Diabetologia 9, 34--42 (1973) by Springer-Verlag 1973

Diuretics and Carbohydrate Metabolism: The Effects of and Amiloride on Blood Glucose, Plasma Insulin and Cations in the Rat*

A. Ayrmley-Green** and K.G. Alberti Nuffield Department of Clinical Medicine, The Radcliffe Infirmary, Oxford, England Received : September 18, 1972, accepted : October 20, 1972

Summary. The effects of two unrelated , mild streptozotocin . Amiloride given with or furosemido and amiloride on blood glucose, plasma insulin 30 min before glucose increased the insulin area without and glucose tolerance in the consCious rat are reported. affecting glucose disappearance. Chronic administration Furosemide (1 mg/kg or 2 mg/kg) given intravenously enhanced glucose disappearance with increased plasma caused an immediate but highly transient 23% and 53% insulin re~aponsc, and caused hyperkalaemia. Chronic fall in plasma insulin followed by a rise in blood glucose. furosomide administration had no effects. Possible mech- The hyperglycaemic effect alone occurred in mild strepto- anisms for these effects are discussed. zotocin diabetic animals. Furosemide given with or 30 rain before intravenous glucose (0.5 G/kg) caused glucose Key words: Diuretics ; glucose intolerance ; furosemide; intolerance with diminished insulin response. None of amiloride ; plasma immunoreactive insulin; blood glucose; these effects were observed in adrenalectomised animals. intravenous glucose tolerance; streptozotocin -- diabetic Amiloride (1, 5 or 10 mg/kg) given I.V. alone immediately rats; adrenalectomised rats; plasma and potas- increased plasma insulin (up to 213 FU/ml) without sium; red cell sodium and ; total body sodium affecting blood glucose. This effect was attenuated by and potassium.

In~oducfion sulphamoyl anthranilic acid structure, but the thiadia- zinc ring is replaced by a furfuryl group (Fig. 1). It Glucose intolerance following therapy has has been suggested that kaliuretic diuretics such as been reported in man and in experimental animals, this may cause glucose intolerance secondary to although the mechanism of the effect, its incidence and potassium depletion, since potassium depletion is clinical importance remain controversial. Benzothia- associated with diminished glucose tolerance [16] diazine diuretics are known to exacerbate hypergly- caemia in diabetic patients (for review, see [1]) and 0 NH N II I frequently to cause impaired carbohydrate tolerance CI~.~ C--NH-- C -- NH2" HCI in hypertensive patients [1]. Prospective studies in 11 .,,.J normal subjects [2] and non-diabetic hypertensive patients [3, 4] have shown that most patients who develop glucose intolerance have a diabetic predisposi- Amiloride tion, although prednisone-primed glucose tolerance tests or tests may reveal abnormalities in others [5, 6]. Animal studies have given conflicting results, showing hyperglycaemia [7, 8, 9], impaired /NH-CH~I~ glucose tolerance [10] or no effect [11, 12]. Acute diuretic induced hyperglycaemia using large doses of diuretics under ether anaesthesia has been described in the rodent [13, 14, 15], but few studies have examined HzN--02S/ Furosernide sequential insulin changes in conscious animals. Further studies using small doses therefore seemed Fig. 1. The structures of amiloride and furosemido necessary, first to establish whether there was a con- sistent effect and second to examine the mechanisms whilst repletion improves tolerance [17]. Amiloride is a involved. -carbonylguanidine which is structurally un- Two 3iuretics were used, furosemide (Lasix) related related to the , has a weaker natriuretic effect to the benzothiadiazines, and amiloride (Midamor) a and does not cause potassium loss [18]. The non- diuretic. Furosemide contains the thiazide group of amiloride is shared by arginine, a potent stimulus to insulin secretion (Fig. 1). It might there- * Presented in part at the 7th Annual meeting of the ~fore be predicted that furosemide and amiloride would European Association for the study of diabetes, South- ampton, 1971. have opposite effects oil insulin secretion and glucose ** Wellcomo Junior Research Fellow. metabolism. A. Aynsley-Green and K.G. Alberti: Diuretics and Carbohydrate Metabolism 35

Results confirming this hypothesis are presented Calculations: The glucose disappearance rate (kg) was below. calculated using the absolute blood glucose values from 2.5-20 rain [22]. Insulin areas were calculated from the areas trader the insulin curves during the first five, ten, or Materials and Methods hi thirty minutes of the test..~-~ ratios were calculated from Special Chemicals: Pttrc furosemide was a gift from the increments of plasma insulin add blood glucose values Hoechst Pharmaceuticals, Ltd., and used at a final con- from the zero value [23]. Statistical significance was centration of 1 and 2 mg/ml in normal saline, pH 7.0-- assessed by the Student 't' test. 7.4. Pure amiloride hydrochloride, a gift from Merck, Sharp and Dohme Ltd., was injected as a suspensionin normal saline at concentrations of I, 5 and 10 mg/ml. Anti-insulin serum was a gift from Dr. P.H. Wright; Results Ir'6-insulin was purchased from Abbott Laboratories, Chicago, U.S.A. Streptozotocin was kindly provided by I. The effects of acute administration of furosemide on the Upjohn Co., Kalamazoo, Michigan, U.S.A. Ultra pure blood glucose and plasma insulin water was obtained by pas.sing distilled water through an Elgaean ion exchange column (Elga Products Ltd., Lane a) Normal animals: A significant decrease in plasma End, Bucks). insulin occurred with both 1 mg and 2 mg/kg furose- A~mlytical methods: Insulin was assayed by a micro- mide (Fig. 2; Table I). Plasma insulin fell by 23% modiiication of the double antibody radioimmunoassay of Soeldner and Slone [19] with ethylene diamine tetracetie acid (EDTA) present throughout the assay [20]. Purified .a_ 15o rat insulin (kindly supplied by Novo Industri A/S Copen- hagen) was used as the insulin standard. The assay was unaffected by furosemide and amiloride and allowed the measurement of I tzU insulin/ml in 50 ~zl plasma samples, derived from EDTA treated blood. Glucose was mea- 100 sured in a noutralised perehloric acid extract of whole blood by a glucose oxidase method [21]. Sodium and potassium were measured by flame photo- "8 0 metry using an E.E.L. integrating flame photometer with 5C 1 i I I I a internal standard. Ammonium heparin plasma 0 5 ~0 20 30 and red cell haemoly'sates were assayed after diluting 1: Time: min I00 with lithium working diluent. No correction was made for plasma trapping. Whole body sodium and p~tassium values were obtained by assaying the nitric acid digest of the minced carcas. = 150 Animals: Male white Wistar rats weighing 250 to 350 gm were used unless otherwise stated. Adrenaloctom- ised Wistar rats (Scientific Products Farm, Ash, Canter- bury), maintained on 0.9~/o saline, were used 5--10 days I00 after adrenaleetomy. Diabetic rats were obtained by ~ intraporitoneal injection ~f 60 mg/kg or 75 mg/kg strepto- zotocin 48 h before the experiments. Experimental model: Conscious overnight-starved rats were used 16--20 h after the insert, ion of small cannulae /,0[ llltl I I I (0.4 mm internal diameter) into the left femoral artery and vein under light ether anaesthesia. The animals were 0 5 10 20 30 kept in restraining cages with free access to water. Can- lime: rain nulae were kept patent with saline. Up to 0.35 ml arterial Fig. 2. The effects of acute administration of furosemide blood was withdrawn at each sample time, the volume on blood glucose and plasma insulin in normal animals being replaced with saline. Up to ten samples could be Furosemido, l mg/kg, shown by A-- A (n = 5), or 2 mg/kg withdrawn witlmut changes in glucose or insulin. shown by -- (n = 4) was given as an acute intravenous Immediately on withdrawing, 100 ~zl of blood were injection after the time 0 sample had been drawn. Control added to 2 ml 2~) perchloric acid for subsequent glucose animals, shown by o--o (n = 6) received an equivalent a~ay and the ~cmaining 0.2--0.25 ml were added to volume of saline. Each point represents the mean of the EDTA for insulin assay. Additional 0.4 ml blood samples values obtained for each group. For clarity the standard were taken where applicable for plasma an(] red cell so- error of the mean is omitted, but significant differences, dium and potassium assay. compared with control values, are shown by *(p < 0.05) Acute experimena~: The diuretics were given as acute or t(P < 0.01). Abscissa: time after injection. Ordinate: IV boluses either alone, with, or thirty minutes before a blood glucose and plasma insulin expresced as percentage standard glucose dose of 0.5 g/kg body weight. The latter of zero (pre-injoction) value was given intravenously as a 50~/o solution (w/v) in 15 see and subsequent timing of samples "*'as made from the end of the glucose infusion. at I rain and 26% at 2.5rain with the lower dose Chronic experiments: Groups of animals were given (p. < 0.025), whereas with 2 mg/kg, there was a 53% de- daily intraperitonoal injections of either furosomide 2 mg/ crease at one minute, the suppression persisting until kg or 10 mg/kg amiloride. Control animals of similar 10 rain after injection, when the plasma insulin was weight were given an equivalent volume of intraperitoneal still 30% (p < 0.05) below the fasting value. A small saline. After 30 days (furosemidc) and 10 days (amiloride) intravenous glucose tolerance tests were performed, 24 h but significant rise in blood glucose occurred at twenty after the last dose of diuretic. minutes with both groups and persisted to 30 min with

3* 36 A. Aynsley-Green and K.G. Alberti: Diuretics and Carbohydrate Metabolism Diabetologia the higher dose. Absolute blood glucose and plasma animals, animals given furoscmide 1 mg/kg and 2 rag/ insulin values showed some variation between groups. kg simultaneously with glucose, and animals pretreated However, changes within groups were consistent. with furosemide 1 mg/kg 30 rain before glucose is shown No significant changes in plasma insulin or blood in Fig. 3. glucose occurred in animals injected with saline alone. Control animals showed a glucose disappearance b) Diabetic animals: No significant change occurred rate of 3.7 ' 0.27 ~ (Table 2). Glucose disappear- in insulin secretion in the "mild" diabetic group after ance was markedly decreased in the animals given furosemide 2 mg/kg and although fasting values were furosemide either with or before glucose (Table 2), within the normal range a sig~.cant rise in blood glu- although the 2.5 min blood glucose value in the low cose of 27% occurred after 20 min (Table 1). No change dose group and the 5 rain value in all the furosemide in blood glucose or plasma insulin occurred in the groups were significantly lower than in control animals ketotic hyperglycaemic animals (Table 1). (Fig. 3). However, the initial insulin response was

Table 1. Blood glucose and plasma insulin after acute administration of furosemide ( 4- S.E.M.)

Furosemide 2 mg/kg Furosemide 1 m~/kg Time Saline Normal Diabetic Adrenalec- Normal (rain) control mild moderate tomised a) Blood glucose rag/100 ml --10 784- 8 103+ 8 80• 2624-47 393=2 103:t:6 0 82+ 2 99i10 824- 8 321• 38• 97• 1 934- 6 97+ 4 81-4- 7 3202=28 36A=4 102=t=4 2.5 91=t= 9 99+ 5 83-4-13 316-4-27 40-4-4 107-4-3 5 78-4- 7 98~ 6 94+22 281+25 38• 110~4 10 73=t= 7 1124- 2 1004-24 3064-24 364-4 1174-3 c 20 754-11 1204- 3 b 1094- 5a 335+28 404-4 1203=6c 30 804-12 1232= 5 b 103111 326+22 38:J:5 ~ 1094-9 n=5 4 3 4 11 5 b) Plasma insulin ~U/rr~ --10 164-3 19+5 144-4 6.04-0.8 3.04-0.5 34-4-8 0 174-4 17-I-3 13-4-4 5.4~0.8 2.8=l=0.5 31• 1 15=1=3 84-2 c 12+3 5.9={=0.9 3.2=t=0.4 24-4-4b 2.5 15• 134-4 114-3 6.54-1.5 3.32=0.4 23• c 5 184-2 12-t-2 c 124-3 5.54-1.3 2.64-0.3 224-6 10 17• 124-3b 12=t=4 3.6-t-0.5 2.63=0.5 314-6 20 16=1=2 18=t=4 16+4 5.23=0.7 3.03=0.4 29-t-6 30 16-4-2 162=5 142=2 4.54-0.8 3.04-0.4 344-4 n=5 4 3 4 6 5

a p < 0.05 compared with control values; b p < 0.05 / e p < 0.01J for paired differences with zero value Mild diabetic animals received 60 mg/kg streptozotocin; moderate diabetic animals received 75 mg/kg stroptozotocin.

c) Adrenalectamised animals: Mean fasting blood much lower in the furosemide-treatcd animals (Table glucose and plasma insulin were significantly lower 2). This was not significant in the pretreated group. than in control animals (Table 1). Furosemide 2 mg/kg AI values were also lower, particularly in the 2 mg/kg did not affect either parameter, implying that the decrease in plasma insulin and hyperglycaemia may group confirming that the plasma insulin at the early have been mediated in normal animals by an adrener- times was inappropriately low for the plasma glucose gic or glucocorticoid effect. Small changes in insulin (Table 2). The thirty minute insulin area was decreased would not have been detected by the methods used. in all furosemide treated groups. However, a 30 to 50% change would have been dis- b) Adrenalectamised animals: There was no signifi- cerned. cant difference ill any parameter between control adrenalectomised animals given glucose alone, and II. The effects of acute administration of furosemide on animals given glucose together with furosemide 2 rag/ intravenous y~ucose tolerance kg (n -----3 for each group). Mean kg for control animals a) Norma/animals: The response of blood glucose was 2.30% /rain and for the furosemide group 2.33%] and plasma insulin to I.V. glucose (0.5 g/kg) in control min. Vol. 9, No. 1, 1973 A. Aynsley-Grccn and K.G. Alberti: Diuretics and Carbohydrate Metabolism 37

Table 2. The effects of acute administration of furosemide and amiloride on intravenous glucose tolerance (all values q- S.EiM.)

Saline control Furosemide Amiloride 1 mg/kg 2 mg/kg 1 mg 30 min 5 mg/kg 10 mg/kg 5 mg/kg before glucose 30 rain before glu- cose a) Glucose disappearance rate (kg) (%/min) 3.71• 2.14• 2.03• b 2.34q- 0.47 b 2.924-0.31 3.46• 3.27• b) Ten minute insulin area (tzU/mi/10 min) 827• 476• a 453• a 550+104 1618• 1753::k405 1896• c) Thirty minute insulin area (izU/ml/30 rain) 1189• 805~108 a 6924-63 b 9244-25 a 2194• b 31574-599 b 2407={=471a d) hi AG 1 min 1.43• 0.78• 0.77q-0.13 a 0.704-0.06 b 2.14• 1.80• 1.62• 2.5 min 1.08 ={=0.09 0.94• 0.63• a 0.83• 3.364- 1.63 1.92• 2.08• 5 min 0.86• 0.42• 0.74• 0.38• 2.17• 2.52• 2.12• 10 min 0.744-0.17 0.634-0.16 0.43• 0.40• 2.04• 1.20 1.71 -{-0.66 0.96• n= 5 5 5 5 4 5 4

a p < 0.05 / b p < 0.01J compared with control values

300 an equivalent daily injection of saline. No significant differences were found. ~_ 250 III. Cation changes after furosemide administration E 200 a) J~tring the glucose tolerance test: When furosemide 8 15o was given 30 rain before glucose, a significant fall in 01 plasma and red cell potassium had occurred at the zero ~ loo sample time. Red cell potassium had returned to con- m ear~ 5O Iii I I I I I trol values at the end of the test, although plasma -30 0 5 10 20 30 potassium was still decreased (Table 3). The group re- Time: min ceiving furosemide simultaneously with glucose showed no significant change. ~200 F b) After chronic administration: There was no significant difference in plasma, red cell or total body cation values. Total body sodium was 100• 8 rag/ 100 G body weight compared with 123 -4- 6 for controls, p : N.S. Total body potassium was 2PA• 17 rag/100 G body weight compared with 255 • 10 for controls, p: N.S. 30 0 5 10 20 30 Time: rain IV. The effect8 of acute administration of amiloride on Fig. 3. The effects of acute administration of furosemide blood glucose and plasma insulin on blood glucose and plasma insulin during the intraven- ous glucose tolerance test. Furosemide 1 mg/kg, shown by a) Normal animals: Amiloride was injected as a /k--/k (n= 5) or 2 mg/kg shown by []--E (n----5) was suspension in normal s~line at 1 mg, 5 mg and 10 mg/kg given by intravenous injection simultaneously with glu- (4, 20 and 40 ~M/kg respectively). A significant dose cose (0.5 g/kg) after time zero sample had been drawn. related increase in insulin secretion occurred one Control animals (o--o) (n= 5) received glucose alone. Furosemide 1 mg/kg, shown by O--@ (n = 5) was given minute after injection in all groups, which persisted to 30 min before glucose. Each point represents the mean of 2.5 rain after injection in the high dose group (Fig. 4). the values obtained for each group The one minute insulin values represent a 2, 5 and 7 fold increase in insulin concentration respectively. c) Chronic administration of furosemide: Intraven- Interestingly, no significant change in blood glucose ous glucose tolerance tests were performed after the occurred. daily intraperitoneal injection of furosemide 2 mg/kg b) Diabetic animals: ~n the milder diabetic group for 30 days, and on a control group which had received (60 mg/kg streptozotocin) amiloride, 10 mg/kg, caused 38 A. Aynsley-Green and K.G. Alberti: Diuretics and Carbohydrate Metabolism Diabetologia

a significant rise in plasma insulin, but this was only peritoneal injection of amiloride 10 mg/kg for 10 days. an increase of 25 FU/ml, compared with 213 FU/ml for Fasting blood glucose was significantly elevated in the the same dose in normal animals (Table 4). No change experimental group with a slight elevation in plasma in plasma insulin occurred in the ketotic hyperglycae- insulin (Fig. 6). Glucose disappearance was markedly mic group. As both diabetic groups contained very enhanced (kg 2.96 • 0.14 for controls, 4.16 4-0.17 for small numbers the results only show a trend and do the amiloride treated group; p<0.005), and was as- not allow statistical comparison. sociated with a significant elevation in peak initial in-

~i~176 250 o 90 g 200 8 = ?oh ~(2,--~'~C--.- ----~ .9.o E 60t ,"- m r ~ I rn 0 5 10 20 30 E 150 Time: min o 250-- i' _~ ioo ( aim _8 50 m 0 ,_.1~: I I I I J -30 0 5 10 20 30 lOON \ Time: rain

t 300 - , I' 0 5 10 2o 30 Time: rain Fig. 4. The effects of acute administration of amiloride on blood glucose and plasma insulhr in normal animals. OoOi) ~ Amiloride 1 mg/kg (n=3), shown by O--I, 5 mg/kg (n-: 6), shown by m-m or 10 mg/kg (n = 5) shown by A--A, was given as an acute intravenous injection after the time 0 sample had been drawn. Control animals, shown by o--o (n = 5) received saline alone. Each point represents the mean of the values for each gzoup. For 0 clarity, the standard error of the mean is omitted, but -30 0 5 lO 20 30 significant differences compared with control values are Time: min shown by *(p < 0.05) or t(P < 0.01) Fig. 5. The effects of acute administration of amiloride on blood glucose and plasma insulin during the intravenous c) Adr~ised animals: A significant increase glucose tolerance test. Amiloride 5 mg/kg, shown by. - in insulin secretion occurred 1 rain after the injection (n = 4) or 10 mg/kg, shown by A--A (n = 5) was given by of 10 mg/kg amiloride (Table 4). There was marked intravenous injection simultaneously with glucose (0.5 G/ attenuation of the absolute response compared with kg) after the time zero sample had been drawn. Control animals, shown by o--o (n = 5) received glucose alone. normal animals, but it represents a seventeen fold in- Amiloride 5 mg/kg shown by Q--O (n = 4) was given crease in plasma insulin compared with a nine fold in- 30 rain before glucose. Each point represents the mean of crease in the normal group. the values for each group. For clarity significant differ- ences in plasma insulin compared with control values are shown only for the 1 min values *(p < 0.05, ~fp < 0.01). V. The effects of acute administration of amiloride on The plasma insulin values in all the amiloride treated iutravenou~ glucose tolerance groups at 2.5 min were also significantly increased (p < 0.05) a) Normal animals: The response of blood glucose and plasma insulin to I.V. glucose (0.5 g/kg) in control sulfa response and 5 min insulin area.(481 +23 ttU animals, animals given amiloride 5 mg/kg and 10 rag/ ml-i 5 min -1 for controls, 845+ 124 izU m1-1 5 min -1 for kg simultaneously with glucose, and animals pretreated the amiloride group; p < 0.025). with amiloride, 5 mg/kg, 30 rain before glucose is shown in Fig. 5. Absolute blood glucose values in the amiloride V I. Cation changes after amiloride administration treated groups were not significantly different from con- trol values. Glucose disappearance rate was not af- a) During the glucose tolerance test after acute ad- fected. However, all the amiloride treated groups ministration of amiloride: No significant change in red showed a highly significant increase in total 30 rain cell or plasma cation values between control IVGTT insulin area (Table 2). and the high dose amiloride IVGTT occurred during b) The effecta of chronic administration of amilorlde the test. on intravenous gluc,~e tolerance: Intravenous glucose b) After chronic administration: Cation values are tolerance tests were performed after the daily intra- shown in Table 5. In contrast with furosemide there Vol. 9, No. 1, 1973 A. Aynsley-Green and K.G. Alberti: Diuretics and Carbohydrate Metabolism 39

is a higMy significant increase in plasma potassium in chronically, could not show any effect. Foy and Furman the experimental group (7.0 mEq/1 compared with [15] found hyperglycaemia, glucose intolerance and 4.2 mEq/l p,~0.01), associated with increased total impaired insulin response in the mouse. Most of these body potassium. studies can be criticized because of the use of very

Table 3. The effect~ of acute administration of furosernide on red cell and plasma sodium and potassium during the intravenous glucose tolerance test (• S.E. ~.)

Time Red cell (tzg/g) Plasma (mEq/1) (min) Sodium Potassium n Sodium Potassium n Control 0 4714- 16 34964-360 36 1374-1 4.84-0.1 36 30 5474- 88 32134-153 6 138=]=1 4.6-t-0.2 6 Furoscmide 2 mg/kg 30 4824- 87 32924- 98 5 13~4-2 4.04-0.1 5 1 mg/kg 30 min before glucose 0 541• 31564-112 a 5 136• 4.0=]=0.2b 5 30 459-}- 39 3139• 77 5 1394-2 3.84-0.1 a 5

a p < 0.05 / b p < 0.01] compared with control values

Table 4. Blood glucose and plazma insulin after acute administration of amiloride (4- S.E.M.)

Amiloride 10 mg/kg Time Saline Normal Diabetic Adrenalectomised (min) control mild moderate a) Blood glucose (rag/100 ml) --I0 784- 4 684-4 704- 8 3204-30 434-5 0 824- 2 724-6 684-17 330• 414-6 1 934- 6 654-2 83-t- 15 315• 564-9 2.5 91-4- 9 734-5 68-4- 5 3614-58 464-7 5 784- 7 794-7 614- 5 3414-48 424-7 10 734- 7 79• 674-12 3624-78 424-7 20 754-11 894-5 724- 7 3494-76 41• 30 804-12 924-6 904- 4 3774-58 434-9 n----5 5 2 3 3 b) Plasma insulin (IzU/ml) --10 16• 27• 6 11• 6-}-1 3• 1 0 17-4-4 334- 3 104-I 74-1 24- 0.4 1 154-3 246-4-18b 354-1 54-2 34___21c 2.5 154-2 142i16 b 22-t- 1 6-~1 23• c 5 184-2 47-{- 12 a 18-4-6 5-f-1 5• 1 10 174-3 41-t-7 a 154-5 64-1 3-*- 1 20 164-2 424-3 a 134-4 4-{-I 34- 1 30 16• 464-5 a 134-5 5• 4,a- 1 n~5 5 2 3 3

a p < 0.05 / b p < O.OlJ compared with controls r p < 0.01 for paired differences with zero value; p values significant but not stated for mild diabetic group as n only 2. Mild diabetic animals received 60 mg/kg streptozotocin; moderate diabetic animals received 75 mg/kg streptozotocin.

Discussion large doses and the use of anaesthesia, which in the rat is known to profoundly affect blood glucose Previous studies in the rat have shown hypergly- and plasma insulin [26, 27, 28]. In addition, early eaemia and impaired glucose tolerance after acute sequential changes in insulin and glucose following administration of furosemide (50--200 mg/kg) [9, 14, diuretic administration have not been studied. 24], while chronic administration (100 mg/kg/day) Our studies in the conscious rat have shown that resulted in glucose intolerance [25]. In contrast, Senft low doses of furosemide suppress plasma insulin in- et al. [12] using up to 200 mg/kg acutely or 200 mg/kg stantly with subsequent hyperglyeaemia. The rate of 40 A. Aynsley-Green and K.O. Alberti: Diuretics and Carbohydrate Metabolism Diabetologia

fall of plasma insulin is of particular interest in that the secretion since it persisted in diabetic animals without normal half-life of insulin quoted in the literature is of a change in plasma insulin. the order of 3--4.8 min [29]. Our data suggest a half- Possible hepaticmechanisms include catecholamine life of about one minute. This disappearance rate stimulation of glycogenolysis or inhibition of phospho- could have been artifically enhanced by an extremely diesterase by furosemide, as suggested by Senft [12]. rapid shift of fluid into the intravascular compartment. This hypothesis would explain the lack of effect on blood glucose in the ketotic hyperglycaemic animals in whom hepatic glycogen stores would be decreased. -~ 2501 Decreased peripheral utilisation of glucose caused by 8 furosemide has also been reported by Weller and Bo- ~-~m 200 E rondy [25]. 150 A change in glucose space is implied by the decreas- o ed absolute blood glucose values during the IVGTT 100 with furosemide. A decreased glucose stimulus at the fl cell could then result in an apparent decrease in insulin _8 5o j-f m secretion. However, the ~ ratio shows that the plasma I I l I I 0 5 10 20 30 insulin response was still reduced. Time: min Insulin release could have been suppressed by a direct effect on the pancreas, through an effect on ~400 - electrolyte exchange which is of major importance in % insulin release [30]. However, red cell and plasma sodium and potassium showed minimal changes in the present study, although this does not preclude effects ~ 200 on the fl cell which need not be reflected in changes in the plasma, nor be analagous to red cell effects. loo The absence of any effect on glucose tolerance after OL chronic administration and the absence of significant 0 5 10 20 30 changes in tissue cations supports the observation in Time: min man that chronic administration of furosemide does Fig. 6. The effects of chronic administration of amiloride not affect glucose tolerance [3]. on blood glucose and plasma insulin during the intra- In contrast, amiloride is a potent insulin secretago- venous glucose tolerance test. Animals received amiloride 10 mg/kg (shown by 0--0) or an equivalent volume of gue, with or without exogenous glucose. When given saline (shown by o--o) by daily intraperitoneal injection acutely alone, it caused a dose related increase in for 10 days. Intravenous glucose (0.5 g/kg) was given 24 h plasma insulin. Between 20 and 40 FtM/kg gave a after the last injection. Each point represents the mean of similar peak insulin response to 2800 [zM/kg of glucose. the values obtained for each group (n = 4 for both groups). Significant differences compared with control values are The insulinogenic effect of a glucose load was poten- shown by *(p < 0.05) or ~(p < 0.01) tiated both by the concurrent injection of amiloride,

Table 5. Red cell, plasma and total body ,od.ium and potassium after chronic admini,s- tra$ion of amiloride ( • S.E.M.)

Red cell (~tg/g) Plasma (mEq/1) Total body (mg/lO0 g B.W.) Sodium Potassium n Sod• Potassium n Sodium Potaasium n Control group 425• 4134• 4 147• 4.2• 4 90• 181• 6 4 Amiloride treated group 491• 3734:E139a4 141• 7.0-t-0.3 b 4 78:t:4 237:h17 a 6

p < o.051 b p < O.O1J compared with controls

Adrenaleetomy abolishes the furosemide effects, im- and by pretreatment. Chronic administration of plying that in normal animals they are secondary amiloride also enhanced glucose stimulated insulin to catecholamine release. Foy and Furman [15] have, secretion. suggested this mechanism in the mouse. However, the Studies in man have given conflicting results on the hyperglycaemia is not due to a decrease in insulin effect of amiloride on blood glucose [31, 32, 33, 34, 35] Vol. 9, No. 1, 1973 A. Aynsley-Green and K.G. Alberti: Diuretics and carbohydrate Metabolism 41 without investigating its effect on plasma insulin. No 7. Meng, K., Kroneberg, G. : Erh6hung des Blutzuckers effect on glucose tolerance was reported by PySr/~l/i der Ratte durch Saluretica. Arch. exp. Path. Phar- makl 247, 351--352 (1964). and Rantanen [36] while McNay et al. [37] showed 8. Meng, K., I~roneberg, G.: Untersuchungen an der increased glucose disappearance and I/G ratios. Ratte zur Frage der diabetogenen Wirkung yon Salu- The lack of early changes in blood glucose suggests retica. Arch. exp. Path. Pharmak. 251, 433--444 direct fl cell stimulation by amiloride. The diuretic (1965). promotes natriuresis and decreases potassium and 9. Wales, J.K., Grant, A., Wolff, F.W.: Studies on the hyperglycaemic effects of nonthiazide diuretics. J. ion secretion by an effect on the renal tubule Pharmacol. exp. Ther. 159, 229:-235 (1968). [18]. In frog skin [38] and toad bladder [39] amiloride 10. Weller, J.M., Borondy, M.: Effects of benzothiadia- inhibits sodium entry on the mucosal side of the cell. zinc drugs on carbohydrate metabolism. Metabolism Inhibition of the sodium pump is not involved [18]. 14, 708--714 (1965). A structural feature common to amiloride and 11. Watson, L.S., van Pelt, S.M., Winter, C.A. : Effect of on blood glucose of rats. Fedn. Proc. other compounds affecting apical cell sodium transport 23, 438 (1964). in amphibia is the presence of a guanidine group, 12. Senft, G., Losert, W, ~Schultz~ G., Sitt, R., Bartelhei- which has a close structural relationship to the triply mer, K.H. : Ursachcn der St6rungen in Kohlchydra~- hydrated sodium ion [40]. An effect of amiloride on stoffwechsel unter dem EinfluI] sulfonamidierter Dinretica. Arch. exp. Path. Pharmak. 255, 369--382 active potassium [41] and sodium [34, 42] transport in (1966). the human red cell has also been described. It is 13. Tabachnik, I.I.A., Gulbenkian, A., Yannell, A. : The possible that the action of amiloride reflects a direct hyperglycaemic activity of benzothiadiazine and other effect on fl cell sodium and/or potassium exchange. diuretics. Life Sci. 4, 1931--1936 (1965). 14. Foy, g. M. : Acute diuretic induced hyperglycaemia in Other guanidine derivatives, including arginine, rats. Life Sci. 6, 897--902 (1967). stimulate insulin secretion in vivo and in vitro [43, 44, 15. Foy, J.M., Furman, B.L.: Effects of diuretics on 45, 46]. Amiloride shows similarities with arginine in mouse blood sugar following single dose administra- that they both have synergistic effects with glucose, tion. Br. g. Pharmaeol. 41, 287--297 (1971). and both inhibit sodium transport in the toad bladder 16. Corm, J.W. : , the potassium ion and impaired carbohydrate tolerance. New Engl. J. Med. (Alberti, K.G.M.M. -- unpublished observations). 27a, 1135~-1143 (1965). Thus it is possible that the stimulation of insulin secre- 17. Rapoport, M. I., Hurd, I-I. 1~. : Thiazide induced glucose tion by all these compounds is due to the ionic effects intolerance treated with potassium. Arch. int. Med. of the guanidine group. exp. 113, 405--408 (1964). 18. Baer, J.E., Jones, C.B., Spitzer, S.A., Russo, H.F.: Acknowledgements. A.A.G. was the recipient of a Well- The potassium sparing and natriuretic activity of N- come Junior Research Fellowship and the Beaney Scholar- amidino- 3,5-diamino- 6-chloropyrazinecarboxamide ship in Materia Medica from Guy's Hospital, London. hydrochloride dihydrate (amiloride hydrochloride). J. K.G.M.M.A. t[mnks the Wellcome Trust for financial Pharmac01. exp. Ther. 157, 472--485 (1967). support. The authors thank Hoechst Pharmaceuticals 19. Soeldner, J.S., Slone, D.: Critical variables in the Ltd., Merck, Sharpe & Dohme Ltd., and the Peel Medical radioimmunoassay of serum insulin using the double Research Trust, for financial grants, Miss H. Kuresova antibody technic. Diabetes 14, 771- 779 (1965). and Miss S. Warne for technical assistance, and Professor 20. Aynsley-Green, A., Alberti, K. G. M. M. : Serum-insulin P. B. Beeson for facilities to carry out this work. or Plasma-insulin ? Lancet 1972 I, 318- 319. 21. Bergmeyer, H.U., Bernt, E.: Methoden der enzyma- tischen Analyse, p. 123. Weinheim: Verlag Chemic l~eferenees 1962. 22. Ikkos, D., Luft, R. : On the intravenous glucose toler- ance test. Acta. endocr. Coperrh. 25, 312--334 (1957). 1. Peters, G., Roch-Ramel, F. : Handbook of Experimen- 23. Seltzer, H.S., Allen, E.W., Herron, A.L., Jr., Bren- tal Pharmacology. Band XXIV Diuretica, p. 337-- nan, M.T. : Insulin secretion in response to glycemic 341. Berlin-Heidelberg-New Yorlr Springer 1969. stimulus: Relation of delayed, initial release to car- 2. Wales, J.K., Viktora, J.K., Wolff, F.W.: The effect of in normal subjects receiving bohydrate intolerance in mild diabetes mellitus. J. high fat or high carbohydrate diets. J. Amer. Med. clim Invest. 46, 323--335 (1967). Sci. 254, 499--505 (1967). 24. Formanek, K., Kenner, T.:BritSP ecial features of the 3. Anderson, J., Godfrey, B. E., Hill, D. M., Munro-Faure, action of a new dim'eric, ,. J. Pharmacol. Chemo- A.D., Sheldon, J.: A comparison of the effects of ther. 2fi, 27--33 (1966). hydrochlorothiazide and of Frusemide in the treat- 25. Weller, J.M., Borondy, M. : Effect of Furosemide on ment of hypertensive patients. Quart. J. Med. 40, glucose metabolism. Metabolism 16, 532--536 (1967). 541--560 (1971). 26. Biebuyck, J.F., Alberti, K.G.M.M.: Influence of 4..Kohner, E.M., Lowy, C., D011ery, C.T., Schumer, B. : altered dietary and endocrine states on the effects of Effects of diuretic therapy on glucose tolerance in anaesthetic agents on serum insulin and blood meta- hypertensive patients. Lancet 1971 I, 986--990. bolites. Submitted for publication (1972). 5. Zamrazil, V., Dvorak, V., O~kera, J., Zamrazilova, E. : 27. Aynsley-Green, A., Biebuyck, J.F., Alberti, K.G.M. The infuence of hydrochlorothiazide upon glucose M. : Anaesthesia and insulin secretion : A comparative tolerance test after prednisone. Metabolism 16, 445-- study on the effects of , , pen- 450 (1967). tobarbitone sodium and hydrochloride on 6. Kadota, I., Nishimura, Y,, Ishigani, R., Yoshida, A. : intravenous glucose tolerance and insulin secretion in Abnormal carbohydrate metabolism induced by the rat. Submitted for publication (1972). thiazide diuretics. Folia endocr, jap. 40, 277--280 28. Hawkins, R.A., Alberti, K. G. M. M., Houghton, C.R. (1964). S., Williamson, D.H, Krebs, H.A.: The effect of 42 A. Aynsley-Green and K.G. Alberti: Diuretics and Carbohydrate Metabolism

acetoacetate on plasma insulin concentration. Bio- active sodium transport by the isolated frog skin: chem. J. 125, 541--544 (1971). Evidence concerning site of action Br. J. Pharmac. 29. Sonksen, P.H., Turner, R.L., Grayburn, J.A., Chemother. 38, 702--718 (1970). Newman, G.B., Nabarro, J.D.N.: Measurement of 39, Bentley, P.J. : Amiloride : a potent inhibitor of sodium insulin delivery rate in man. J. elin. Endoe. Metab. 33, transport across the toad bladder. J. Physiol. (Lend.) 279--286 (1971). 195, 317--330 (1968). 30. Hales, C.N., Milner, R.D.G.: Cations and the secre- 40. Fastier, F. 1~. : Structure-activity relationships of ami- tion of insulin from rabbit pancreas in vitro. J. dine derivatives Pharmacol. Rev. 14, 37 -- 90 (1962). Physiol. (Lend.) 199, 177--187 (1968). 41. Sachs, J.R., ~'elt, L.G.: Concentration dependence 31. Rorive, G., Hagemeijer, F., Luyckx, A., Van Cawen- of active potassium transport in the human red blood berge, H. : Etude elinique d'un nouveau diur~tique cell in the presence of inhibitors. J. clin. Invest. 47, doud d'un effet d'epargne potassique: le chlorhydrate 949--959 (1968). d'amiloride (MK 870). Presse Mdd. 76 (23), 1127- 1128 42. Wessels, F. : Der EinfluB yon Etacryns~ure und Ami- (1968). lorid auf den Natriumtransport der Erythrocyten des 32. Esch, I.: Die Behandlung dekompensierter Herzkran- Menschen in Vitro. Arzneimittel-Forseh. 19, 1959-- ker mit einem neuen Kalium konservierenden Diureti- 1961 (1969). kum. Wien med. Wschr. 118 (50), 1074--1078 (1968). 43. Alsever, R.N., Georg, R.H., Sussman, K.E. : Stimula- 33. Brailovsky, D., Kukolj, S., Rebolledo, H.: Use del tion of insulin secretion by Guanidinoacetic acid and clorhidrato de amilorida (MK 870) en el.tratamiento y other Guanidine Derivatives. Endoerinqlogy 86, 332 prevencion de la deplecion de potasio. Revta. reed. 336 (1970). Chile 95, 299--303 (1967). 44. Malaisse, W.J., Tinant, A., Ooms, H.A., Franckson, 34. Gross, W. : Wechselwirkungen zwischen Kalium und J. R.M. : Metabolic effects of ~-guanidinobutyramide. Glukose in Erythrocyten und Serum nach Gabe yon I. In vivo study in anaesthetised dogs. Hormone Amilorid und Etacryns~ure. Sixth Symp. Ges. Ne- Metab. Res. 1, 258--265 (1969). phrol. Vienna 1968, p. 323--327. Wien: Verlag Wien 45. Malaisse, W.J., Mandelbaum, I.M., Frahekson, J.R. Med. Akad. 1969. M.: Metabolic effects of ~-guanidinobutyramide. II. 35. Lant, A., Smith, A.J., Wilson, G.M.: Clinical evalua- In vitro studies on Muscle, Adipose tissue and the tion of amiloride, a potassium sparing diuretic. Clin. Endocrine pancreas. Hormone Metab. Res. 2, 21 : 27 Pharmac. Ther. 1O, 50--63 (1969). (1970). 36. PySr/~l/~, K., Rantanen, I. : Effect of amiloride hydro- 46. Blazquez, E., Lopez-Quijada, C., Candela, J.L.: The chloride (MK 870) on blood pressure, serum electroly- effect of ~-guanidinobutyramide on the secretion and tes and glucose tolerance in patients with mild hyper- synthesis of insulin in vitro. Diabetologia 6, 447" 448 tension. An. Med. intern. Ferm. 57, 91--97 (1968). (1970). 37. McNay, J., Hornbrook, K.R., Oran, E.: Intolerance A. Aynsley-Green, M.B. of diabetia patients to positive K~ balance despite The United Oxford Hospitals enhaneemv.,t of glucose disappearance rate. J. clin. Nuffield Dept. of Clinical Medicine Invest. 47, 68A (1968). The Radcliffe Infirmary 38. Salako, L.A., Smith, A.J.: Effects of amiloride on Oxford England