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Glucose oxidation in skeletal muscle from the spontaneously hypertensive rat
Life Sciences Vol . 19, pp . 1921-1924, 1976 . Printed in the II .S .A .
Pergamon Preoa
GLUCOSE OXIDATION IN SKELETAL MUSCLE FROM THE SPONTANF~USLY HYPERTENSIVE RAT Phillip M . Hutchips and Carol C . Cunningham Departments of Physiology and Biochemistry Bowman Gray School of Medicine Winston-Salem, North Carolina 27103 (Received is final form November 5, 1976) S[AMARY The metabolic activity of the cremaster muscle, as measured by glucose oxidation, was lower (16 .7$) in the spontaneously hypertensive rat (SHR) than in the normotensive Wistar-Kyoto rat (WKY) . This alteration in metabolic activity was accompanied by a reduction of the respiratory rate (12 .5$) in the SfBt rats when compared to WKY animals . The decreased respiratory rate and the lowered metabolic activity of the cremaster muscle occurred at a time when blood pressure is increasing in SHR animals . A reduction in arteriolar density has been observed in skeletal muscle of young spontaneously hypertensive rats (SHR) (1) . The possibility exists that this rarefaction of arterioles is associated with a reduction in metabolic rate of the observed muscle (cremaster) . We have shown previously a differential reactivity of skeletal muscle arterioles to isoproterenol, a beta agonist (2) . This alteration in vascular beta-receptors in SHR skeletal muscle also . suggested that the metabolic activity of such tissue in the hypertensive animal might be affected . To test this possibility we have compared the respiratory rate and metabolic activity of the cremaster muscle in spontaneously hypertensive and normotensive control rats . In order to assess metabolic activity of this muscle we have measured the rate of oxidation of glucose under aerobic conditions . METHJDS The microcirculatory model was the cremaster muscle of 100 gm spontaneously hypertensive rats (3) . The preparation used was a "closed" adaptation of the Baez open cremaster technique (4) . The controls for the spontaneously hypertensive rats (SHR) were normotensive Wistar Kyoto rats (WKY) . A total of 20 animals (10-SHR ; 10-WKY) weighing approximately 100 grams were anesthetized with a warm solution of 2$ chloralose dissolved in 10$ urethane (6 ml/kg) . In this size animal, the.cremaster is roughly 250 microns thick and contains two layers of skeletal muscle with an interposed blood supply(5) . In additïon, very little connective tissue is found attached to the cremaster in this size animal . After a tracheostomy was performed, the carotid artery was cannulated for the registration of systemic blood pressure . Heart râte and respiratory rate were determined from the pulsatile pressure recording . (1) .
The crea~aster was dissected according to previously described techniques The cremaster muscle, separated away from the testicle, was excised from 1921
192 2
Glucose Oxidation and Hypertension
Vol . 19, No . 12
the animal and then placed immediately in ice cold Krebs-Ringer phosphate solution, pH 7 .4 (6) . The muscle was split into two pieces surgically and each half was weighed after being blotted on tissue paper to renoue adhering sus pension medium . The glucose oxidation activity of each half was determined by an assay described by the following . The assay mixture, added to a 50 ml erlenmeyer Mask, consisted of 6 mg of non-radioactive glucose, 0 .5 u curies of glucose-U- 1 C, 1 .5 I .U . of insulin, and enough Krebs-Ringer phosphate solution to bring the final volume to 3 .0 ml . This high level of insulin was employed to insure maximum rate of glucose uptake in the skeletal tissue . Previous work with rat hemidiaphragms (7) demonstrate that this level is appropriate . A vial containing 0 .5 ml of hyamine hydroxide was placed in the center of the flask, after which the assay was initiated by addition of the muscle segment . The flask was immediately stoppered and the assay mixture was incubated forl ~ hour at 37° in a shaker bath operated at 1 cycle/sec . During this time the C02 evolved was collected in the hyamine hydroxide . Termination of the assay and radioactivity measurements were carried out as described by Iiolmes, et al (8) . The specific activity of the muscle segments was expressed as mg glucose oxi dized/hr~g wet weight of tissue . The specific activities obtained for both miscle segments were averaged together to obtain a specific activity for the whole muscle . This number was utilized for comparison of the specific activities of muscles from the hypertensioe rats with those from the control animals . The above assay has been compared with a measurmient of glucose oxidation determined by oxygen consumption . Oxygen utilization was measured polarographically as described by Estabrook (9) in an air saturated assay solution . Qucose oxidation of crenaster muscle from two control animals, as measured by oxygen utilization fell within the range of values obtained for control animals reported in Table I . This correlation between the two methods for measuring glucose utilization supports the validity of the 00 Z trapping assay described above . This latter assay was utilized because it allowed for activity measurements immediately after muscles were excised from several animals . The insulin used was Regular Iletin (F1i Lilly) . The glucose-U- 14 C and hyamine hydroxide were obtained from New England Nuclear, and the non-radioactive glucose from Sigma Chemical Compazry . Other materials were analytical reagent grade . Exact statistical probabilities (Table I) were calculated using a standard Student's "t" test piogram available in the Statistical Package for the HewlettPackard 65 programmable calculator . RESULTS Table I illustrates that when the SHR is compared to WKY on a weight basis the SHR's are slightly older than the normal animals . This further corrobor ates reports of retarded growth in the SHR . Even at this early stage. in the development of hypertension, there is already a significant increase in blood pressure . This finding is consistent with recent reports that the SHR is exhibiting statistically significant elevations in blood pressure at the fourth week of age . A 16 .7$ reduction in the glucose oxidation rate of the cremaster muscle was observed in the SHR . We feel that the p value of 0 .056 obtained in the Student's "t" test is sufficiently close to the cornentianally accepted level of statistical significance (p = 0 .05) that the differences noted between the rates of glucose oxidation in the cremaster muscle can be considered significant . This diminished metabolic activity of cremaster muscle fran SHR
Vol . 19, Po. 12
Glucose Ozidation and i~ypertension
1923
192 4
Glucose Oxidation and Hyperteaeion
Vol . 19, No . 12
animals is accompanied by a significant 12 .5$ decrease in respiratory rate . Upon scanning data from five years of experiments with the SHR, it was noted that the decreased respiratory rate was evident in all previous studies . DISCUSSION We have demonstrated that the metabolic rate of the cremaster muscle as measured by the rate of glucose oxidation is lower in the spontaneously hypertensive rats (SHR) than in the normotensive Wistar-Kyoto rat (WKY) . This find ing is accompanied by the observation of a reduced respiratory rate in these experiments and tt~se of previous years . Both of these findings are consistent with a decreased utilization of 0 2 by the skeletal muscle tissue . Although this study confirms a possible connection between the previously reported decreased arteriolar vascularity (1) and a diminished metabolic rate, it does not establish a cause and effect relationship . Likewise, it does not indicate that the hypertension is a result of or results in the reduced metabolic activity and vascularity . It does, however, reveal that the lowered metabolic activity and vascularity are occurring at the time when arterial pressure is rising in the SHR . Further studies are indicated to determine whether : 1) the hypertension results from the reduced vascularity and metabolic activity, 2) the reduced metabolic activity and vascularity results frown the hypertension, 3) the three parameters are independently related .
This work was supported by U . S . Public Health Service Grant No .HL-13936 . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
P .M . fITPC[iINS and A .E . DARNELL, Circ .Res .34 (Supp1 .I) :I-161-I-165 (1974) . P .M . H.TI~HINS, . A .W . GREED, and irk It~iN~ Microvas .Res .9 :101-106 (1975) . K . OKAMJTO and K . AOKI, J~a . Circ . J .27 :282S . BAEZ, Microvas . Res .T:384~d~ (r9T~ . G . MA.TNO ~~ P~bE, J . Bi h sic and Biochem . 01 .11 :571-605 (1961) . H . DELUCA and P . COI-~N, Manometri ues~d -T33 (1964) . P . RANDLE, P . GARLAND, G .~,î~ ßFNI1DN, and C . POGSON, Recent Pro . in Hormone Res . 22 :1-44 (1966) . $~P .~[Ji HORSI', and R . GOOD, Lancet _1 :1225-1228 (1966) . R .W . ESTABROOK, Methods Enzymol . 10 :41-47 (1967j.
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Pergamon Preoa
GLUCOSE OXIDATION IN SKELETAL MUSCLE FROM THE SPONTANF~USLY HYPERTENSIVE RAT Phillip M . Hutchips and Carol C . Cunningham Departments of Physiology and Biochemistry Bowman Gray School of Medicine Winston-Salem, North Carolina 27103 (Received is final form November 5, 1976) S[AMARY The metabolic activity of the cremaster muscle, as measured by glucose oxidation, was lower (16 .7$) in the spontaneously hypertensive rat (SHR) than in the normotensive Wistar-Kyoto rat (WKY) . This alteration in metabolic activity was accompanied by a reduction of the respiratory rate (12 .5$) in the SfBt rats when compared to WKY animals . The decreased respiratory rate and the lowered metabolic activity of the cremaster muscle occurred at a time when blood pressure is increasing in SHR animals . A reduction in arteriolar density has been observed in skeletal muscle of young spontaneously hypertensive rats (SHR) (1) . The possibility exists that this rarefaction of arterioles is associated with a reduction in metabolic rate of the observed muscle (cremaster) . We have shown previously a differential reactivity of skeletal muscle arterioles to isoproterenol, a beta agonist (2) . This alteration in vascular beta-receptors in SHR skeletal muscle also . suggested that the metabolic activity of such tissue in the hypertensive animal might be affected . To test this possibility we have compared the respiratory rate and metabolic activity of the cremaster muscle in spontaneously hypertensive and normotensive control rats . In order to assess metabolic activity of this muscle we have measured the rate of oxidation of glucose under aerobic conditions . METHJDS The microcirculatory model was the cremaster muscle of 100 gm spontaneously hypertensive rats (3) . The preparation used was a "closed" adaptation of the Baez open cremaster technique (4) . The controls for the spontaneously hypertensive rats (SHR) were normotensive Wistar Kyoto rats (WKY) . A total of 20 animals (10-SHR ; 10-WKY) weighing approximately 100 grams were anesthetized with a warm solution of 2$ chloralose dissolved in 10$ urethane (6 ml/kg) . In this size animal, the.cremaster is roughly 250 microns thick and contains two layers of skeletal muscle with an interposed blood supply(5) . In additïon, very little connective tissue is found attached to the cremaster in this size animal . After a tracheostomy was performed, the carotid artery was cannulated for the registration of systemic blood pressure . Heart râte and respiratory rate were determined from the pulsatile pressure recording . (1) .
The crea~aster was dissected according to previously described techniques The cremaster muscle, separated away from the testicle, was excised from 1921
192 2
Glucose Oxidation and Hypertension
Vol . 19, No . 12
the animal and then placed immediately in ice cold Krebs-Ringer phosphate solution, pH 7 .4 (6) . The muscle was split into two pieces surgically and each half was weighed after being blotted on tissue paper to renoue adhering sus pension medium . The glucose oxidation activity of each half was determined by an assay described by the following . The assay mixture, added to a 50 ml erlenmeyer Mask, consisted of 6 mg of non-radioactive glucose, 0 .5 u curies of glucose-U- 1 C, 1 .5 I .U . of insulin, and enough Krebs-Ringer phosphate solution to bring the final volume to 3 .0 ml . This high level of insulin was employed to insure maximum rate of glucose uptake in the skeletal tissue . Previous work with rat hemidiaphragms (7) demonstrate that this level is appropriate . A vial containing 0 .5 ml of hyamine hydroxide was placed in the center of the flask, after which the assay was initiated by addition of the muscle segment . The flask was immediately stoppered and the assay mixture was incubated forl ~ hour at 37° in a shaker bath operated at 1 cycle/sec . During this time the C02 evolved was collected in the hyamine hydroxide . Termination of the assay and radioactivity measurements were carried out as described by Iiolmes, et al (8) . The specific activity of the muscle segments was expressed as mg glucose oxi dized/hr~g wet weight of tissue . The specific activities obtained for both miscle segments were averaged together to obtain a specific activity for the whole muscle . This number was utilized for comparison of the specific activities of muscles from the hypertensioe rats with those from the control animals . The above assay has been compared with a measurmient of glucose oxidation determined by oxygen consumption . Oxygen utilization was measured polarographically as described by Estabrook (9) in an air saturated assay solution . Qucose oxidation of crenaster muscle from two control animals, as measured by oxygen utilization fell within the range of values obtained for control animals reported in Table I . This correlation between the two methods for measuring glucose utilization supports the validity of the 00 Z trapping assay described above . This latter assay was utilized because it allowed for activity measurements immediately after muscles were excised from several animals . The insulin used was Regular Iletin (F1i Lilly) . The glucose-U- 14 C and hyamine hydroxide were obtained from New England Nuclear, and the non-radioactive glucose from Sigma Chemical Compazry . Other materials were analytical reagent grade . Exact statistical probabilities (Table I) were calculated using a standard Student's "t" test piogram available in the Statistical Package for the HewlettPackard 65 programmable calculator . RESULTS Table I illustrates that when the SHR is compared to WKY on a weight basis the SHR's are slightly older than the normal animals . This further corrobor ates reports of retarded growth in the SHR . Even at this early stage. in the development of hypertension, there is already a significant increase in blood pressure . This finding is consistent with recent reports that the SHR is exhibiting statistically significant elevations in blood pressure at the fourth week of age . A 16 .7$ reduction in the glucose oxidation rate of the cremaster muscle was observed in the SHR . We feel that the p value of 0 .056 obtained in the Student's "t" test is sufficiently close to the cornentianally accepted level of statistical significance (p = 0 .05) that the differences noted between the rates of glucose oxidation in the cremaster muscle can be considered significant . This diminished metabolic activity of cremaster muscle fran SHR
Vol . 19, Po. 12
Glucose Ozidation and i~ypertension
1923
192 4
Glucose Oxidation and Hyperteaeion
Vol . 19, No . 12
animals is accompanied by a significant 12 .5$ decrease in respiratory rate . Upon scanning data from five years of experiments with the SHR, it was noted that the decreased respiratory rate was evident in all previous studies . DISCUSSION We have demonstrated that the metabolic rate of the cremaster muscle as measured by the rate of glucose oxidation is lower in the spontaneously hypertensive rats (SHR) than in the normotensive Wistar-Kyoto rat (WKY) . This find ing is accompanied by the observation of a reduced respiratory rate in these experiments and tt~se of previous years . Both of these findings are consistent with a decreased utilization of 0 2 by the skeletal muscle tissue . Although this study confirms a possible connection between the previously reported decreased arteriolar vascularity (1) and a diminished metabolic rate, it does not establish a cause and effect relationship . Likewise, it does not indicate that the hypertension is a result of or results in the reduced metabolic activity and vascularity . It does, however, reveal that the lowered metabolic activity and vascularity are occurring at the time when arterial pressure is rising in the SHR . Further studies are indicated to determine whether : 1) the hypertension results from the reduced vascularity and metabolic activity, 2) the reduced metabolic activity and vascularity results frown the hypertension, 3) the three parameters are independently related .
This work was supported by U . S . Public Health Service Grant No .HL-13936 . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
P .M . fITPC[iINS and A .E . DARNELL, Circ .Res .34 (Supp1 .I) :I-161-I-165 (1974) . P .M . H.TI~HINS, . A .W . GREED, and irk It~iN~ Microvas .Res .9 :101-106 (1975) . K . OKAMJTO and K . AOKI, J~a . Circ . J .27 :282S . BAEZ, Microvas . Res .T:384~d~ (r9T~ . G . MA.TNO ~~ P~bE, J . Bi h sic and Biochem . 01 .11 :571-605 (1961) . H . DELUCA and P . COI-~N, Manometri ues~d -T33 (1964) . P . RANDLE, P . GARLAND, G .~,î~ ßFNI1DN, and C . POGSON, Recent Pro . in Hormone Res . 22 :1-44 (1966) . $~P .~[Ji HORSI', and R . GOOD, Lancet _1 :1225-1228 (1966) . R .W . ESTABROOK, Methods Enzymol . 10 :41-47 (1967j.
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