, to assess achievement of the steady state (data not shown). Thereafter, between the 120th and 180th minute, four blood and expired air samples were collected 20 minutes apart, for measurements of isotope enrichments, plasma substrate and hormone concentrations, and 13CO2 enrichment in the expired air. Between the 90th and 120th minute, total CO2 production rate was also determined by a calorimeter (Deltatrac; Datex Italia, Milan, Italy). Thereafter, a euglycemic, hyperinsulinemic clamp was started. Regular insulin (Humulin R; Eli Lilly, Indianapolis, IN) was infused at the rate of 1.9 mU/kg BW per minute, for 180 minutes. In the first 10 minutes, the insulin infusion was doubled to rapidly achieve stable plasma insulin concentrations. Plasma glucose was maintained between 85 and 90 mg/dL by a variable exogenous 20% dextrose infusion. The source of the infused dextrose was maize starch. After 120 minutes from the start of insulin, blood and expired air samples were again taken every 20 minutes up to the 180th minute, for the measurements at the steady state in the hyperinsulinemic condition. Between the 160th and 180th minute, the rate of total CO2 production was again determined by calorimetry. Because the maize-derived starch from which the infused glucose is produced may be naturally enriched in 13C above the subjects’ basal enrichment, and this enrichment may affect the expired total 13CO2 because of oxidation of the infused glucose, control studies were separately performed, at least 1 month apart from the l-[methyl-2H3,1-13C]methionine kinetic study, in two hypertensive and two normotensive subjects under the same euglycemic, hyperinsulinemic clamp conditions. From these studies, the contribution of the rate of infused glucose to 13CO2 expiration could be calculated and used to correct the total 13CO2 production in the studies performed with the [methyl-2H3,1-13C]methionine tracer infusion. The mean factor calculated in the two hypertensive subjects was 0.021 µmol/kg LBM per hour of expired 13CO2 per g/kg LBM per hour of infused glucose; in the two normotensive controls this factor was 0.024 µmol/kg LBM per hour. In these four subjects, who participated also in the control studies, individually determined correction factors were applied to each subject. In the remaining 10 subjects (6 hypertensive, 4 normotensive) the mean of the two correction factors (0.0225) was used. Analytical measurements Plasma enrichments of [methyl-2H3,1-13C] (M+4) and of [1-13C] (M+1) methionine were measured by gas chromatography–mass spectrometry (model 5790; Agilent, Palo Alto, CA), as tert-butyldimethylsilyl derivatives and electron impact ionization (17). The monitored fragments have mass/charge (m/z) ratios of 324/320 and 321/320, respectively. Enrichment was expressed as a tracer/tracer ratio (TTR) (18). Plasma homocysteine concentration was determined by fluorescence polarized immunoassay (Abbott, Abbott Park, IL). Plasma homocysteine enrichment was measured with modifications of the method reported previously (19). Major modifications were the extraction method, the reducing agent used (dithiothreitol rather than mercaptoethanol), and the resulting derivatized compound (a tert-silyl derivative rather than the previous di-silyl derivative). In the electron impact mode, the ion fragments monitored were 420 m/z for native homocysteine and 421 m/z for [M+1] homocysteine. The intraassay coefficient of variation was <2%. The expired 13CO2 enrichment was determined by gas chromatography–isotope ratio mass spectrometer (Delta Plus GC-C-IRMS; Thermoquest, Bremen, Germany). Plasma insulin concentration was measured by radioimmunoassay, as referenced elsewhere (13). The concentrations of plasma vitamin B12 (20) and folate (using the Folate III Elecsys assay; Roche Diagnostics GmbH, Mannheim, DE) were determined by ELISA methods in chemiluminescence using ruthenium. The US folate reference values (21) were adjusted to European standards. Plasma vitamin B6 concentration was determined using an enzyme immune assay (LSBio; LifeSpan BioSciences, Inc., Seattle, WA). Calculations The rate of insulin-mediated glucose disposal was calculated as the mean value of the last 60 minutes of the euglycemic, hyperinsulinemic clamp. The insulin resistance homeostatic model assessment (HOMA) index was calculated according to Matthews et al. (22). The average of plasma methionine enrichments, determined in four samples of each of the two steady-state periods (i.e., in the last 60 minutes of the basal, p.a. state and in the last 60 minutes of the insulin clamp) were used for the kinetic calculations. Whole-body plasma and methionine kinetics were calculated according to the model of Storch et al. (15). In this model, rates of methionine transmethylation, homocysteine remethylation, and trans-sulfuration can be determined. In addition, the methionine/homocysteine kinetic data were calculated also with plasma homocysteine enrichment used as precursor pool, as reported previously (13). Statistical analysis All data are expressed as means ± standard errors (SEs). The kinetic data were expressed in kilograms of LBM. The statistical comparisons between the clamp and the basal data were performed with two-way analyses of variance (ANOVAs) for repeated measurements. Student t tests for unpaired data were used to compare single data sets between the two steady-state groups. Linear regression analysis was used to test correlations between pairs of variables. Statistica® Software (version 10; TIBCO Software Inc., Palo Alto, CA) was used. A P value of <0.05 was considered statistically significant. Results Postabsorptive state In hypertensive subjects, plasma homocysteine concentrations were ~30% greater (13.6 ± 1.1 µmol/L) than in normotensive subjects (10.3 ± 0.6 µmol/L) (P < 0.04), whereas plasma methionine concentrations were not different between the two groups (hypertensive, 21.6 ± 1.7 µmol/L; normotensive, 23.6 ± 1.1 µmol/L). In the hypertensive subjects, p.a. plasma vitamin B12 (402 ± 40 ng/L) and folate (6.2 ± 1.2 µg/L) concentrations were not different from values in normotensive subjects (353 ± 63 ng/L and 5.9 ± 0.9 µg/L, respectively). However, in the hypertensive subjects, p.a. plasma vitamin B6 concentrations (12.2 ± 2.5 ng/mL) were below the manufacturer’s reported lower normal limit (20 ng/mL) and also significantly lower (P < 0.045) than those of normotensive controls (23.9 ± 5.3 ng/mL). In the hypertensive subjects, the HOMA index was not significantly different from values of the normotensive subjects (Table 1). In Table 2, the data for methionine plasma enrichment and expired 13CO2 are reported. Table 2. Plasma Isotope Enrichments, Expressed as TTR, of [methyl-2H3,1-13C]Methionine and of [1-13C]Methionine and the Rates of 13CO2 Expiration in the Basal and the Clamp Periods, for Hypertensive and Normotensive Subjects   [methyl-2H3,1-13C]Methionine TTR  [1-13C]Methionine TTR  Expired 13CO2, Corrected (µmol/kg LBM per hour)  Hypertensive subjects (n = 8)         Basal  0.103 ± 0.004  0.019 ± 0.007  22.54 ± 2.33   Clamp  0.144 ± 0.012a  0.026 ± 0.005a  44.59 ± 2.58a  Normotensive subjects (n = 6)         Basal  0.095 ± 0.007  0.012 ± 0.002  21.73 ± 5.19   Clamp  0.132 ± 0.014a  0.019 ± 0.002a  66.47 ± 7.19a    [methyl-2H3,1-13C]Methionine TTR  [1-13C]Methionine TTR  Expired 13CO2, Corrected (µmol/kg LBM per hour)  Hypertensive subjects (n = 8)         Basal  0.103 ± 0.004  0.019 ± 0.007  22.54 ± 2.33   Clamp  0.144 ± 0.012a  0.026 ± 0.005a  44.59 ± 2.58a  Normotensive subjects (n = 6)         Basal  0.095 ± 0.007  0.012 ± 0.002  21.73 ± 5.19   Clamp  0.132 ± 0.014a  0.019 ± 0.002a  66.47 ± 7.19a  Clamp data for 13CO2 expiration have been corrected for the contribution by the exogenously infused glucose to total 13CO2 expiration (see text). Data are expressed as means ± SE. a P < 0.001, clamp vs basal. View Large In the hypertensive subjects, methionine rate of appearance (Ra), determined by either 2H3-methyl or the 13C-methionine TTR, was not different from that of normotensive controls (Table 2). In contrast, methionine disposal to protein synthesis was significantly lower in the hypertensive than in normotensive subjects (Table 3). Methionine transmethylation, homocysteine trans-sulfuration, remethylation, and clearance were also not different from the corresponding values of normotensive subjects (Table 3; Fig. 1). Table 3. Methionine Ra, Calculated With Either the [methyl-2H3,1-13C] TTR or the [1-13C-carbon] TTR, Transmethylation, Homocysteine Remethylation, and Methionine Disposal to Protein Synthesis, in the Basal and Clamp Periods, in Hypertensive and Normotensive Subjects   Methionine Ra [methyl-2H3, 1-13C]  Methionine Ra [1-13C-carbon]  Methionine Transmethylation  Homocysteine Remethylation  Methionine to Protein Synthesis  Hypertensive subjects (n = 8)             Basal  21.53 ± 0.86  18.19 ± 1.17  5.81 ± 1.05  3.34 ± 0.97  15.71 ± 1.07a   Clamp  15.29 ± 1.36b  12.42 ± 0.93b  6.94 ± 0.85  2.87 ± 0.85  7.72 ± 0.65b  Normotensive subjects (n = 6)             Basal  25.16 ± 1.79  22.14 ± 1.40  6.15 ± 1.36  3.03 ± 0.85  19.02 ± 1.23   Clamp  18.08 ± 2.14b  15.30 ± 1.54b  9.35 ± 1.19b  2.77 ± 0.62  7.48 ± 1.31b    Methionine Ra [methyl-2H3, 1-13C]  Methionine Ra [1-13C-carbon]  Methionine Transmethylation  Homocysteine Remethylation  Methionine to Protein Synthesis  Hypertensive subjects (n = 8)             Basal  21.53 ± 0.86  18.19 ± 1.17  5.81 ± 1.05  3.34 ± 0.97  15.71 ± 1.07a   Clamp  15.29 ± 1.36b  12.42 ± 0.93b  6.94 ± 0.85  2.87 ± 0.85  7.72 ± 0.65b  Normotensive subjects (n = 6)             Basal  25.16 ± 1.79  22.14 ± 1.40  6.15 ± 1.36  3.03 ± 0.85  19.02 ± 1.23   Clamp  18.08 ± 2.14b  15.30 ± 1.54b  9.35 ± 1.19b  2.77 ± 0.62  7.48 ± 1.31b  All rates are expressed as µmol/kg LBM per hour. The data are reported as means ± SE. a P < 0.01, hypertensive vs normotensive, by two-way ANOVA for repeated measurements (interaction effect). b P < 0.025 or less, clamp vs basal period within each group (by the two-tailed Student t test for paired data). View Large Figure 1. View largeDownload slide Rates of homocysteine trans-sulfuration (in µmol/kg LBM per hour) in subjects with essential hypertension (EH) and in normotensive controls (NT) in basal state and after the hyperinsulinemic, euglycemic clamp. *Different from normotensive subjects, NT, P < 0.003, interaction effect. #Different within groups, clamp from basal (i.e., treatment effect), P < 0.0001. The statistical analysis was performed by the two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. Figure 1. View largeDownload slide Rates of homocysteine trans-sulfuration (in µmol/kg LBM per hour) in subjects with essential hypertension (EH) and in normotensive controls (NT) in basal state and after the hyperinsulinemic, euglycemic clamp. *Different from normotensive subjects, NT, P < 0.003, interaction effect. #Different within groups, clamp from basal (i.e., treatment effect), P < 0.0001. The statistical analysis was performed by the two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. Total CO2 production in the hypertensive subjects (199 ± 6 mL/kg LBM per hour) was lower (P = 0.032) than that of the normotensive subjects (220 ± 6 mL/kg LBM per hour). Euglycemic hyperinsulinemia Insulin was raised to similar concentrations in both hypertensive (from 51 ± 10 to 1166 ± 150 pmol/L) and normotensive subjects (from 33 ± 8 to 992 ± 97 pmol/L). Euglycemia was maintained in both groups (between 4.7 and 5 mmol/L). In the hypertensive subjects, insulin-mediated glucose disposal (460 ± 44 mg/kg LBM per hour) was lower, by ~40%, than that in normotensive subjects (792 ± 67 mg/kg LBM per hour, P < 0.0005). Homocysteine concentration did not change in either the hypertensive or the normotensive group after hyperinsulinemia (to 13.2 ± 0.8 µmol/L and to 10.2 ± 0.8 µmol/L, respectively), whereas methionine concentration was markedly decreased (by ~40%), although to a similar extent in both groups (to 13.3 ± 1 µmol/L and 14.9 ± 2.2 µmol/L, respectively). Plasma concentrations of other amino acids were suppressed, although to a similar extent in both groups (data not shown). Methionine Ra, determined with either 2H3-methyl or 13C-methionine enrichment, was suppressed as compared with baseline to similar absolute values in both groups, by approximately −20% in hypertensive subjects and by −25% to −30% in normotensive subjects (Table 3). Transmethylation was significantly stimulated in the normotensive subjects, whereas in the hypertensive subjects its stimulation was blunted and insignificant (Table 3). Remethylation remained substantially unchanged in both groups (Table 3). Insulin stimulated homocysteine trans-sulfuration in both groups but to a lower extent (by −50%, P < 0.005) and to lower absolute values (~40% less, P < 0.01) in hypertensive than in the normotensive subjects (Fig. 1). When all data were analyzed together, a significant positive correlation was found between insulin-mediated glucose disposal (the M value) and the relative increase of trans-sulfuration compared with baseline (r = 0.77, P < 0.0015) (Fig. 2). Figure 2. View largeDownload slide Relationship between the insulin-mediated glucose disposal (M value) and changes (Δ) in trans-sulfuration during the clamp vs the basal values for all subjects together. Black diamonds indicate subjects with essential hypertension; white diamonds indicate normotensive controls. Figure 2. View largeDownload slide Relationship between the insulin-mediated glucose disposal (M value) and changes (Δ) in trans-sulfuration during the clamp vs the basal values for all subjects together. Black diamonds indicate subjects with essential hypertension; white diamonds indicate normotensive controls. Protein synthesis was suppressed to similar absolute values in both groups but by less (P < 0.015) in hypertensive subjects (by −7.99 ± 0.95 µmol/kg LBM per hour) than in normotensive subjects (by −11.54 ± 0.26 µmol/kg LBM per hour) (Table 3). Total homocysteine clearance was stimulated by insulin in both groups (P < 0.0015 vs baseline), although to a lower, insignificant extent (P = 0.08) in the hypertensive subjects (Fig. 3). Homocysteine clearance through trans-sulfuration was significantly lower (by ~50%) and was less stimulated (by −65%, P < 0.002) in hypertensive subjects than in normotensive subjects. Data calculation with plasma homocysteine enrichment used as a precursor pool (13) yielded substantially the same results (data not reported). Figure 3. View largeDownload slide Rates of homocysteine clearance (in L/kg LBM per hour) in subjects with essential hypertension (EH, dotted bars) and normotensive controls (NT, white bars) in basal state and after the hyperinsulinemic, euglycemic clamp. Clearance is reported as either total, through trans-sulfuration (TS), or through remethylation (RM). *Different from normotensive subjects, P = 0.005 as group effect. #Different within the groups, clamp from basal (i.e., treatment effect), P ≤ 0.001. The statistical analysis was performed by two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. Figure 3. View largeDownload slide Rates of homocysteine clearance (in L/kg LBM per hour) in subjects with essential hypertension (EH, dotted bars) and normotensive controls (NT, white bars) in basal state and after the hyperinsulinemic, euglycemic clamp. Clearance is reported as either total, through trans-sulfuration (TS), or through remethylation (RM). *Different from normotensive subjects, P = 0.005 as group effect. #Different within the groups, clamp from basal (i.e., treatment effect), P ≤ 0.001. The statistical analysis was performed by two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. During hyperinsulinemia, total CO2 production was increased in both groups. However, in hypertensive subjects total CO2 production (225 ± 7 mL/kg LBM per hour) remained lower (P < 0.01) than in normotensive subjects (265 ± 11 mL/kg LBM per hour). When the CO2 data were analyzed by ANOVA, total CO2 production in the hypertensive subjects was significantly lower (P < 0.01, group effect) than in normotensive controls. Discussion This study shows that in subjects with essential hypertension and hyperhomocysteinemia, homocysteine trans-sulfuration is lower than in controls, both in the p.a. and in the insulin-stimulated state. In the hypertensive subjects, the stimulation by insulin of trans-sulfuration was about half that of normotensive subjects, and their insulin-mediated glucose disposal was markedly reduced. A positive correlation was found between glucose disposal (the M value) and the increase of trans-sulfuration after hyperinsulinemia (Fig. 2). This observation suggests that in hypertension the concept of insulin resistance should be extended beyond glucose metabolism to include homocysteine catabolism. These data show defects in the methionine/homocysteine cycle in essential hypertension. Because trans-sulfuration is an irreversible catabolic step of homocysteine, its impairment in hypertension, particularly during hyperinsulinemia, may be one cause for the increased homocysteine concentrations observed in this condition. Furthermore, the combined decrease of trans-sulfuration and of glucose oxidation (as indicated by the lower CO2 production after hyperinsulinemia) indicates an overall impairment in insulin-mediated oxidative pathways in essential hypertension. However, it cannot be excluded that, in hypertensive subjects, the direct relationship between the increase in trans-sulfuration and insulin sensitivity was due just to insulin sensitivity and not to hypertension itself. Additional studies in hypertensive subjects with normal insulin sensitivity may be needed to investigate this question. The reduction of both insulin-mediated glucose disposal and trans-sulfuration in the hypertensive subjects, as well as the tight correlation between these two parameters, suggests either a common step for insulin resistance involving both glucose and methionine metabolism or multiple steps being simultaneously, differently, and specifically involved in hypertension. As regards glucose, its utilization in response to hyperinsulinemia takes place predominantly in skeletal muscle, through a variety of mechanisms, such as increased blood flow, recruitment of glucose transporters on the plasma membrane, and tyrosine kinase activation at the insulin receptor level (23), ultimately resulting in the stimulation of both oxidative and nonoxidative glucose disposal. In this respect, in hypertension multiple defects in insulin-mediated glucose metabolism have been reported (12, 24). Conversely, homocysteine trans-sulfuration takes place predominantly in the liver (25) and, to a lower extent, in other tissues or organs such as the gastrointestinal tract (26) and is mediated by the heme-containing enzyme cystathionine β-synthase (CBS). Decreased CBS expression, deficiencies of the vitamin B6–dependent cofactor pyridoxal phosphate, changes in the redox state of CBS, the allosteric regulation of CBS by adenosylmethionine (27), all can modify CBS activity and homocysteine trans-sulfuration. At first glance, all these steps or mechanisms are different from those regulating glucose utilization, with the exception of the redox state, which can also affect glucose oxidation at the level of pyruvate dehydrogenase (28). Thus, although there was a direct correlation between glucose disposal and the increase of trans-sulfuration after hyperinsulinemia (Fig. 2), the sites and the causes of such an effect are probably not the same. These complex issues therefore warrant further investigation. Another feature of homocysteine disposal, clearance (a parameter related to substrate concentration), was impaired in the hypertensive subjects, specifically in the insulin-stimulated state and restricted to the trans-sulfuration pathway (Fig. 3). In contrast, remethylation-mediated homocysteine clearance was unchanged by insulin and was not different between the groups. However, such a difference in homocysteine trans-sulfuration clearance between the groups might have been overestimated, because clearance is inversely (and not linearly) related to concentration, as previously reported for leucine (29). Nevertheless, although in the basal state there was a ~30% difference in homocysteine concentration between normotensive and hypertensive subjects that can account for the difference in trans-sulfuration-related clearance (by ~30% too), after the clamp the differences between groups were ~50% or greater, despite unchanged homocysteine concentrations in both groups. This observation suggests a specific defect in homocysteine removal from plasma through trans-sulfuration in essential hypertension. The reasons for the unchanged homocysteine concentrations after hyperinsulinemia, at variance with the reduction of methionine and of most amino acids, as previously reported (13, 14), are unknown. It is possible that the simultaneous stimulation of transmethylation (which increases homocysteine production) and of trans-sulfuration (which increases homocysteine disposal) by insulin resulted in no net change in homocysteine concentrations. In an in vitro cell model, exposure to insulin increased homocysteine concentration and reduced trans-sulfuration (30), in contrast with the present findings. How these previous in vitro data can be compared with the in vivo ones of the current study cannot be determined. Indeed, in in vivo studies several metabolic changes occur simultaneously, and all body tissues are analyzed together, leading to experimental conditions quite different from those of an in vitro setting. The methionine/homocysteine cycle is vitamin dependent. Whereas remethylation is sensitive to the methyl supply by vitamin B12 and folate (6), trans-sulfuration may be moderately sensitive to vitamin B6 concentrations, leading to an increased fractional synthesis rate of cystathionine (31, 32). In our study, no differences between the hypertensive and the normotensive subjects were detected in plasma vitamin B12 and folate concentrations, and remethylation rates were also similar. In contrast, vitamin B6 was marginally lower in the hypertensive subjects. No specific dietary differences between the groups that could justify the lower vitamin B6 values in the hypertensive subjects were apparent. Nevertheless, trans-sulfuration in the hypertensive subjects was decreased (i.e., not increased), as opposed to what could be expected from previously reported results involving mild vitamin B6 deficiency (32). Thus, either moderate changes in vitamin B6 concentrations do not affect trans-sulfuration, or hypertension per se is the major determinant of the observed reduced rates of trans-sulfuration also in response to insulin. Between ~75% and ~80% of whole body methionine uptake is used for protein synthesis (Table 2) (33), and the remainder is used for catabolism after conversion to homocysteine. In our hypertensive subjects, p.a. methionine disposal to whole body protein synthesis was decreased. The data on the effects of hypertension on whole body protein turnover are scarce. We have previously reported normal leucine turnover, increased fibrinogen production, normal whole body amino acid disposal, and normal insulin-mediated suppression of protein degradation (34). It may be possible that in hypertension the synthesis of specific proteins in increased, whereas that of others is decreased. The clinical and metabolic significance of these findings warrants investigation. Notably, protein synthesis was suppressed, and to the same absolute value, in both groups after systemic hyperinsulinemia (Table 3), in agreement with the literature [see reference (35) for a review]. Such a “paradoxical” effect is probably due to the concomitant decrease in amino acid concentrations, as commonly observed (13). However, absolute rates of protein synthesis were no longer different between the two groups, because in the hypertensive subjects the relative suppression compared with baseline was lower than that in normotensive subjects. This observation might unveil a sort of insulin resistance to the suppression of whole body protein synthesis in hypertension, which might have favorable significance (i.e., to maintain protein synthesis). The hypertensive subjects were treated with various hypotensive medications, mostly with ACE-Is and ARBs. The effects of these drugs on the observed findings should be discussed. Treating subjects with essential hypertension with ACE-Is decreased plasma homocysteine concentration (36), possibly by improving endothelial and renal function. β-Blockers can decrease homocysteine concentration too (36). As possible mechanisms, β-adrenergic receptor stimulation of cultures of astrocytes increased the secretion of homocysteinic acid, whereas atenolol reduced it (37). Whatever the mechanisms could be, it is likely that if untreated our subjects would have exhibited homocysteine concentrations greater than those measured here. Under the hypothesis of decreased trans-sulfuration as one possible mechanism of increased homocysteine, it is also possible that ACE-Is or ARBs decrease this metabolic step too. Additional studies are needed to directly test this hypothesis. Conversely, thiazide diuretics increased homocysteine concentration (37). However, only two of our subjects were treated with these medications, so the impact of the overall effect here reported, if any, should be minor. Thus study was performed in male subjects. Because some (albeit minor) sex-related differences in methionine metabolism have been previously reported (38), our data should be confirmed in female subjects. Conclusions We report here that homocysteine disposal through trans-sulfuration is reduced in essential hypertension, in both p.a. and insulin-stimulated states. This observation provides a possible explanation for the increased homocysteine concentration often observed in this condition. Such a defect seems to be part of the insulin resistance syndrome often associated with essential hypertension. Abbreviations: ACE-I angiotensin-converting enzyme inhibitor ANOVA analysis of variance ARB angiotensin receptor blocker BMI body mass index BW body weight CBS cystathionine β-synthase HbA1c hemoglobin A1c HOMA homeostatic model assessment LBM lean body mass m/z mass/charge p.a. postabsorptive Ra rate of appearance SE standard error TTR tracer/tracer ratio. Acknowledgments We thank Dr. Carlo Artusi of the Central Laboratory of the Padova University Hospital for the analyses of plasma homocysteine, vitamin B12, and folates, and Dr. Elisabetta Iori of the Department of Medicine, Metabolism Unit, for the analyses of plasma vitamin B6. Financial Support: This work was supported by a Fondo per gli Investimenti della Ricerca di Base (FIRB) Research Project (years 2002–2003), titled Molecular Mechanisms in the Pathogenesis and Progression of Diabetic Nephropathy and of Kidney Ageing—Subproject: Homocysteine and Arginine. Author Contributions: P.T. designed the study, recruited the subjects, contributed to the in vivo experiments, collected and analyzed the data, and wrote the manuscript. D.C. recruited the subjects and contributed to the experiments. M.V. contributed to data collection and analysis and reviewed the manuscript. L.P. contributed to data collection and analysis. A.C. recruited the subjects and contributed to the experiments. E.K. contributed to the experiments and to data collection and reviewed the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Guba SC, Fink LM, Fonseca V. Hyperhomocysteinemia. 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Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship With Insulin Resistance

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-01076
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Abstract

Abstract Context Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown. Objective To investigate whole body methionine and homocysteine kinetics and the effects of insulin in essential hypertension. Design and Setting Eight hypertensive male subjects and six male normotensive controls were infused with l-[methyl-2H3,1-13C]methionine for 6 hours. In the last 3 hours a euglycemic, hyperinsulinemic clamp was performed. Steady-state methionine and homocysteine kinetics were determined in postabsorptive and hyperinsulinemic conditions. Results Postabsorptive hypertensive subjects had elevated homocysteine concentrations (+30%, P = 0.035) and slightly (by 15% to 20%) but insignificantly lower methionine rates of appearance (Ras) (P = 0.07 to P = 0.05) and utilization for protein synthesis (P = 0.06) than postabsorptive normotensive controls. Hyperinsulinemia suppressed methionine Ra and protein synthesis, whereas it increased homocysteine trans-sulfuration, clearance, and methionine transmethylation (the latter only in the normotensive subjects). However, in the hypertensive subjects trans-sulfuration was significantly lower (P < 0.05) and increased ~50% less [by +1.59 ± 0.34 vs +3.45 ± 0.52 µmol/kg lean body mass (LBM) per hour, P < 0.005] than in normotensive controls. Homocysteine clearance through trans-sulfuration was ~50% lower in hypertensive than in normotensive subjects (P < 0.005). In the hypertensive subjects, insulin-mediated glucose disposal was ~45% lower (460 ± 44 vs 792 ± 67 mg/kg LBM per hour, P < 0.0005) than in normotensive controls and was positively correlated with the increase of trans-sulfuration (P < 0.0015). Conclusions In subjects with essential hypertension, hyperhomocysteinemia is associated with decreased homocysteine trans-sulfuration and probably represents a feature of insulin resistance. Homocysteine is an independent vascular risk factor (1, 2), is often elevated in arterial hypertension (3), and has been proposed as an independent risk factor for hypertension (4). Hyperhomocysteinemia has been associated with tissue and organ toxicity through a variety of possible mechanisms (5). Homocysteine is produced within the methionine cycle from S-adenosyl homocysteine demethylation (6) and is removed through either remethylation to methionine or trans-sulfuration to cystathionine and subsequent oxidation. Homocysteine removal is therefore important to prevent its accumulation in plasma and tissues. The methionine cycle is strictly associated with the provision of methylene tetrahydrofolate via the one-carbon methyl cycle and is a controlling step for numerous methylation reactions (7). The one-carbon methyl cycle is relevant in several physiological and pathological conditions, such as nutrition, growth, aging, and metabolic diseases (8–10). Methionine is an essential amino acid and therefore also important for protein synthesis. It is abundant in meat proteins and a precursor of both homocysteine and glutathione (6). Homocysteine is an amino acid not present in the proteins but is involved in oxidative processes that can modify proteins and mediate a number of adverse reactions, including vascular damage (5, 11). The mechanisms whereby homocysteine concentration is elevated in hypertension are not fully understood. Homocysteine can accumulate in blood because of increased production, decreased removal, or both. Whether any of these metabolic steps is altered in subjects with essential hypertension has not yet been investigated. Hypertensive subjects are often insulin-resistant in respect to glucose utilization (12). Insulin in turn affects some methionine metabolic steps also in vivo. We have previously reported that euglycemic hyperinsulinemia stimulates methionine transmethylation, as well as homocysteine trans-sulfuration and clearance, in healthy subjects (13). This insulin effect was impaired in an insulin-resistant condition such as type 2 diabetes mellitus (14). Therefore, one hypothesis is that in hypertensive subjects insulin resistance is extended to some methionine/homocysteine metabolic steps, possibly representing a cause of hyperhomocysteinemia. The methionine–homocysteine cycle can be investigated in vivo with stable isotope techniques (15). Therefore, the aim of this study was to measure methionine and homocysteine kinetics in human subjects affected by essential hypertension, both in the postabsorptive (p.a) state and after the insulin stimulus. Subjects and Methods Subjects Eight male subjects with essential hypertension and six male normotensive controls were enrolled. In the hypertensive subjects, age, body weight (BW), estimated lean body mass (LBM) (16), and body mass index (BMI) were not different from those of the normotensive controls (Table 1). Subjects had no history of diabetes or impaired glucose tolerance, and their fasting plasma glucose concentration and hemoglobin A1c (HbA1c) (data not reported) were normal. All subjects were adapted to a standard mixed, balanced diet containing ~50%–55% calories as carbohydrates, ~15%–20% as proteins, and ~30% as lipids. No specific dietary assessment was performed. However, no patient reported any major dietary deviation from usual, after a brief inquiry at the enrollment visit in the outpatient unit. All but one of the eight hypertensive subjects were treated with one or more hypotensive medications [five with angiotensin-converting enzyme inhibitors (ACE-Is), of whom two were also treated with diuretics and two with β-blockers; two with angiotensin receptor blockers (ARBs); one with calcium antagonists]. One subjects was not being treated yet because his hypertension was recently discovered. Two hypertensive and three normotensive subjects were also treated for hypercholesterolemia (with statins). All drugs were suspended the night before the study day. Table 1. Subjects’ Clinical and Biochemical Characteristics   Hypertensive Subjects (N = 8)  Normotensive Subjects (N = 6)  P  Age, y  59 ± 5  48 ± 4  0.13  Weight, kg  87.8 ± 5.1  79.7 ± 3.5  0.31  BMI, kg  27.6 ± 1.0  26.2 ± 1.1  0.39  LBM, kg  56.2 ± 2.6  54.8 ± 1.6  0.27  % LBM  68.2 ± 1.3  69.1 ± 1.7  0.54  Fasting glucose, mmol/L  4.9 ± 0.1  4.9 ± 0.1  0.69  Fasting insulin, pmol/L  53 ± 10  33 ± 8  0.16  HOMA index  1.98 ± 0.38  1.20 ± 0.32  0.19    Hypertensive Subjects (N = 8)  Normotensive Subjects (N = 6)  P  Age, y  59 ± 5  48 ± 4  0.13  Weight, kg  87.8 ± 5.1  79.7 ± 3.5  0.31  BMI, kg  27.6 ± 1.0  26.2 ± 1.1  0.39  LBM, kg  56.2 ± 2.6  54.8 ± 1.6  0.27  % LBM  68.2 ± 1.3  69.1 ± 1.7  0.54  Fasting glucose, mmol/L  4.9 ± 0.1  4.9 ± 0.1  0.69  Fasting insulin, pmol/L  53 ± 10  33 ± 8  0.16  HOMA index  1.98 ± 0.38  1.20 ± 0.32  0.19  Values are expressed as means ± standard error. View Large The aims of the protocol were explained in detail to each subject, and each subject signed an informed consent form before the study. The protocol was approved by the Ethics Committee of the Medical Faculty at the University of Padova, Italy, and was performed according to the Helsinki Declaration (as revised in 1983). The studies began in 2010. Experimental design The study was performed as described in detail elsewhere (13). Briefly, subjects were admitted to the Metabolism Unit at 7 am on the study day after an overnight fast. Two 18-gauge polyethylene catheters were placed intravenously, one in an antecubital vein of the right arm, another in a contralateral wrist vein in a retrograde fashion, the hand being maintained in a Plexiglas box heated at 55°C, for arterialized venous blood sampling. After preinfusion blood and expired air sampling, a primed, continuous infusion of l-[methyl-2H3,1-13C]methionine (Isotech, Miamisburg, OH; isotope purity 99% for both the methyl-2H3 and the 13C isotopes) was started. The priming dose was equivalent to 45 times the constant infusion rate per minute, or ~0.03 µmol/kg BW per minute. Blood samples were initially drawn every 30 minutes for 120 minutes, to assess achievement of the steady state (data not shown). Thereafter, between the 120th and 180th minute, four blood and expired air samples were collected 20 minutes apart, for measurements of isotope enrichments, plasma substrate and hormone concentrations, and 13CO2 enrichment in the expired air. Between the 90th and 120th minute, total CO2 production rate was also determined by a calorimeter (Deltatrac; Datex Italia, Milan, Italy). Thereafter, a euglycemic, hyperinsulinemic clamp was started. Regular insulin (Humulin R; Eli Lilly, Indianapolis, IN) was infused at the rate of 1.9 mU/kg BW per minute, for 180 minutes. In the first 10 minutes, the insulin infusion was doubled to rapidly achieve stable plasma insulin concentrations. Plasma glucose was maintained between 85 and 90 mg/dL by a variable exogenous 20% dextrose infusion. The source of the infused dextrose was maize starch. After 120 minutes from the start of insulin, blood and expired air samples were again taken every 20 minutes up to the 180th minute, for the measurements at the steady state in the hyperinsulinemic condition. Between the 160th and 180th minute, the rate of total CO2 production was again determined by calorimetry. Because the maize-derived starch from which the infused glucose is produced may be naturally enriched in 13C above the subjects’ basal enrichment, and this enrichment may affect the expired total 13CO2 because of oxidation of the infused glucose, control studies were separately performed, at least 1 month apart from the l-[methyl-2H3,1-13C]methionine kinetic study, in two hypertensive and two normotensive subjects under the same euglycemic, hyperinsulinemic clamp conditions. From these studies, the contribution of the rate of infused glucose to 13CO2 expiration could be calculated and used to correct the total 13CO2 production in the studies performed with the [methyl-2H3,1-13C]methionine tracer infusion. The mean factor calculated in the two hypertensive subjects was 0.021 µmol/kg LBM per hour of expired 13CO2 per g/kg LBM per hour of infused glucose; in the two normotensive controls this factor was 0.024 µmol/kg LBM per hour. In these four subjects, who participated also in the control studies, individually determined correction factors were applied to each subject. In the remaining 10 subjects (6 hypertensive, 4 normotensive) the mean of the two correction factors (0.0225) was used. Analytical measurements Plasma enrichments of [methyl-2H3,1-13C] (M+4) and of [1-13C] (M+1) methionine were measured by gas chromatography–mass spectrometry (model 5790; Agilent, Palo Alto, CA), as tert-butyldimethylsilyl derivatives and electron impact ionization (17). The monitored fragments have mass/charge (m/z) ratios of 324/320 and 321/320, respectively. Enrichment was expressed as a tracer/tracer ratio (TTR) (18). Plasma homocysteine concentration was determined by fluorescence polarized immunoassay (Abbott, Abbott Park, IL). Plasma homocysteine enrichment was measured with modifications of the method reported previously (19). Major modifications were the extraction method, the reducing agent used (dithiothreitol rather than mercaptoethanol), and the resulting derivatized compound (a tert-silyl derivative rather than the previous di-silyl derivative). In the electron impact mode, the ion fragments monitored were 420 m/z for native homocysteine and 421 m/z for [M+1] homocysteine. The intraassay coefficient of variation was <2%. The expired 13CO2 enrichment was determined by gas chromatography–isotope ratio mass spectrometer (Delta Plus GC-C-IRMS; Thermoquest, Bremen, Germany). Plasma insulin concentration was measured by radioimmunoassay, as referenced elsewhere (13). The concentrations of plasma vitamin B12 (20) and folate (using the Folate III Elecsys assay; Roche Diagnostics GmbH, Mannheim, DE) were determined by ELISA methods in chemiluminescence using ruthenium. The US folate reference values (21) were adjusted to European standards. Plasma vitamin B6 concentration was determined using an enzyme immune assay (LSBio; LifeSpan BioSciences, Inc., Seattle, WA). Calculations The rate of insulin-mediated glucose disposal was calculated as the mean value of the last 60 minutes of the euglycemic, hyperinsulinemic clamp. The insulin resistance homeostatic model assessment (HOMA) index was calculated according to Matthews et al. (22). The average of plasma methionine enrichments, determined in four samples of each of the two steady-state periods (i.e., in the last 60 minutes of the basal, p.a. state and in the last 60 minutes of the insulin clamp) were used for the kinetic calculations. Whole-body plasma and methionine kinetics were calculated according to the model of Storch et al. (15). In this model, rates of methionine transmethylation, homocysteine remethylation, and trans-sulfuration can be determined. In addition, the methionine/homocysteine kinetic data were calculated also with plasma homocysteine enrichment used as precursor pool, as reported previously (13). Statistical analysis All data are expressed as means ± standard errors (SEs). The kinetic data were expressed in kilograms of LBM. The statistical comparisons between the clamp and the basal data were performed with two-way analyses of variance (ANOVAs) for repeated measurements. Student t tests for unpaired data were used to compare single data sets between the two steady-state groups. Linear regression analysis was used to test correlations between pairs of variables. Statistica® Software (version 10; TIBCO Software Inc., Palo Alto, CA) was used. A P value of <0.05 was considered statistically significant. Results Postabsorptive state In hypertensive subjects, plasma homocysteine concentrations were ~30% greater (13.6 ± 1.1 µmol/L) than in normotensive subjects (10.3 ± 0.6 µmol/L) (P < 0.04), whereas plasma methionine concentrations were not different between the two groups (hypertensive, 21.6 ± 1.7 µmol/L; normotensive, 23.6 ± 1.1 µmol/L). In the hypertensive subjects, p.a. plasma vitamin B12 (402 ± 40 ng/L) and folate (6.2 ± 1.2 µg/L) concentrations were not different from values in normotensive subjects (353 ± 63 ng/L and 5.9 ± 0.9 µg/L, respectively). However, in the hypertensive subjects, p.a. plasma vitamin B6 concentrations (12.2 ± 2.5 ng/mL) were below the manufacturer’s reported lower normal limit (20 ng/mL) and also significantly lower (P < 0.045) than those of normotensive controls (23.9 ± 5.3 ng/mL). In the hypertensive subjects, the HOMA index was not significantly different from values of the normotensive subjects (Table 1). In Table 2, the data for methionine plasma enrichment and expired 13CO2 are reported. Table 2. Plasma Isotope Enrichments, Expressed as TTR, of [methyl-2H3,1-13C]Methionine and of [1-13C]Methionine and the Rates of 13CO2 Expiration in the Basal and the Clamp Periods, for Hypertensive and Normotensive Subjects   [methyl-2H3,1-13C]Methionine TTR  [1-13C]Methionine TTR  Expired 13CO2, Corrected (µmol/kg LBM per hour)  Hypertensive subjects (n = 8)         Basal  0.103 ± 0.004  0.019 ± 0.007  22.54 ± 2.33   Clamp  0.144 ± 0.012a  0.026 ± 0.005a  44.59 ± 2.58a  Normotensive subjects (n = 6)         Basal  0.095 ± 0.007  0.012 ± 0.002  21.73 ± 5.19   Clamp  0.132 ± 0.014a  0.019 ± 0.002a  66.47 ± 7.19a    [methyl-2H3,1-13C]Methionine TTR  [1-13C]Methionine TTR  Expired 13CO2, Corrected (µmol/kg LBM per hour)  Hypertensive subjects (n = 8)         Basal  0.103 ± 0.004  0.019 ± 0.007  22.54 ± 2.33   Clamp  0.144 ± 0.012a  0.026 ± 0.005a  44.59 ± 2.58a  Normotensive subjects (n = 6)         Basal  0.095 ± 0.007  0.012 ± 0.002  21.73 ± 5.19   Clamp  0.132 ± 0.014a  0.019 ± 0.002a  66.47 ± 7.19a  Clamp data for 13CO2 expiration have been corrected for the contribution by the exogenously infused glucose to total 13CO2 expiration (see text). Data are expressed as means ± SE. a P < 0.001, clamp vs basal. View Large In the hypertensive subjects, methionine rate of appearance (Ra), determined by either 2H3-methyl or the 13C-methionine TTR, was not different from that of normotensive controls (Table 2). In contrast, methionine disposal to protein synthesis was significantly lower in the hypertensive than in normotensive subjects (Table 3). Methionine transmethylation, homocysteine trans-sulfuration, remethylation, and clearance were also not different from the corresponding values of normotensive subjects (Table 3; Fig. 1). Table 3. Methionine Ra, Calculated With Either the [methyl-2H3,1-13C] TTR or the [1-13C-carbon] TTR, Transmethylation, Homocysteine Remethylation, and Methionine Disposal to Protein Synthesis, in the Basal and Clamp Periods, in Hypertensive and Normotensive Subjects   Methionine Ra [methyl-2H3, 1-13C]  Methionine Ra [1-13C-carbon]  Methionine Transmethylation  Homocysteine Remethylation  Methionine to Protein Synthesis  Hypertensive subjects (n = 8)             Basal  21.53 ± 0.86  18.19 ± 1.17  5.81 ± 1.05  3.34 ± 0.97  15.71 ± 1.07a   Clamp  15.29 ± 1.36b  12.42 ± 0.93b  6.94 ± 0.85  2.87 ± 0.85  7.72 ± 0.65b  Normotensive subjects (n = 6)             Basal  25.16 ± 1.79  22.14 ± 1.40  6.15 ± 1.36  3.03 ± 0.85  19.02 ± 1.23   Clamp  18.08 ± 2.14b  15.30 ± 1.54b  9.35 ± 1.19b  2.77 ± 0.62  7.48 ± 1.31b    Methionine Ra [methyl-2H3, 1-13C]  Methionine Ra [1-13C-carbon]  Methionine Transmethylation  Homocysteine Remethylation  Methionine to Protein Synthesis  Hypertensive subjects (n = 8)             Basal  21.53 ± 0.86  18.19 ± 1.17  5.81 ± 1.05  3.34 ± 0.97  15.71 ± 1.07a   Clamp  15.29 ± 1.36b  12.42 ± 0.93b  6.94 ± 0.85  2.87 ± 0.85  7.72 ± 0.65b  Normotensive subjects (n = 6)             Basal  25.16 ± 1.79  22.14 ± 1.40  6.15 ± 1.36  3.03 ± 0.85  19.02 ± 1.23   Clamp  18.08 ± 2.14b  15.30 ± 1.54b  9.35 ± 1.19b  2.77 ± 0.62  7.48 ± 1.31b  All rates are expressed as µmol/kg LBM per hour. The data are reported as means ± SE. a P < 0.01, hypertensive vs normotensive, by two-way ANOVA for repeated measurements (interaction effect). b P < 0.025 or less, clamp vs basal period within each group (by the two-tailed Student t test for paired data). View Large Figure 1. View largeDownload slide Rates of homocysteine trans-sulfuration (in µmol/kg LBM per hour) in subjects with essential hypertension (EH) and in normotensive controls (NT) in basal state and after the hyperinsulinemic, euglycemic clamp. *Different from normotensive subjects, NT, P < 0.003, interaction effect. #Different within groups, clamp from basal (i.e., treatment effect), P < 0.0001. The statistical analysis was performed by the two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. Figure 1. View largeDownload slide Rates of homocysteine trans-sulfuration (in µmol/kg LBM per hour) in subjects with essential hypertension (EH) and in normotensive controls (NT) in basal state and after the hyperinsulinemic, euglycemic clamp. *Different from normotensive subjects, NT, P < 0.003, interaction effect. #Different within groups, clamp from basal (i.e., treatment effect), P < 0.0001. The statistical analysis was performed by the two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. Total CO2 production in the hypertensive subjects (199 ± 6 mL/kg LBM per hour) was lower (P = 0.032) than that of the normotensive subjects (220 ± 6 mL/kg LBM per hour). Euglycemic hyperinsulinemia Insulin was raised to similar concentrations in both hypertensive (from 51 ± 10 to 1166 ± 150 pmol/L) and normotensive subjects (from 33 ± 8 to 992 ± 97 pmol/L). Euglycemia was maintained in both groups (between 4.7 and 5 mmol/L). In the hypertensive subjects, insulin-mediated glucose disposal (460 ± 44 mg/kg LBM per hour) was lower, by ~40%, than that in normotensive subjects (792 ± 67 mg/kg LBM per hour, P < 0.0005). Homocysteine concentration did not change in either the hypertensive or the normotensive group after hyperinsulinemia (to 13.2 ± 0.8 µmol/L and to 10.2 ± 0.8 µmol/L, respectively), whereas methionine concentration was markedly decreased (by ~40%), although to a similar extent in both groups (to 13.3 ± 1 µmol/L and 14.9 ± 2.2 µmol/L, respectively). Plasma concentrations of other amino acids were suppressed, although to a similar extent in both groups (data not shown). Methionine Ra, determined with either 2H3-methyl or 13C-methionine enrichment, was suppressed as compared with baseline to similar absolute values in both groups, by approximately −20% in hypertensive subjects and by −25% to −30% in normotensive subjects (Table 3). Transmethylation was significantly stimulated in the normotensive subjects, whereas in the hypertensive subjects its stimulation was blunted and insignificant (Table 3). Remethylation remained substantially unchanged in both groups (Table 3). Insulin stimulated homocysteine trans-sulfuration in both groups but to a lower extent (by −50%, P < 0.005) and to lower absolute values (~40% less, P < 0.01) in hypertensive than in the normotensive subjects (Fig. 1). When all data were analyzed together, a significant positive correlation was found between insulin-mediated glucose disposal (the M value) and the relative increase of trans-sulfuration compared with baseline (r = 0.77, P < 0.0015) (Fig. 2). Figure 2. View largeDownload slide Relationship between the insulin-mediated glucose disposal (M value) and changes (Δ) in trans-sulfuration during the clamp vs the basal values for all subjects together. Black diamonds indicate subjects with essential hypertension; white diamonds indicate normotensive controls. Figure 2. View largeDownload slide Relationship between the insulin-mediated glucose disposal (M value) and changes (Δ) in trans-sulfuration during the clamp vs the basal values for all subjects together. Black diamonds indicate subjects with essential hypertension; white diamonds indicate normotensive controls. Protein synthesis was suppressed to similar absolute values in both groups but by less (P < 0.015) in hypertensive subjects (by −7.99 ± 0.95 µmol/kg LBM per hour) than in normotensive subjects (by −11.54 ± 0.26 µmol/kg LBM per hour) (Table 3). Total homocysteine clearance was stimulated by insulin in both groups (P < 0.0015 vs baseline), although to a lower, insignificant extent (P = 0.08) in the hypertensive subjects (Fig. 3). Homocysteine clearance through trans-sulfuration was significantly lower (by ~50%) and was less stimulated (by −65%, P < 0.002) in hypertensive subjects than in normotensive subjects. Data calculation with plasma homocysteine enrichment used as a precursor pool (13) yielded substantially the same results (data not reported). Figure 3. View largeDownload slide Rates of homocysteine clearance (in L/kg LBM per hour) in subjects with essential hypertension (EH, dotted bars) and normotensive controls (NT, white bars) in basal state and after the hyperinsulinemic, euglycemic clamp. Clearance is reported as either total, through trans-sulfuration (TS), or through remethylation (RM). *Different from normotensive subjects, P = 0.005 as group effect. #Different within the groups, clamp from basal (i.e., treatment effect), P ≤ 0.001. The statistical analysis was performed by two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. Figure 3. View largeDownload slide Rates of homocysteine clearance (in L/kg LBM per hour) in subjects with essential hypertension (EH, dotted bars) and normotensive controls (NT, white bars) in basal state and after the hyperinsulinemic, euglycemic clamp. Clearance is reported as either total, through trans-sulfuration (TS), or through remethylation (RM). *Different from normotensive subjects, P = 0.005 as group effect. #Different within the groups, clamp from basal (i.e., treatment effect), P ≤ 0.001. The statistical analysis was performed by two-way ANOVA for repeated measures. Values are means ± standard errors of the mean. During hyperinsulinemia, total CO2 production was increased in both groups. However, in hypertensive subjects total CO2 production (225 ± 7 mL/kg LBM per hour) remained lower (P < 0.01) than in normotensive subjects (265 ± 11 mL/kg LBM per hour). When the CO2 data were analyzed by ANOVA, total CO2 production in the hypertensive subjects was significantly lower (P < 0.01, group effect) than in normotensive controls. Discussion This study shows that in subjects with essential hypertension and hyperhomocysteinemia, homocysteine trans-sulfuration is lower than in controls, both in the p.a. and in the insulin-stimulated state. In the hypertensive subjects, the stimulation by insulin of trans-sulfuration was about half that of normotensive subjects, and their insulin-mediated glucose disposal was markedly reduced. A positive correlation was found between glucose disposal (the M value) and the increase of trans-sulfuration after hyperinsulinemia (Fig. 2). This observation suggests that in hypertension the concept of insulin resistance should be extended beyond glucose metabolism to include homocysteine catabolism. These data show defects in the methionine/homocysteine cycle in essential hypertension. Because trans-sulfuration is an irreversible catabolic step of homocysteine, its impairment in hypertension, particularly during hyperinsulinemia, may be one cause for the increased homocysteine concentrations observed in this condition. Furthermore, the combined decrease of trans-sulfuration and of glucose oxidation (as indicated by the lower CO2 production after hyperinsulinemia) indicates an overall impairment in insulin-mediated oxidative pathways in essential hypertension. However, it cannot be excluded that, in hypertensive subjects, the direct relationship between the increase in trans-sulfuration and insulin sensitivity was due just to insulin sensitivity and not to hypertension itself. Additional studies in hypertensive subjects with normal insulin sensitivity may be needed to investigate this question. The reduction of both insulin-mediated glucose disposal and trans-sulfuration in the hypertensive subjects, as well as the tight correlation between these two parameters, suggests either a common step for insulin resistance involving both glucose and methionine metabolism or multiple steps being simultaneously, differently, and specifically involved in hypertension. As regards glucose, its utilization in response to hyperinsulinemia takes place predominantly in skeletal muscle, through a variety of mechanisms, such as increased blood flow, recruitment of glucose transporters on the plasma membrane, and tyrosine kinase activation at the insulin receptor level (23), ultimately resulting in the stimulation of both oxidative and nonoxidative glucose disposal. In this respect, in hypertension multiple defects in insulin-mediated glucose metabolism have been reported (12, 24). Conversely, homocysteine trans-sulfuration takes place predominantly in the liver (25) and, to a lower extent, in other tissues or organs such as the gastrointestinal tract (26) and is mediated by the heme-containing enzyme cystathionine β-synthase (CBS). Decreased CBS expression, deficiencies of the vitamin B6–dependent cofactor pyridoxal phosphate, changes in the redox state of CBS, the allosteric regulation of CBS by adenosylmethionine (27), all can modify CBS activity and homocysteine trans-sulfuration. At first glance, all these steps or mechanisms are different from those regulating glucose utilization, with the exception of the redox state, which can also affect glucose oxidation at the level of pyruvate dehydrogenase (28). Thus, although there was a direct correlation between glucose disposal and the increase of trans-sulfuration after hyperinsulinemia (Fig. 2), the sites and the causes of such an effect are probably not the same. These complex issues therefore warrant further investigation. Another feature of homocysteine disposal, clearance (a parameter related to substrate concentration), was impaired in the hypertensive subjects, specifically in the insulin-stimulated state and restricted to the trans-sulfuration pathway (Fig. 3). In contrast, remethylation-mediated homocysteine clearance was unchanged by insulin and was not different between the groups. However, such a difference in homocysteine trans-sulfuration clearance between the groups might have been overestimated, because clearance is inversely (and not linearly) related to concentration, as previously reported for leucine (29). Nevertheless, although in the basal state there was a ~30% difference in homocysteine concentration between normotensive and hypertensive subjects that can account for the difference in trans-sulfuration-related clearance (by ~30% too), after the clamp the differences between groups were ~50% or greater, despite unchanged homocysteine concentrations in both groups. This observation suggests a specific defect in homocysteine removal from plasma through trans-sulfuration in essential hypertension. The reasons for the unchanged homocysteine concentrations after hyperinsulinemia, at variance with the reduction of methionine and of most amino acids, as previously reported (13, 14), are unknown. It is possible that the simultaneous stimulation of transmethylation (which increases homocysteine production) and of trans-sulfuration (which increases homocysteine disposal) by insulin resulted in no net change in homocysteine concentrations. In an in vitro cell model, exposure to insulin increased homocysteine concentration and reduced trans-sulfuration (30), in contrast with the present findings. How these previous in vitro data can be compared with the in vivo ones of the current study cannot be determined. Indeed, in in vivo studies several metabolic changes occur simultaneously, and all body tissues are analyzed together, leading to experimental conditions quite different from those of an in vitro setting. The methionine/homocysteine cycle is vitamin dependent. Whereas remethylation is sensitive to the methyl supply by vitamin B12 and folate (6), trans-sulfuration may be moderately sensitive to vitamin B6 concentrations, leading to an increased fractional synthesis rate of cystathionine (31, 32). In our study, no differences between the hypertensive and the normotensive subjects were detected in plasma vitamin B12 and folate concentrations, and remethylation rates were also similar. In contrast, vitamin B6 was marginally lower in the hypertensive subjects. No specific dietary differences between the groups that could justify the lower vitamin B6 values in the hypertensive subjects were apparent. Nevertheless, trans-sulfuration in the hypertensive subjects was decreased (i.e., not increased), as opposed to what could be expected from previously reported results involving mild vitamin B6 deficiency (32). Thus, either moderate changes in vitamin B6 concentrations do not affect trans-sulfuration, or hypertension per se is the major determinant of the observed reduced rates of trans-sulfuration also in response to insulin. Between ~75% and ~80% of whole body methionine uptake is used for protein synthesis (Table 2) (33), and the remainder is used for catabolism after conversion to homocysteine. In our hypertensive subjects, p.a. methionine disposal to whole body protein synthesis was decreased. The data on the effects of hypertension on whole body protein turnover are scarce. We have previously reported normal leucine turnover, increased fibrinogen production, normal whole body amino acid disposal, and normal insulin-mediated suppression of protein degradation (34). It may be possible that in hypertension the synthesis of specific proteins in increased, whereas that of others is decreased. The clinical and metabolic significance of these findings warrants investigation. Notably, protein synthesis was suppressed, and to the same absolute value, in both groups after systemic hyperinsulinemia (Table 3), in agreement with the literature [see reference (35) for a review]. Such a “paradoxical” effect is probably due to the concomitant decrease in amino acid concentrations, as commonly observed (13). However, absolute rates of protein synthesis were no longer different between the two groups, because in the hypertensive subjects the relative suppression compared with baseline was lower than that in normotensive subjects. This observation might unveil a sort of insulin resistance to the suppression of whole body protein synthesis in hypertension, which might have favorable significance (i.e., to maintain protein synthesis). The hypertensive subjects were treated with various hypotensive medications, mostly with ACE-Is and ARBs. The effects of these drugs on the observed findings should be discussed. Treating subjects with essential hypertension with ACE-Is decreased plasma homocysteine concentration (36), possibly by improving endothelial and renal function. β-Blockers can decrease homocysteine concentration too (36). As possible mechanisms, β-adrenergic receptor stimulation of cultures of astrocytes increased the secretion of homocysteinic acid, whereas atenolol reduced it (37). Whatever the mechanisms could be, it is likely that if untreated our subjects would have exhibited homocysteine concentrations greater than those measured here. Under the hypothesis of decreased trans-sulfuration as one possible mechanism of increased homocysteine, it is also possible that ACE-Is or ARBs decrease this metabolic step too. Additional studies are needed to directly test this hypothesis. Conversely, thiazide diuretics increased homocysteine concentration (37). However, only two of our subjects were treated with these medications, so the impact of the overall effect here reported, if any, should be minor. Thus study was performed in male subjects. Because some (albeit minor) sex-related differences in methionine metabolism have been previously reported (38), our data should be confirmed in female subjects. Conclusions We report here that homocysteine disposal through trans-sulfuration is reduced in essential hypertension, in both p.a. and insulin-stimulated states. This observation provides a possible explanation for the increased homocysteine concentration often observed in this condition. Such a defect seems to be part of the insulin resistance syndrome often associated with essential hypertension. Abbreviations: ACE-I angiotensin-converting enzyme inhibitor ANOVA analysis of variance ARB angiotensin receptor blocker BMI body mass index BW body weight CBS cystathionine β-synthase HbA1c hemoglobin A1c HOMA homeostatic model assessment LBM lean body mass m/z mass/charge p.a. postabsorptive Ra rate of appearance SE standard error TTR tracer/tracer ratio. Acknowledgments We thank Dr. Carlo Artusi of the Central Laboratory of the Padova University Hospital for the analyses of plasma homocysteine, vitamin B12, and folates, and Dr. Elisabetta Iori of the Department of Medicine, Metabolism Unit, for the analyses of plasma vitamin B6. Financial Support: This work was supported by a Fondo per gli Investimenti della Ricerca di Base (FIRB) Research Project (years 2002–2003), titled Molecular Mechanisms in the Pathogenesis and Progression of Diabetic Nephropathy and of Kidney Ageing—Subproject: Homocysteine and Arginine. Author Contributions: P.T. designed the study, recruited the subjects, contributed to the in vivo experiments, collected and analyzed the data, and wrote the manuscript. D.C. recruited the subjects and contributed to the experiments. M.V. contributed to data collection and analysis and reviewed the manuscript. L.P. contributed to data collection and analysis. A.C. recruited the subjects and contributed to the experiments. E.K. contributed to the experiments and to data collection and reviewed the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Guba SC, Fink LM, Fonseca V. Hyperhomocysteinemia. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Jan 1, 2018

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