Oral vanadyl sulfate in treatment of diabetes mellitus in rats

Oral vanadyl sulfate in treatment of diabetes mellitus in rats SASANKA RAMANADHAM, JEAN JACQUES MONGOLD, ROGER W. BROWNSEY, GERARD H. CROS, AND JOHN H. McNEILL Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, and Department of Biochemistry, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia V6T 1 W5, Canada; Laboratoire de Pharmacodynamie, Faculte de Pharmacie, 34060 Montpellier, France

RAMANADHAM,

SASANKA, JEAN JACQUES MONGOLD,

ROGER

W. BROWNSEY, GERARD H. CROS, AND JOHN MCNEILL. vanadyl

sulfate in treatment

of

diabetes mellitus

Oral in rats. Am. J.

Physiol. 257 (Heart Circ. Physiol. 26): H904-H911, 1989.Recent reports have suggested that vanadium in the form of vanadyl (+IV) possessesinsulin-like activity. Therefore, in the present study we examined the effects of administering oral vanadyl to diabetic animals. Wistar rats made diabetic with streptozotocin and age-matched controls were maintained for 10 wk in the absence and presence of vanadyl sulfate trihydrate in the drinking water. In the presence of vanadyl, decreases in rate of growth and circulating levels of insulin were the only significant alterations recorded in control animals. In contrast, diabetic animals treated with vanadyl, despite having lower body weights and insulin levels, had normal plasma concentrations of glucose, lipid, creatinine, and thyroid hormone. In addition, abnormalities in isolated working heart function and glycerol output from adipose tissue of diabetic animals were also corrected after vanadyl treatment. These results suggest that vanadium when used in the vanadyl form is effective in diminishing the diabetic state in the rat by substituting for and replacing insulin or possibly by enhancing the effects of endogenous insulin. streptozotocin; hyperglycemia; heart function; glycerol output

VANADIUM, a group V trace element (mol wt 50.9) has been reported to be essential for normal growth in the chick and rat (16,17,33). Although the normal intake of vanadium in humans has been reported to be between 12.4 and 28.0 fig/day (34), at present it is not known what harmful effects, if any, are caused by reduced intakes of vanadium in humans. Vanadium, because of its existence in several valence states (+I1 to +V), exhibits discrete physiological effects. For example, it has been reported that the vanadate (+V) form inhibits Na+-K+ transport and that the vanadyl (+IV) form produces insulin-like effects (4, 5, 31). The insulin-like effects have been suggested to be produced by alteration of the protein kinase activity associated with the insulin receptor (38) and/or inhibition of phosphotyrosine phosphatase activity (6). The recognized insulin-like effects of vanadium include inhibition of hepatic cholesterol and phospholipid synthesis (8, 32), activation of glucose transport and H904

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$1.50

Copyright

oxidation in rat adipocytes and skeletal muscle (7, 13, 30), activation of glycogen synthase activity in rat adipocytes (36), and enhancement of glycogen synthesis in the liver and diaphragm (37). The insulin-like effects appear to coincide with the presence or formation in vivo of vanadyl from vanadate (4, 9, 13, 20, 23, 31). Thus vanadium may potentially offer a novel approach to treatment for diabetes, especially in light of the development of resistance in the diabetic population to insulin after prolonged treatment. As early as 1899, Lyonnet et al. (22) observed a decrease in the urinary output of glucose in two of three diabetic patients given sodium vanadate. This glucoselowering effect of vanadate in diabetes was “rediscovered” in our laboratory by Heyliger et al. (15). In the latter study, elevated plasma glucose levels in female rats made diabetic with streptozotocin were returned to control levels after daily oral administration of sodium ovanadate for 4 wk. Although not noted in the published study of Heyliger et al. (X5),later work in our laboratory and that by others revealed that the concentration of vanadate used in the drinking water (0.80 mg/ml) was toxic to some animals, resulting in severe diarrhea and death because of dehydration. Meyerovitch et al. (24) recently reported that the concentration of sodium o-vanadate that could be tolerated in the drinking water without significant toxicity was 0.2 mg/ml. However, Paulson et al. (26) have reported that a higher concentration of vanadate (0.80 mg/ml) was not toxic when the solution administered to rats was buffered to pH 7.0. At this neutral pH, some of the vanadate would be expected to exist as vanadyl. Furthermore, the 50% lethal dose (LD& of sodium ovanadate in the rabbit, guinea pig, mouse, and rat has been found to be 6-10 times lower than that of vanadyl sulfate (18, 37). The observations described above suggest that the vanadyl rather than the vanadate form may be more appropriate for chronic use in diabetic rats. We therefore investigated the effectiveness of chronic vanadyl treatment on metabolic and hormone status, as assessed by plasma parameters, and on the function of cardiac muscle and adipose tissue in streptozotocin-diabetic Wistar rats.

0 1989 the American

Physiological

Society

ORAL

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blotted, and extracted in ice-cold buffer (pH 7.4) containing tris(hydroxymethyl)aminomethane (Tris; 10.00 Treatment and Maintenance mM), 3-( N-morpholino)propranesulfonic acid (MOPS; For these studies two groups of male Wistar rats (175- 20.00 mM), sucrose (250.00 mM), ethylene glycol-bis(Pacid (EGTA; 200g) were used. One group of animals was injected with aminoethyl ether)-N,N,N’,N’-tetraacetic 2.00 mM), glutathione (7.50 mM), pepstatin and leupepstreptozotocin (STZ, 60 mg/kg iv) to induce diabetes tin (1.00 pg/ml each), benzamidine (2.50 mM), and somellitus, and the second group received only the vehicle dium azide (0.02% wt/vol). Extractions were carried out (0.1 M citrate buffer, pH 4.5) and served as age-matched control animals (CON). After 3 days, blood glucose levels using 3 ml buffer/g wet weight tissue with a Polytron homogenizer at a setting for 6 for 5 s. Homogenates were were measured using a glucometer, and animals with immediately centrifuged (12,000 g for 5 min) at 4”C, and levels X3.75 mM were considered diabetic. The control fractions were separated from fat cake and and diabetic animals were each then subdivided into two infranatant of 32P incorporation into further groups: one group was given plain tap water to pellet before determination drink (CON and STZ), and the other group was given proteins (described in detail below). Glycerol determinations. After the removal of incudrinking water containing vanadyl sulfate trihydrate (VST, 1 mg/ml; CON-T and STZ-T). The animals were bated adipose tissue, samples (1 ml) of bicarbonatecaged in groups of two or three and were maintained for buffered incubation medium were heated at 95°C for 10 10 wk with free access to food and drink. During this min and after chilling on ice were centrifuged (12,000 g for 5 min) before storage (-80°C for up to 3 days). period, body weight and average fluid intake were monwere carried out on deproteinitored. The average amount of vanadyl intake by each Glycerol determinations kits employing the animal in the treated group was then calculated by ized samples, using commercial multiplying the average volume consumed (by each ani- method of Garland and Randle (14). Incorporation of 32P into proteins of adipose tissue mal) by the concentration of vanadyl used (1 mg/ml). supernatant fractions. Samples of supernatant fractions (20-30 ~1 containing -100 pg protein) prepared from Working Heart Perfusion adipose tissue as described above were incubated in a Myocardial dysfunction is a well-known consequence final volume of 50 ~1 containing MgC1, (5 mM) for 2 min of diabetes. Therefore, the working heart preparation at 30°C and for a further 10 min at 30°C after addition was used to assess the ability of chronic vanadyl treatof [T-~~P]ATP (100 PM; 500-1,000 disintegrations ment to prevent abnormalities in myocardial performmine1 pmol-l). Reactions were stopped by addition of ance. After 10 wk of maintenance in the absence and 0.5 ml ice-cold trichloroacetic acid (10% wt/vol). After presence of vanadyl, the animals were killed by decapiincubation at 0°C for 60 min, protein pellets were retation. The heart was immediately isolated and placed covered by centrifugation (12,000 g for 2 min) and diin a well-oxygenated (95% 02-5% COz) buffer containing gested (10 min at 95°C) in sample buffer (pH 6.8) con(in mM) 120.0NaCl, 5.6 KCl, 2.2 MgCl,, 19.0NaHC03, taining Tris HCl (125 mM), sodium dodecyl sulfate 2.4 CaC12, and 10.0 glucose. Heart function was moni- (SDS; 5% wt/vol), and bromophenol blue (0.02% wt/ tored using a working heart apparatus as described pre- vol). Digested protein samples were applied to separate viously (37). The heart was allowed to equilibrate at 37°C tracks (1 cm) of SDS-polyacrylamide slab gels and dewhile being paced (300 beats/min) for 15 min. Measureveloped at 15 mA/slab for 1 h followed by 30 mA/slab ments of left ventricular developed force (LVDP), rate for the remaining 3-4 h, according to the discontinuous of force development (+dp/dt), and rate of relaxation pH gel system (19). After electrophoresis, the gels were (-dp/dt) were then obtained at various left atria1 filling fixed, stained, and destained as described previously (3). pressures (5.0-22.5cmH20). Radioautographs were obtained from gels by exposure at -70°C to preflashed X-omat XAR film for 2-5 days, using “rigid form” cassettes (Halsey Products, Brooklyn, Incubation of Epididymal Adipose Tissue NY) containing Du Pont intensifer screens (Cronex Hiand Preparation of Extracts plus). Incorporation of 32P into phosphoproteins was The effects of vanadyl treatment on the lipolytic activestimated by densitometric scanning using a Bio-Rad ity of adipose tissue (assessed by measuring rates of video densitometer, and maximum peak absorbances glycerol release) and on protein phosphorylation were were within the range of 0.5-1.5 units. Protein loading determined in vitro. Epididymal fat pads were removed of gels was determined by also scanning the Coomassie at the time of death and were immediately incubated at blue-stained gels. 37°C (with shaking) in Krebs-Henseleit bicarbonatebuffered medium that contained glucose (11 mM) and Plasma Analyses CaC12 (1.25 mM) as described previously (1). This preincubation was carried out for 45 min with -10 ml medium/ Various plasma parameters, which are known to be g wet wt tissue by which time adipose tissue metabolism affected in the diabetic rat, were measured to determine reaches a basal steady state. The pads were then transthe effectiveness of chronic vanadyl treatment in preferred to fresh medium of the same composition for venting these alterations. At the time of death, blood was experimental incubation in the absence or presence of collected into heparinized tubes and centrifuged (3,000g insulin (0.5 pg/ml, as described in Ref. 1). After incubafor 25 min) at 4°C. The plasma fraction was removed tion for an additional 45 min, the pads were removed, and frozen at -2OOC until the following assays were METHODS

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l

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performed: glucose, triglyceride, cholesterol, lipid, creatinine, insulin, and Td.

SULFATE

AND

phospho-

Statistical Methods

All results were expressed as means and standard error of the mean. Comparisons between means of the different groups were made using multi-way analysis of variance followed by the Duncan’s test. Comparisons between means within each group (glycerol output t insulin) were made using the Students’ unpaired t test. A probability value of P < 0.05 was considered to indicate significant difference between means.

DIABETES

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Materials

Streptozotocin was kindly donated by Upjohn. The kits for glucose, triglyceride, cholesterol, phospholipid, creatinine, and glycerol were obtained from Boehringer Mannheim. The radioimmunoassay kits for insulin and T4 and [T-~~P]ATP were obtained from Amersham International (Oakville, Ontario). Biochemical reagents were obtained from Sigma Chemical (St. Louis, MO). Laboratory grade chemicals and vanadyl sulfate trihydrate were obtained from BDH or Fisher Scientific (both of Vancouver, BC). Materials for gel electrophoresis were obtained from Bio-Rad (Mississauga, Ontario). RESULTS

Body and Heart Weights and Fluid and Vanadyl Intake

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Body weight and fluid and vanadyl intake values recorded in the CON and STZ animals during the lo-wk maintenance period in the absence and presence (T) of vanadyl sulfate trihydrate (1 mg/ml) are shown in Fig. 1. The rate of growth in all groups was decreased relative to the CON group. Vanadyl treatment significantly decreased the growth of the CON-T group but not of the STZ-T group (Fig. 1A and Table 1) relative to their agematched counterparts (CON and STZ). Intakes of fluid and vanadyl by the animals are presented in Fig. 1, B and C. Fluid intake, as expected, was markedly increased in the STZ group given only water compared with the CON group. During treatment with vanadyl, fluid intake was unaffected in the CON-T group but was lowered in the STZ-T group to the level of the CON group. Vanadyl intake (expressed as average selfadministered dose per animal) was higher in the STZ-T group than in the CON-T group in the first week of treatment, but during the remaining period of monitoring, vanadyl intakes were similar in the two groups. Measurements recorded in the CON and STZ animals at the end of the 10 wk of maintenance in the absence or presence (T) of vanadyl sulfate trihydrate are described in Table 1. Body and heart weights recorded in the STZ, CON-T, and STZ-T groups were significantly decreased relative to the CON group. The ratio between the two weights, however, was elevated only in the untreated diabetic (STZ) group as compared with the control group.

(T) 31

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DAYS FIG.

during treated period 1 mg/ml) followed intake.

1. Body weight and fluid and vanadyl intake measurements study period. Data were obtained from streptozotocin (STZ)and age-matched control (CON) animals during maintenance in absence and presence (T) of vanadyl sulfate trihydrate (VST, in drinking water and were analyzed using analysis of variance by Duncan’s test. A: body weight; B: fluid intake; C: vanadyl or, Significantly different from CON-T group (P < 0.05).

Plasma Glucose and Insulin

As expected, the elevation in glucose levels of the STZ group was accompanied by a decrease in circulating levels of insulin (Fig. 2). The concentration of glucose in plasma of rats after vanadyl treatment was unchanged in the CON-T group, although interestingly, a decrease in the insulin levels were observed. In contrast, the concentration of glucose in the plasma of diabetic animals treated for 10 wk with vanadyl (STZ-T) was normalized, although insulin levels remained decreased. Other Plasma Measurements

The concentrations of thyroid hormone, creatinine, and lipid measured in samples of plasma are shown in Table 1. In the STZ group plasma levels of T4 were significantly decreased, whereas creatinine values were significantly increased relative to the CON group. After treatment with vanadyl, plasma levels of T4 and creatinine remained unchanged in the CON-T group, and they were returned to control levels in the STZ-T group. Plasma concentrations of triglyceride, cholesterol, and

H907

ORAL VANADYL SULFATE AND DIABETES TABLE 1. Various parameters recorded in control and streptozotocin-diabetic in absence and presence of vanadyl treatment CON

Body weight, Heart weight, Heart-to-body Plasma ‘L nM Triglyceride, Cholesterol, Phospholipid, Creatinine,

g g weight

CON-T

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(7)

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370t6* (11) 1.17tO.O4* 3.19kO.07 (9) 51t4 (10) 1.49kO.28 1.3620.11 1.56t0.13 46k4

Values are means t SE for no. of animals given in parentheses. CON, control; CON-T, control treated; STZ, streptozotocin diabetic; STZ-T, diabetic treated. Treatment (T), vanadyl sulfate trihydrate (1 mg/ml), was included in drinking water for 10 wk. Statistical significance was determined using analysis of variance followed by Duncan’s test. * Significantly different from all groups (P c 0.05); t significantly different from CON and STZ-T groups (P < 0.05); $ significantly different from CON-T and STZ-T groups (P < 0.05).

-120

100

of glycerol observed during in vitro incubation of epididymal adipose tissue isolated from each of the experimental groups (Fig. 4). Rates of glycerol release in the control group were similar to values reported previously (-1 pm1 8-l. h-l). The anticipated increase in glycerol release from adipose tissue of the diabetic group was observed with rates approximately twice those observed with tissue from control animals. After treatment of controls with vanadyl, no alteration of glycerol release was observed, but corresponding treatment of diabetic rats with vanadyl substantially attenuated the glycerol release observed in vitro. Increased rates of lipolysis induced during diabetes were therefore corrected by treatment with vanadyl. In vitro exposure of tissue isolated from all four experimental groups to insulin led to a similar degree of inhibition of glycerol output, suggesting that the responsiveness of tissues was similar with the high concentration of insulin employed. The inhibition observed in response to insulin was 32-38% in all four groups, and all changes were statistically significant (P < 0.05). l

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FIG. 2. Plasma concentration of glucose and insulin measured at 10 wk. Data were obtained from streptozotocin (STZ)-treated and agematched control (CON) animals maintained for 10 wk in absence and presence (T) of vanadyl sulfate trihydrate (1 mg/ml) in drinking water and were analyzed using analysis of variance followed by Duncan’s test. *, Significantly different from control group (P < 0.05).

phospholipid in the CON-T group were unchanged from control levels. In contrast, after treatment with vanadyl, the lipid levels in the STZ-T group were significantly decreased relative to the untreated diabetic group (STZ) and were similar to the levels measured in the control group* Heart Function

Three indexes of heart function were assessed during in vitro perfusion of isolated hearts, and the results are shown in Fig. 3, A-C. LVDP, +dP/dt, and -dP/dt in all groups studied responded similarly with increasing left atria1 filling pressures (5.0-15.0 cmHzO). However, at higher filling pressures (17.5-22.5cmHeO), a decline in performance of the hearts from the STZ group in comparison to the CON group was observed. In the vanadyltreated animals, myocardial performance at all filling pressures was unaffected in the CON-T group and normalized in the STZ-T group. Glycerol Output

To assess the metabolic status of an insulin-sensitive tissue, rates of lipolysis were estimated from the release

Protein Phosphorylation

A large number of studies in recent years have provided strong support for the contention that changes in phosphorylation of key regulatory proteins play a significant role in the actions of hormones, including epinephrine and insulin, on adipose tissue (for review, see Ref. 10). The effects of hormones on protein phosphorylation are explained at least in part through changes in activity of cellular protein kinases and/or protein phosphatases that may be detected after cell fractionation (3). To gain insight into the mechanisms by which vanadium may exert its effects on fat cell metabolism, we investigated protein phosphorylation in supernatant fractions from adipose tissue from each of the treatment groups. These studies involved incubation of fresh tissue extracts in the presence of [T-~~P]ATP to allow phosphorylation of endogenous substrates by protein kinases present in the same extracts. This approach has been validated in a number of previous studies (for review, see Ref. 3). Seven major 32P-labeled protein subunits were consistently observed with approximate subunit M, values (X 10m3)of 120,105,90,73,65,54, and 46 (Table 2). A large

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3. Mechanical performance of isolated perfused hearts. Heart function of streptozotocin (STZ)-treated and age-matched control (CON) animals was studied using isolated working heart system. Animals were maintained for 10 wk in absence and presence (T) of vanadyl sulfate trihydrate (1 mg/ml) in drinking water. Hearts were perfused in a working heart system, and left ventricular developed pressure (A; LVDP), rate of force development (B; +dP/dt), and rate of relaxation (C; -dP/dt) were recorded. Data were analyzed using analysis of variance followed by Duncan’s test. *, STZ group significantly different from control groups (P < 0.05); *, STZ group significantly different from all other group@ < 0.05). FIG.

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FIG. 4. Glycerol output from epididyml fat pads incubated in vitro in absence or presence of insulin (0.50 pg/ml). Epididymal fat pads were obtained from streptozotocin (STZ)-treated and age-matched control (CON) animals maintained for 10 wk in absence and presence (T) of vanadyl sulfate trihydrate (1 mg/ml) in drinking water. Fat pads were removed immediately from animals on death, allowed to equilibrate in bicarbonate-buffered medium, then preincubated in fresh medium in absence and presence of insulin for 45 min. After removal of tissue, incubation medium was deproteinized and glycerol concentrations determined. *, STZ groups significantly different from other groups (P < 0.05) by analysis of variance followed by Duncan’s test. In each group, glycerol output in presence of insulin was significantly different from output in absence of insulin (P c 0.05) by Student’s t test.

number of minor 32P-labeled protein subunits were also observed. Incorporation of 32P into the majority of bands was similar in all the experimental groups, but incorporation into the subunits of M, 65,000 and 46,000 were elevated in the STZ group (148 t 6 and 153 t 4% of control, respectively, P < 0.05). These STZ-induced changes were abolished in the STZ-T group after vanadyl treatment. The phosphoprotein profile detected by autoradiography of proteins separated by SDS-polyacrylamide gel electrophoresis differed substantially from profiles observed previously with younger rats weighing MO-220 g (3). For example, in the present studies we observe very little incorporation of 32P into the M, 230,000 subunit of the lipogenic enzyme acetyl-CoA carboxylase. Indeed the total activity of acetyl-CoA carboxylase determined in all groups was ~20 mU/g (compared with values of lOO120 mU/g in rats weighing 180-220 g), indicating the low lipogenic capacity of tissue from all four groups. DISCUSSION

In view of the lower toxicity and insulin-like properties of vanadyl (+IV) compared with vanadate (+V), in the present study we investigated the effects of vanadyl in the treatment of STZ-diabetic male Wistar rats. A range of established abnormalities associated with the diabetic state were assessed after 10 wk of daily oral administration of vanadvl sulfate trihvdrate.

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2. Incorporation of 32P from [T-~~P]ATP into proteins in supernatant fractions from epididymal adipose tissue

TABLE

Approximate Subunit M

120,000 105,000

90,000 73,000 65,000 54,000 46,000 n

CON

CON-T

STZ

STZ-T

28k4 35k7 31t5 46t6 45k5 235t33 32k5 6

27+4 25t3 29*4 41*5 54tlO 200t24 30t5 4

30s 32t5 31t4 50t5 65*6* 24Ok30 49t7* 6

25k4 26k3 23k3 44t4 47t6 180t24 29*5 5

Values are means t SE; n, no. of animals. Supernatant fractions were obtained from homogenates of epididymal fat pads by centrifugation (12,000 g) and then incubated in presence of [T-~~P]ATP as described in METHODS. Proteins were separated by SDS-polyacrylamide gel electrophoresis and autoradiographs obtained from fixed, stained, and dried gels. Incorporation of 32P into individual protein subunits of indicated subunit M, was estimated by scanning densitometry. Absorbance values were corrected for protein loading by scanning Coomassie blue-stained gels. CON, control; CON-T, control treated; STZ, streptozotocin diabetic; STZ-T, diabetic treated. Treatment (T), vanadyl sulfate trihydrate (1 mg/ml), was included in drinking water for 10 wk. * Values in STZ group significantly different from other 3 groups (P < 0.05).

Administration of STZ to rats, as expected, resulted in hyperglycemia, hypoinsulinemia, lowered thyroid hormone levels, elevations in plasma lipid and creatinine levels, relative cardiac hypertrophy, depressed cardiac function, and elevations in glycerol output from epididymal fat pads when compared with age-matched control groups (CON and CON-T). Of the parameters studied, the most apparent effects observed after treatment of nondiabetic animals with vanadyl (CON-T) were decreases in body and heart weights and in the plasma concentrations of insulin. All other plasma parameters measured including glucose concentration were found to be similar to the untreated CON group. In contrast, treatment with oral vanadyl for 10 wk produced profound beneficial effects in the diabetic animals (STZ-T). After vanadyl treatment plasma glucose levels of STZ were indistinguishable from values in control animals, although circulating insulin levels remained low. Although body and heart weights of the STZ-T rats were not normalized, the ratio of heart to body weight in this group was similar to both CON and CON-T groups. The concentrations of thyroid hormone, creatinine, and lipid in plasma from animals in the STZT group were also found to be similar to control values. Finally, the abnormalities in cardiac function and lipolytic activity of adipose tissue induced by STZ were found to be corrected subsequent to treatment with vanadyl. The chronic vanadyl regimen used in the present study was unable to reverse the decrease in the rate of body weight gain of the STZ group, and this confirms the results observed previously in studies in which similar concentrations of vanadate were used (24, 26). The vanadyl treatment, however, did not further decrease the rate of growth (already suppressed by the induction of diabetes) in the STZ-T group as it did in the CON-T group, despite similar intakes of vanadium by the two

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groups. It may be noted that although there was similar vanadium consumption, plasma levels of glucose were normalized in the STZ-T group but remained unaffected in the CON-T group. This is in contrast to the observations of Meyerovitch et al. (24) who reported hypoglycemia in diabetic animals 4 days after the initiation of treatment with equimolar concentrations of vanadate. The observation in the present study of a markedly higher intake of vanadyl by the STZ-T group during the first week of treatment without an increase in body weight suggests that the hypoglycemia reported by Meyerovitch et al. (24) at 4 days may have been a consequence of short-term malnutrition associated with a high initial consumption of vanadate. A further interesting and important observation made in the present study was the lowering of plasma glucose levels after vanadyl treatment of diabetic animals (STZT) in the absence of increases in the endogenous levels of insulin. This leads to the suggestion that vanadyl is able to substitute for insulin under in vivo conditions for a prolonged period, analagous to the insulin-like effects of vanadium compounds observed under acute in vitro conditions (8, 13, 31, 32, 36, 37). Alternatively, vanadyl may be able to enhance responsiveness of tissues to low circulating levels of insulin. In contrast to the absence of change in the STZ-T group, circulating levels of insulin were decreased in the CON-T group while euglycemia was being maintained. This observation of a decrease in insulin levels while plasma glucose levels remained unaffected in the CON-T group is similar to the finding in a previous study in which vanadate was used (15). It seems attractive to speculate that by contributing to the maintenance of a euglycemic state, vanadyl causes a feedback inhibition of insulin release in the control animals, although the possibility of more direct actions on pancreatic insulin release cannot be eliminated. To examine the possible mechanism(s) by which vanadyl treatment may modify the diabetic state, properties of adipose tissue were studied in vitro. In agreement with previous studies, we observed a substantial increase in basal lipolysis in tissues from diabetic rats (29). No suppression of lipogenic capacity was indicated by total activities of acetyl-CoA carboxylase that were very low even in the control group. The changes in lipolytic rate in the STZ group and the correction to near-control values with vanadyl treatment offer a reasonable explanation for the corresponding changes in circulating lipid levels, since alterations in the supply of free fatty acids is an important determinant of hepatic lipid synthesis and release. The mechanism by which vanadyl may affect triglyceride hydrolysis cannot be directly deduced from the evidence available thus far. However, it may be noted that the M, 65,000 protein observed in the protein phosphorylation studies has previously been shown to be a substrate for adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase. This suggests that the CAMP-mediated protein phosphorylation cascade is activated during diabetes and that vanadyl treatment leads to restored regulation of this cascade. This will provide an important rationale for further studies on the

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mechanism of action of vanadyl. In agreement with a previous report (39), the present study revealed depressed mechanical function of hearts from diabetic rats which was most apparent at high atria1 filling pressures (X5 cmHz0). After treatment with vanadyl for 10 wk, the contractile performance of hearts from the STZ-T animals was similar to that of hearts from CON and significantly higher than hearts from STZ animals. Direct inotropic effects similar to those of insulin (30) have been observed with vanadium (35) but seem unlikely to explain the normalization of cardiac function in the diabetic animals observed in the present study, since myocardial performance in the CON-T animals was not significantly affected by vanadyl treatment. Alternatively, it may be suggested that the ability of chronic vanadyl treatment to suppress adipose tissue lipolysis (and circulating levels of lipids) or to increase thyroid hormone levels may play an important role in the normalization of heart function in diabetic animals. The well-known switch of myocardial myosin isoform from V1 to Vs in diabetic rats has been associated with hypothyroidism (1). In support of this, treatment of diabetic rats with pharmacological doses of Ta reestablished the normal predominance of the V1-isoform (11). Similarly, methyl palmoxirate also promoted a switch back to the &-form but in the absence of alleviation of the hypothyroid state in diabetic rats (12). However, similar dose regiments of methyl palmoxirate (an inhibitor of fatty acid oxidation) and thyroid hormone were found to be insufficient to lower lipid levels or improve cardiac function in diabetic rats when used alone and, in combination, were able to improve cardiac performance only (34). In contrast, our laboratory has recently reported that hydralazine was able to lower plasma lipid levels and normalize cardiac function in diabetic rats (28). These observations suggest that the mechanism(s) leading to cardiac abnormalities in diabetes are complex and that it is not yet possible to explain the beneficial effects of chronic vanadyl treatment on cardiac function in terms of any one specific action. The concentration of vanadium in the vanadyl (+IV) solutions used in the present study (1 mg/ml, 1.08 mM) was similar to the concentration present in the vanadate (+V) solutions (0.80 mg/ml, 1.20 mM) used in previous studies (15, 24, 26). However, in rats treated with the vanadyl form, severe diarrhea and death were recorded in only one control and diabetic animal. Thus the overt toxicity including possible damage to the kidneys (as assessed by the measurement of plasma creatinine levels) reported with similar levels of vanadate (24) were not observed with vanadyl. In addition, the lack of significant changes in myocardial function and lipolytic activity or insulin responsiveness in CON-T animals indicates that the amounts of vanadyl employed in the present study do not produce direct toxic effects on the heart or adipose tissue. These findings therefore suggest that the vanadyl form may be more suitable than vanadate for long-term use in diabetic animals. Finally, an important observation made in diabetic animals after vanadyl treatment was a striking absence

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of cataracts (or blindness) as compared with untreated diabetic animals. Surprisingly, this remarkable effect of vanadyl was also observed in an earlier preliminary study in which the treatment regimen used was not able to reverse the hyperglycemic state. This observation has significant clinical relevance, since retinopathy is a major manifestation of diabetes in humans (21) and will be further addressed in future studies. In summary, vanadium administered as oral vanadyl sulfate trihydrate was well tolerated by both CON and STZ rats and was able to prevent or attenuate a number of abnormalities associated with the diabetic state. The single most important consequence of treating diabetic animals with vanadyl is undoubtedly the normalization of plasma glucose levels normally exquisitely dependent on insulin. The fact that euglycemia was achieved with no apparent alterations in circulating levels of insulin highlights the therapeutic potential of vanadyl in type I diabetes, which is associated with severely impaired synthesis and secretion of insulin. In parallel, vanadyl appears to diminish the requirement for insulin, since endogenous levels of insulin were reduced in the CON-T group with maintenance of normal circulating glucose concentrations. In conclusion, the ability of vanadyl to diminish the diabetic state demonstrates its effectiveness to act in an insulin-like fashion and its potential usefulness in the diabetic animal under chronic in vivo conditions. However, the possibility that vanadium compounds may prove to be as beneficial in treating human diabetes remains to be determined. We thank Judy Wyne for the excellent preparation of the figures, Sylvia Chan for her careful typing of the manuscript, and Ahmad Doroudian and Gordon Dong for their excellent technical assistance. This work was supported in part by grants from the Canadian Diabetes Foundation, Alfred and Agnes Woods Research Fund, Medical Research Council of Canada, and British Columbia Health Care Research Foundation. Address for reprint requests: J. H. McNeill, Faculty of Pharmaceutical Sciences, The Univ. of British Columbia, 2146 East Mall, British Columbia, Vancouver V6T lW5, Canada. Received

2 November

1987;

accepted

in final

form

10 May

1989.

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