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【摘要】 Mineralocorticoids enhance expression and insulin stimulates activity of the serum- and glucocorticoid-inducible kinase SGK1, which activates the renal epithelial Na + channel (ENaC). Under a salt-deficient diet, SGK1 knockout mice ( sgk1 -/- ) excrete significantly more NaCl than their wild-type littermates ( sgk1 +/+ ) and become hypotensive. The present experiments explored whether SGK1 participates in the hypertensive effects of a high-fat diet and high-salt intake. Renal SGK1 protein abundance of sgk1 +/+ mice was significantly elevated after a high-fat diet. Under a control diet, fluid intake, blood pressure, urinary flow rate, and urinary Na +, K +, and Cl - excretion were similar in sgk1 -/- and sgk1 +/+ mice. Under a standard diet, high salt (1% NaCl in the drinking water for 25 days) increased fluid intake, urinary flow rate, and urinary Na +, K +, and Cl - excretion similarly in sgk1 -/- and sgk1 +/+ mice without significantly altering blood pressure. A high-fat diet alone (17 wk) did not significantly alter fluid intake, urinary flow rate, urinary Na +, K +, or Cl - excretion, or plasma aldosterone levels but increased plasma insulin, total cholesterol, triglyceride concentrations, and systolic blood pressure to the same extent in both genotypes. Additional salt intake (1% NaCl in the drinking water for 25 days) on top of a high-fat diet did not affect hyperinsulinemia or hyperlipidemia but increased fluid intake, urinary flow rate, and urinary NaCl excretion significantly more in sgk1 -/- than in sgk1 +/+ mice. Furthermore, in animals receiving a high-fat diet, additional salt intake increased blood pressure only in sgk1 +/+ mice (to 132 ± 3 mmHg) but not in sgk1 -/- mice (120 ± 4 mmHg). Thus lack of SGK1 protects against the hypertensive effects of a combined high-fat/high-salt diet.
【关键词】 insulin blood pressure aldosterone mineralocorticoids ENaC kidney
EXPRESSION OF THE SERUM - AND glucocorticoid-inducible kinase SGK1 ( 33 ) is strongly upregulated by mineralocorticoids ( 6, 14, 19, 26, 48, 52, 58, 62, 69 ). For activation, SGK1 requires phosphorylation, which is accomplished by the phosphoinositide-dependent kinase PDK1 ( 7, 42, 57 ). Stimulators of SGK1 activity include insulin, which activates SGK1 through a signaling pathway involving phosphatidylinositide 3 kinase and PDK1 ( 42 ).
SGK1 interacts with the epithelial Na + channel ENaC ( 73 ), and coexpression of SGK1 markedly enhances the activity of ENaC heterologously expressed in Xenopus laevis oocytes ( 1, 11, 19, 46, 52, 72 ) and in A6 cells ( 3, 31, 60, 69 ). SGK1 increases ENaC, in part, by phosphorylation of the ubiquitin ligase Nedd4-2 ( 23, 63 ), which ubiquitinates ENaC, thus preparing the channel protein for clearance from the cell membrane and subsequent degradation ( 66 ). Phosphorylation of Nedd4-2 by SGK1 reduces the affinity of the enzyme to the target proteins and thus disrupts the ubiquitination of ENaC, leading to enhanced ENaC channel protein abundance in the cell membrane ( 1, 46, 72 ). Beyond that, SGK1 may regulate ENaC activity by more direct interaction ( 24 ).
SGK1 is expressed in the aldosterone-sensitive distal nephron ( 2, 48 ), and the stimulation of ENaC by SGK1 is considered to participate in the regulation of renal Na + excretion by aldosterone, insulin, and IGF-1 (8-10, 73). Besides ENaC, SGK1 enhances the activity of further renal transport systems, including the apical K + channel (ROMK) ( 56, 76 ), the Na + -K + -ATPase ( 36, 61, 77 ), the Na + -K + - 2Cl - cotransporter (NKCC2) ( 46 ), the epithelial Ca 2+ channel (TRPV5) ( 30, 54 ), the Na + /H + exchanger type 3 (NHE3) ( 75, 76 ), and the K + channel (KCNE1) ( 29 ).
Considering the significance of renal Na + transport for blood pressure regulation ( 47, 49 ), the effects of SGK1 on ENaC were expected to influence blood pressure. As a matter of fact, moderately enhanced blood pressure is observed in individuals carrying a variant of the SGK1 gene affecting as many as 5% of unselected Caucasians ( 15, 16 ). Most recent evidence disclosed a relatively strong correlation between insulinemia and blood pressure in individuals carrying this SGK1 gene variant, suggesting a particular role of SGK1 in the hypertension paralleling hyperinsulinemia ( 70 ).
Gene-targeted mice lacking SGK1 ( sgk1 -/- ) show normal Na + excretion and blood pressure under normal-salt intake, but their ability to retain Na + and maintain blood pressure under a salt-deficient diet is impaired ( 74 ). In contrast to the mild phenotype of the sgk1 -/- mouse, targeted disruption of the mineralocorticoid receptor in mice leads to severe salt wasting ( 5 ), and the ENaC knockout mouse is not viable ( 39 ). Thus SGK1 is not required for the function of ENaC but significantly contributes to, yet does not fully account for, the effects of mineralocorticoids in the kidney. The role of SGK1 in preventing a decrease in blood pressure following salt depletion does not necessarily imply that the lack of SGK1 protects from an increase in blood pressure following excessive salt intake, which was expected to decrease plasma aldosterone concentrations and thus to decrease SGK1 expression in wild-type mice. Thus a high-salt diet could dissipate the difference between sgk1 -/- mice and wild-type mice. Thus the question arises whether and under which conditions SGK1 may be relevant for blood pressure increase.
The present study has been performed to explore the influence of SGK1 on renal function and systolic blood pressure during a high-fat and high-salt diet, both of which have been shown to favor an increase in blood pressure ( 13, 20, 21, 27, 34, 41, 44, 53, 59 ). Surprisingly, lack of SGK1 does not affect systolic blood pressure after a high-fat diet alone, but it completely abrogates the increase in systolic blood pressure after additional high-salt treatment.
METHODS
Animals. All animal experiments were conducted according to the guidelines of the American Physiological Society as well as the German law for the welfare of animals and were approved by local authorities. Mice deficient in SGK1 ( sgk1 -/- ) were generated and bred as previously described ( 74 ). Heterozygous sgk1-deficient mice were backcrossed to 129/SvJ wild-type mice (Charles River, Sulzfeld, Germany) for 10 generations and then intercrossed to generate homozygous SGK1 mice. Male and female SGK1 knockout mice ( sgk1 -/- ) and their wild-type littermates ( sgk1 +/+ ) were fed either a control diet (1310/1314, 4% kcal fat, 0.25% Na +, 0.36% Cl -, 0.71% K +, Altromin, Heidenau, Germany) or a high-fat diet (C1000, 45% kcal fat, 0.25% Na +, 0.36% Cl -, 0.71% K +, modified according D12451 from Research Diet, Altromin). Mice were maintained on standard diet until the age of 5 wk, when for one-half of the animals the diet was switched to a high-fat diet. Within the first 17 wk, mice were allowed free access to tap water. After 17 wk, tap water was replaced by a 1% NaCl solution.
Metabolic cages. For evaluation of renal excretion, both sgk1 -/- and sgk1 +/+ mice were placed individually in metabolic cages (Tecniplast, Hohenpeissenberg, Germany) for 1 wk ( 68 ) with free access to a standard mouse diet or a high-fat diet, both containing 0.25% Na +, 0.36% Cl -, and 0.71% K + (Altromin). The inner wall of the metabolic cages was siliconized, and urine was collected for 24 h under water-saturated oil every second day when water and food intake were measured. Urinary excretion was determined again in metabolic cages as described above 25 days after exposure to 1% NaCl in the drinking water under both a standard or a high-fat diet.
Glucose tolerance and insulin tolerance test. For determination of glucose tolerance, mice were starved overnight and glucose (3 g/kg body wt) was injected intraperitoneally (ip). Then, a drop of blood was drawn from the tail onto a test strip of a glucometer (Accutrend, Roche, Mannheim, Germany) for measurement of blood glucose levels before and 15, 30, 45, 60, 75, 90, 120, 150, and 180 min after the injection. In another series of experiments, long-acting insulin (Novo Nordisk, Mainz, Germany) was injected (0.15 U/kg body wt ip), and plasma glucose concentrations were determined at 0, 15, 30, 45, 60, and 90 min, as described above.
Blood and urinary concentrations. To obtain a blood specimen, animals were lightly anesthetized with isoflurane (Abbott, Wiesbaden, Germany) and 300 µl of blood were withdrawn into heparinized capillaries by puncturing the retroorbital plexus. For determination of nonfasted blood glucose levels and plasma insulin concentrations, blood was drawn at 9 AM in the morning and centrifuged immediately after collection. Plasma and urinary concentrations of Na + and K + were measured by flame photometry (ELEX 6361, Eppendorf, Germany), and Cl - concentrations were determined by electrometric titration (Chloridometer 6610, Eppendorf, Germany). Plasma concentrations of insulin were determined using an enzyme immunoassay kit (Mercodia, Uppsala, Sweden), and plasma aldosterone concentrations were measured using a commercial RIA kit (Beckman Coulter, Krefeld, Germany). Total cholesterol, triglycerides, and free fatty acids were analyzed using a colorimetric assay (BioVision Research, Sigma, Wako Chemicals).
Blood pressure. Systolic arterial blood pressure was determined by the tail-cuff method 17 wk after exposure to the control or high-fat diet, as well as 9 and 25 days after exposure to 1% NaCl in the drinking water under a continued high-fat diet and 16 and 25 days after exposure to 1% NaCl in the drinking water under a continued control diet. We do not provide blood pressure values during the night, which may be preferable in nocturnal animals ( 43 ). Moreover, as reviewed recently ( 50 ), the tail-cuff approach to determine arterial blood pressure requires certain precautions to reduce the stress of the animals, including appropriate training of the mice over multiple days, prewarming to an ambient temperature of 29°C, measurement in a quiet, semidark, and clean environment, and performance of the measurements by one person and during a defined daytime period, when blood pressure is stable (between 1 and 3 PM). All these precautions were taken in the present study. The tail-cuff method has the advantage of being noninvasive and can provide reproducible results of systolic blood pressure if the above-mentioned precautions are taken into account ( 43 ).
SGK1 protein abundance. To explore whether salt loading decreases SGK1 protein abundance, sgk1 +/+ mice were divided into four groups (5 mice/group) and treated separately with 1 ) a control diet for 3 mo, 2 ) a control diet for 3 mo plus 1% NaCl in the drinking water for 4 wk, 3 ) a high-fat diet for 3 mo, and 4 ) a high-fat diet for 3 mo plus 1% NaCl in the drinking water for 4 wk. Mice were anesthetized with ketamine plus xylazine. Kidneys were removed and immediately shock frozen in liquid nitrogen. Total protein was analyzed from kidneys by Western blot. The kidneys were homogenized in lysis buffer containing 50 mM Tris·HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, and a protease inhibitor cocktail (Complete mini EDTA-free, Roche, Mannheim, Germany). The homogenates were centrifuged at 7,000 g at 4°C for 15 min, and the supernatant was removed and used for Western blotting. Total proteins (100 µg) were separated by SDS-PAGE (10% Tris-Glycine), transferred to nitrocellulose membranes, blocked overnight in blocking buffer (5% fat-free milk in PBS containing 0.1% Tween) at 4°C, and incubated for 1 h with a polyclonal anti-SGK1 antibody (diluted 1:1,000 in blocking buffer) kindly provided by Nicola Perrotti (Catanzaro, Italy), who generated the antibody by immunizing rabbits with the peptide EVLHQPYDRTVDW (SGK1 residues 267-280, Zymed Laboratories, South San Francisco, CA) and subsequently characterized the specificity of the antibody ( 31, 51 ). In a single experiment, we tested the antibody in renal tissue from sgk1 -/- mice, where the 48-kDa band was missing. However, the antibody binds to further proteins, as evidenced by additional bands in the Western blot. After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Amersham, Freiburg, Germany), the bands were visualized with enhanced chemiluminescence according to the manufacturer's instructions. Homogenates were also probed with a primary (GAPDH, Santa Cruz Biotechnology, Heidelberg, Germany) antibody as a loading control. Densitometric analysis of SGK1 was performed by using Quantity One software (Bio-Rad Laboratories) and normalized using GAPDH.
Calculations. Data are provided as means ± SE; n represents the number of independent experiments. All data were tested for significance using ANOVA, and only results with P < 0.05 were considered statistically significant.
RESULTS
Plasma insulin and aldosterone concentrations. Under the standard diet, plasma insulin concentrations ( Fig. 1 A ) and blood glucose levels ( sgk1 -/- : 108 ± 10 mg/dl; sgk1 +/+ : 111 ± 3 mg/dl, n = 7 each) were similar. The replacement of tap water by 1% NaCl in the drinking water did not significantly alter plasma insulin levels in either genotype. Plasma aldosterone concentrations were significantly higher in sgk1 -/- than in sgk1 +/+ mice under the standard diet and high-fat diet alone ( Fig. 1 B ). High-salt intake significantly decreased plasma aldosterone concentrations to similarly low values in both genotypes on the control diet. Treatment of the mice for 17 wk with a high-fat diet did not significantly affect plasma aldosterone concentrations but led to an approximately fourfold increase in insulin concentrations ( Fig. 1 A ) and significantly increased blood glucose levels ( sgk1 -/- : 165 ± 16 mg/dl; sgk1 +/+ : 152 ± 4 mg/dl, n = 7) in both groups of mice. Subsequent replacement of tap water with 1% saline in animals treated with the high-fat diet did not affect the plasma insulin concentration ( Fig. 1 A ) or blood glucose levels ( sgk1 -/- : 150 ± 6 mg/dl; sgk1 +/+ : 152 ± 8 mg/dl, n = 7) but decreased plasma aldosterone concentration. Nevertheless, the plasma aldosterone concentration remained significantly higher in sgk1 -/- mice during the high-fat diet plus 1% NaCl treatment ( Fig. 1 B ).
Fig. 1. Plasma insulin ( A ) and aldosterone ( B ) concentrations in SGK1 knockout mice ( sgk1 -/-; filled bars) and wild-type littermates ( sgk1 +/+; open bars) on a standard (SD) and high-fat diet (HFD) before and after additional 1% NaCl in the drinking water. Values are means ± SE; n = 7/group. # P < 0.05 vs. respective value under an SD. P < 0.05 vs. respective value before 1% NaCl. * P < 0.05 vs. sgk1 +/+.
Hypoglycemic effect of insulin and glucose tolerance in mice fed a high-fat diet. As illustrated in Fig. 2, after 14 wk on the high-fat diet, the decline of plasma glucose concentration following an injection of insulin (0.15 U/kg body wt ip) was significantly decreased in these mice ( Fig. 2 ). Moreover, the increase in plasma glucose concentration after glucose loading (3 g/kg body wt ip) was significantly greater in animals treated with the high-fat diet than in animals fed the control diet ( Fig. 3 ). Accordingly, a high-fat diet leads to insulin resistance of cellular glucose uptake. The peak plasma glucose concentration after a glucose load was significantly higher and the decline of plasma glucose concentration after insulin administration was significantly slower in sgk1 -/- than in sgk1 +/+ mice ( Figs. 2 and 3 ).
Fig. 2. Plasma glucose concentrations after intraperitoneal injection of insulin (0.15 U/kg body wt) in sgk1 +/+ ( ) and sgk1 -/- mice ( ). Values are means ± SE; n = 7/group. Experiments were performed in animals on a control diet ( A ) and after 14 wk on a HFD ( B ). * P < 0.05 vs. sgk1 +/+. # P < 0.05 vs. respective value under an SD.
Fig. 3. Plasma glucose concentrations following intraperitoneal glucose injection (3 g/kg body wt) in sgk1 +/+ ( ) and sgk1 -/- mice ( ). Values are means ± SE; n = 7/group. Experiments were performed in animals on a control diet ( A ) and 14 wk on a HFD ( B ). * P < 0.05 vs. sgk1 +/+. # P < 0.05 vs. respective value under an SD.
Plasma lipid concentrations. No significant differences were observed between the genotypes in plasma total triglyceride ( Fig. 4 ), total cholesterol ( Fig. 4 ), or free fatty acid concentrations (1.4 ± 0.2 mM, n = 6, in sgk1 +/+ mice and 1.2 ± 0.2 mM, n = 6, in sgk1 -/- mice) under the control diet. The high-fat diet increased total plasma triglyceride concentrations 2.5-fold in both genotypes ( Fig. 4 ). Additional 1% NaCl on top of the high-fat diet significantly reduced total plasma triglyceride concentrations only in sgk1 -/- mice. Plasma concentrations of free fatty acids were not significantly altered by either the high-salt diet alone ( sgk1 -/- : 1.5 ± 0.1 mM, n = 6; sgk1 +/+ : 1.2 ± 0.2 mM, n = 6), the high-fat diet alone ( sgk1 -/- : 1.5 ± 0.2 mM, n = 7, sgk1 +/+ 1.4 ± 0.1 mM, n = 7), or the combined high-fat/high-salt treatment ( sgk1 -/- : 1.6 ± 0.2, n = 6, sgk1 +/+ 1.5 ± 0.2, n = 6). Total serum cholesterol concentrations were significantly elevated in both sgk1 -/- and sgk1 +/+ mice during the high-fat and high-fat/high-salt treatments ( Fig. 4 A ).
Fig. 4. Plasma total cholesterol ( A ) and total triglyceride ( B ) concentrations in sgk1 +/+ (open bars) and sgk1 -/- mice (filled bars) on an SD or HFD before and after additional 1% NaCl in the drinking water. Values are means ± SE; n = 7/group. # P < 0.05 vs. respective value under an SD. P < 0.05 vs. respective value before 1% NaCl.
SGK1 protein abundance. As illustrated in Fig. 5, the high-fat diet significantly increased the SGK1 protein abundance in kidneys from sgk1 +/+ mice. Additional treatment with the high-salt diet led to a decrease in SGK1 protein levels which, however, tended to remain higher than under control conditions despite a marked decrease in plasma aldosterone concentration ( Fig. 1 B ). Under the standard diet, replacement of tap water with 1% NaCl significantly suppressed SGK1 protein abundance and thus confirmed the previous observations ( 32 ).
Fig. 5. Effect of an HFD and salt excess on renal SGK1 protein expression. A : representative Western blot of renal SGK1 protein abundance under SD, SD+1% NaCl, HFD, and HFD+1% NaCl. B : values are means ± SE; n = 5/group. Shown is renal SGK1 protein abundance in sgk1 +/+ mice under SD, SD+1% NaCl, HFD, and HFD+1% NaCl. * P < 0.05 vs. control group.
Body weight, plasma electrolytes, and intake of food, fluid, and electrolytes. Body weight was not significantly affected by replacement of tap water with 1% NaCl in sgk1 -/- or sgk1 +/+ mice under the standard diet. The high-fat diet alone caused a similar increase in body weight of both genotypes. The additional salt load (1% NaCl) during the high-fat diet led to a further increase in body weight, an effect reaching statistical significance only in sgk1 +/+ mice ( Table 1 ). Replacement of tap water by 1% NaCl increased fluid intake in sgk1 -/- mice under both the control diet and the high-fat diet, but in sgk1 +/+ mice only under the control diet. Fluid intake did not significantly increase in sgk1 +/+ mice fed the high-fat diet after an additional salt load. Thus during the high-fat diet, fluid intake after replacement of tap water with 1% saline was significantly higher in sgk1 -/- than in sgk1 +/+ mice. The high-fat diet decreased and 1% NaCl increased food intake in both genotypes. Under the combined high-fat/high-salt diet, daily intake of Na + and Cl - was significantly larger in sgk1 -/- than in sgk1 +/+ mice. Replacement of tap water with 1% saline did not significantly affect packed cell volume or plasma Na + and Cl - concentrations. However, the high-fat diet alone significantly increased plasma Cl - concentrations in both genotypes ( Table 1 ). Under the combined high-fat/high-salt diet, plasma K + concentrations were significantly higher in sgk1 -/- mice than in sgk1 +/+ despite the higher plasma aldosterone concentrations in sgk1 -/- mice ( Table 1 ).
Table 1. Body weight, intake of fluid, food, Na +, Cl -, and K +, packed cell volume, Na +, Cl -, and K + concentrations in plasma before and after 25 days of 1% NaCl intake in mice treated with a standard diet or high-fat diet for 17 wk
Systolic blood pressure. Under the standard diet, systolic blood pressure was similar in sgk1 -/- and sgk1 +/+ mice ( Fig. 6 ). Replacement of tap water with 1% NaCl in the drinking water did not significantly alter blood pressure in either genotype ( Fig. 6 ). Exposure to the high-fat diet led to a marked and statistically significant increase in blood pressure, which was similar in both genotypes ( Fig. 6 ). Subsequent exposure to 1% NaCl in the drinking water at the continued high-fat diet was followed by a significant increase in blood pressure in sgk1 +/+ but not in sgk1 -/- mice. Accordingly, blood pressure was virtually identical in sgk1 -/- and sgk1 +/+ animals during the high-fat diet and access to tap water but was significantly higher in sgk1 +/+ than in sgk1 -/- after a 9- or 25-day additional exposure to 1% NaCl in the drinking water.
Fig. 6. Systolic blood pressure in sgk1 +/+ ( ) and sgk1 -/- mice ( ) on an SD or HFD before and after additional 1% NaCl in the drinking water. Values are means ± SE; n = 7/group before and 9, 16, or 25 days after addition of 1% NaCl to the drinking water. # P < 0.05 vs. respective value before 1% NaCl. * P < 0.05 vs. sgk1 +/+.
Urinary excretion. The increased fluid and NaCl intake after replacement of tap water with 1% saline was paralleled by significant increases in urinary flow rate and the urinary excretions of NaCl in both genotypes under both the standard and high-fat diets ( Fig. 7 ). However, fluid and NaCl intake ( Table 1 ) and urinary NaCl excretion under combined treatment with the high-fat diet/high-salt intake were significantly larger in sgk1 -/- than in sgk1 +/+ mice ( Fig. 7 ). Fluid intake and urinary excretion of NaCl were similar in sgk1 +/+ and in sgk1 -/- mice under the standard diet plus 1% NaCl ( Fig. 7 ).
Fig. 7. Urinary excretion of water and electrolytes (Na +, K +, and Cl - ) in sgk1 +/+ (open bars) and sgk1 -/- mice (filled bars) on an SD or HFD before and after additional 1% NaCl in the drinking water. Values are means ± SE; n = 7/group. P < 0.05 vs. respective value before 1% NaCl. * P < 0.05 vs. sgk1 +/+.
DISCUSSION
The present observations confirm the previous observations ( 74 ) that under a standard diet urinary salt excretion and blood pressure are similar in sgk1 -/- and sgk1 +/+ mice. However, the elevated aldosterone plasma concentrations in sgk1 -/- mice point to the functional significance of SGK1-dependent regulation of renal salt conservation even under those dietary conditions. The enhanced plasma aldosterone concentration overrides the lack of SGK1 and allows the maintenance of normal blood pressure. The maintenance of blood pressure in sgk1 -/- mice is consistent with SGK1-independent regulation of renal Na + reabsorption ( 74 ).
The extracellular volume expansion during high-salt intake is expected to decrease aldosterone release and thus to decrease the expression of SGK1 ( 6, 14, 19, 26, 48, 52, 58, 62, 69 ). Accordingly, salt excess should lower SGK1 expression in wild-type mice and thus dissipate the differences in SGK1-dependent stimulation of renal tubular Na + transport between sgk1 +/+ and sgk1 -/- mice.
Under a standard diet, saline drinking indeed lowers plasma aldosterone concentrations and does not lead to differences in blood pressure between sgk1 -/- and sgk1 +/+ mice. Interestingly, plasma aldosterone levels remain elevated in sgk1 -/- mice even under a high-fat diet, which is paralleled by an increase in blood pressure. Presumably, the increase in blood pressure during a high-fat diet does not translate into enhanced renal perfusion and thus remains without effect on renal renin release. Additional saline drinking decreased plasma aldosterone concentrations in both sgk1 -/- and sgk1 +/+ mice. Again, the aldosterone plasma levels remained significantly higher in sgk1 -/- mice. The difference under this experimental condition could be due to increased plasma K + concentrations (see above), a well-known stimulator of aldosterone release ( 65 ). Plasma K + concentrations in sgk1 -/- mice are at least partially enhanced due to impaired renal K + elimination ( 38, 65 ).
In contrast to sgk1 +/+ mice on a standard diet, sgk1 +/+ mice on a high-fat diet were sensitive to additional salt intake, possibly because the additional salt intake did not lead to an appropriate downregulation of SGK1 transcript levels.
A high-fat diet markedly increased the expression of SGK1 in the kidney. The present paper does not address the mechanism underlying enhanced renal expression of SGK1. It is noteworthy, however, that a high-fat diet may lead to activation of the peroxisome proliferator-activated receptor (PPAR ) ( 40 ), which, in turn, has been shown to enhance SGK1 transcription ( 37 ).
Moreover, a high-fat diet leads to increased insulin plasma concentrations. The hyperinsulinemia is presumably a result of insulin resistance, as reflected by the decreased glucose tolerance and the blunted hypoglycemic effect of insulin. Irrespective of the diet, the glucose tolerance and hypoglycemic effect of insulin are significantly decreased in sgk1 -/- mice, pointing to a role of SGK1 in the regulation of cellular glucose uptake. As a matter of fact, SGK1 indeed stimulates GLUT1 ( 55 ) and glucose uptake into several tissues is blunted in sgk1 -/- mice ( 12 ).
The hyperinsulinemia is expected to stimulate SGK1 and, subsequently, ENaC (8-10, 31, 73). Obesity is associated with peripheral insulin resistance, abnormal glucose metabolism, and subsequent development of hyperinsulinemia, which, in turn, favors the development of salt-sensitive hypertension by insulin-dependent stimulation of salt and water reabsorption ( 18, 22, 28, 35, 64 ). Thus the hyperinsulinism following a high-fat diet may override the effects of decreasing plasma aldosterone concentrations during a high-salt diet. A high-fat diet itself increases blood pressure in both genotypes, an observation pointing to the participation of SGK1-independent mechanisms. However, the additional salt load further increases blood pressure in sgk1 +/+ but not in sgk1 -/- mice, an observation disclosing the role of SGK1 in hypertension during a combined excess of fat and salt. Thus lack of SGK1 abrogates a salt-induced blood pressure increase only under a high-fat diet. The particular salt sensitivity of blood pressure during a high-fat diet may reflect SGK1-dependent stimulation of renal Na + reabsorption by insulin ( 8 ).
Interestingly, under a high-fat diet, the plasma triglyceride levels tend to be higher in sgk1 -/- mice than in sgk1 +/+ mice, a difference reaching statistical significance under an additional high-salt diet. The presently available evidence does not allow any safe statements on underlying mechanisms. It is noteworthy, though, that SGK1 modifies a number of transport processes ( 45 ), and lack of SGK1 may in theory affect intestinal absorption of lipids.
During a high-fat diet, the additional administration of a salt load increased body weight only in sgk1 +/+ mice. Under those experimental conditions, the sgk1 +/+ mice may retain more NaCl than the sgk1 -/- mice, even though NaCl intake was larger in sgk1 -/- than in sgk1 +/+ mice. As the electrolyte balance could not be fully determined throughout the treatment, however, no safe conclusions can be derived in regard to differences in extracellular fluid volume.
The present observations demonstrate that SGK1 is not only important for the prevention of hypotension during salt depletion but may also contribute to hypertension during hyperinsulinism and salt excess. As amplified in the introduction, SGK1 participates in the regulation of ENaC and thus renal Na + excretion by aldosterone, insulin, and IGF-1 ( 8, 9, 73 ). While the effect of aldosterone is only partially dependent on the presence of SGK1 and effects of aldosterone and SGK1 are additive, ADH or insulin does not further stimulate ENaC in cells expressing active SGK1 ( 4 ). Thus activation of ENaC by ADH or insulin fully depends on SGK1. The SGK1-dependent stimulation of ENaC is expected to increase extracellular fluid volume and thus blood pressure. DOCA treatment increases blood pressure similarly in sgk1 -/- mice and in sgk1 +/+ mice ( 67 ), indicating that SGK1 is not required for the hypertensive effects of acute mineralocorticoid excess.
On the other hand, SGK1 transcript and protein abundance were less in Sprague-Dawley rats and greater in Dahl salt-sensitive rats on 8 vs. 0.3% NaCl diets, suggesting that SGK1 may play a role in the pathogenesis of hypertension ( 32 ). In addition, moderately enhanced blood pressure is observed in individuals carrying a variant of the SGK1 gene affecting as many as 5% of unselected Caucasians ( 15 ). In the same individuals, increased body mass index ( 25 ) and a shortening of the QT interval ( 15, 16 ) have been observed in addition to increased blood pressure. The increased body mass index may partially be due to enhanced stimulation of the intestinal glucose transporter SGLT1 ( 25 ), the accelerated cardiac repolarization being due to enhanced activation of the cardiac K + channel KCNE1 ( 17, 29 ). Thus altered regulation of carriers and channels by SGK1 could account for the coincidence of obesity, hypertension, and altered cardiac action potential ( 45 ). Those observations are supported by a most recent observation revealing a relatively strong correlation between insulinemia and blood pressure in individuals carrying this SGK1 gene variant ( 71 ). The results of this study are in perfect agreement with our observations that SGK1 plays a particular role in the hypertensive effects of hyperinsulinemia.
GRANTS
This work was supported by grants from Deutschen Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung (to D. Kuhl, F. Lang, and V. Vallon).
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作者单位：1 Departments of Pharmacology and Toxicology, 2 Physiology, and 3 Internal Medicine, University of Tübingen, Tübingen; 4 Department of Clinical Neurobiology, University Hospital of Neurology, Heidelberg; 5 Department of Biology, Chemistry, and Pharmacy, Free University Berlin, Berlin, Germ