Diabetes
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marti, L.
Right arrow Articles by Zorzano, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marti, L.
Right arrow Articles by Zorzano, A.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Diabetes 50:2061-2068, 2001
© 2001 by the American Diabetes Association, Inc.

Combined Treatment With Benzylamine and Low Dosages of Vanadate Enhances Glucose Tolerance and Reduces Hyperglycemia in Streptozotocin-Induced Diabetic Rats

Luc Marti1, Anna Abella1, Christian Carpéné2, Manuel Palacín1, Xavier Testar1, and Antonio Zorzano1

1 Departament de Bioquìmica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain
2 Institut National de la Santé et de la Recherche Médicale (INSERM U 317), Toulouse, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO, such as benzylamine, in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in 3T3-L1 and rat adipocytes. Here we examined whether acute and chronic administration of benzylamine and vanadate in vivo enhances glucose tolerance and reduces hyperglycemia in diabetic rats. Acute intravenous administration of these drugs enhanced glucose tolerance in nondiabetic rats and in streptozotocin (STZ)-induced diabetic rats. This occurred in the absence of changes in plasma insulin concentrations. However, the administration of benzylamine or vanadate alone did not improve glucose tolerance. The improvement caused by benzylamine plus vanadate was abolished when rats were pretreated with the SSAO-inhibitor semicarbazide. Chronic administration of benzylamine and vanadate exerted potent antidiabetic effects in STZ-induced diabetic rats. Although daily administration of vanadate alone (50 and 25 µmol · kg-1 · day-1 i.p.) for 2 weeks had little or no effect on glycemia, vanadate plus benzylamine reduced hyperglycemia in diabetic rats, enhanced basal and insulin-stimulated glucose transport, and upregulated GLUT4 expression in isolated adipocytes. In all, our results substantiated that acute and chronic administration of benzylamine with low dosages of vanadate have potent antidiabetic effects in rats.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The semicarbazide-sensitive amine oxidase (SSAO) belongs to the large family of copper-containing amine oxidases (EC 1.4.3.6) that convert primary amines to aldehydes, with the concomitant production of hydrogen peroxide and ammonia. These proteins are characterized by broad substrate selectivity among species, which makes it difficult to ascertain their biological function. The enzyme readily oxidizes exogenous (e.g., benzylamine, tyramine) or endogenous (e.g., phenylethylamine, histamine) aromatic primary amines, but also endogenous (e.g., methylamine, aminoacetone) aliphatic primary amines (1). More recently, a new function has been assigned to SSAO: the vascular adhesion protein-1 (VAP-1), found to be identical to SSAO, belongs to the family of adhesive proteins implicated in processes like inflammation or cell-to-cell interaction (2,3,4). The relation between the enzymatic and adhesive functions of SSAO/VAP-1 remains to be determined.

Adipose tissue contains high levels of SSAO (5,6,7,8,9), and an increase in the expression of the membrane-bound SSAO has been reported in adipocyte differentiation (8,10). As to the function of adipocyte SSAO, hydrogen peroxide, one of the reaction products, has insulinomimetic properties (11). Moreover, membrane fractionation and vesicle immunoisolation analysis have shown that a portion of the SSAO protein or enzymatic activity colocalizes with intracellular GLUT4-containing vesicles (5,12). We recently reported that substrates of SSAO can stimulate glucose transport in rat adipocytes (12,13,14). The amine-induced stimulation of glucose transport was observed in the presence of ineffective concentrations of vanadate and was abolished by semicarbazide and catalase (12,13). These observations suggested that the SSAO-dependent generation of hydrogen peroxide is responsible for the increased stimulation of glucose transport via a chemical interaction. The combination of vanadate and hydrogen peroxide can form peroxovanadate, a powerful insulinomimetic agent that may be partly responsible for this effect (13,15). In addition, the combination of SSAO substrates with vanadate stimulates glucose transport through translocation of GLUT4 to the adipocyte cell surface (12,13). Furthermore, the combination of SSAO substrates and vanadate stimulates phosphatidylinositol (PI) 3-kinase activity and tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and -3 in conditions in which the insulin receptors themselves are only slightly activated (14). All these data support the view that the combination of SSAO substrates and vanadate stimulates glucose transport via a pathway that does not involve the insulin receptor.

In this study, we examined the acute and chronic effects of benzylamine and vanadate on control and diabetic rats. We demonstrated potent antidiabetic properties of benzylamine and vanadate in streptozotocin (STZ)-induced diabetic rats.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
We obtained 2-deoxy-D-[1,2-3H]glucose (2-DG; 26 Ci/mmol) from NEN Life Science Products and [14C]benzylamine (59 Ci/mmol) from Amersham Pharmacia Biotech (Arlington Heights, IL). Purified porcine insulin was a kind gift from Eli Lilly (Indianapolis, IN). STZ, semicarbazide hydrochloride, benzylamine hydrochloride, sodium orthovanadate, and other chemicals were purchased from Sigma Aldrich (St. Louis, MO). Ketamine was obtained from Mérieux (Imalgene, Mérieux, France) and collagenase type I from Worthington. The osmotic minipumps used in the chronic studies were from Alza. All electrophoresis reagents and molecular weight markers were obtained from Bio-Rad. Enhanced chemiluminescence reagents (super signal substrate) were obtained from Amersham. Anti-GLUT4 antibody (OSCRX) was produced from rabbit, as previously reported (15). Rabbit polyclonal antibodies against rat â1-integrin were kindly given by Dr. C. Enrich (Universitat de Barcelona).

Animals.
Male Wistar rats weighing 180–220 g were purchased from Harlan (Interfauna Ibèrica S.A., Spain). The animals were housed in animal quarters at 22°C with a 12-h light/dark cycle and were fed ad libitum, unless otherwise stated. Type 1 diabetes was induced by a single intraperitoneal injection of a freshly prepared solution of STZ (45–70 mg/kg body wt dissolved in 50 mmol/l citrate buffer [pH 4.5]). Only diabetic animals with glycemia >300 mg/dl were used. Inhibition of SSAO activity was obtained in vivo by administration of semicarbazide (5 mg · kg-1 · day-1 i.p.) for 3 days. All procedures used were approved by the Ethics Committee of the University of Barcelona.

Glucose tolerance tests.
Anesthetized (pentobarbital 80 mg/kg body wt) control rats or nonanesthetized diabetic rats were injected intravenously via the tail vein with vehicle (phosphate-buffered saline [PBS], pH 7.4) or with 7 µmol/kg body wt benzylamine and/or 20 µmol/kg sodium orthovanadate. In anesthetized control rats, an intravenous (saphenous vein) glucose tolerance test (0.8 g/kg body wt) (16) was carried out 30 min after administration of benzylamine, vanadate, or both. Blood samples were collected before (0 min) and 5, 10, 15, 20, 30, and 45 min after glucose administration. In other studies of diabetic rats, an oral glucose tolerance test (2 g/kg body wt) (17) was carried out 15 min after injection of benzylamine, vanadate, or both. Blood samples were collected before and 15, 30, 45, 60, 90, and 150 min after glucose administration. Plasma was obtained for the determination of glucose and insulin concentrations. Food was withdrawn at 8:00 A.M. to ensure a 3-h fast before the beginning of the study.

Chronic treatment of diabetic animals.
Osmotic minipumps containing benzylamine (84 µmol · kg-1 body wt · day-1) were implanted subcutaneously in STZ-induced diabetic rats anesthetized by ketamine hydrochloride (95 mg/kg) and xylazine (10 mg/kg). Animals that did not receive benzylamine were sham-operated. A single intraperitoneal injection of vanadate (25 or 50 µmol/kg body wt) or the vehicle (PBS) was administered daily at 9:00 A.M. Food was removed after the administration of vanadate and replaced at 2:00 P.M. Glycemia was measured in arteriovenous blood collected from the tail vessels at 9:00 A.M., before the administration of vanadate, for 14 days. Insulin concentrations were determined before and after treatment.

Glucose transport measurements in isolated rat adipocytes and preparation of membrane extracts.
Adipocytes were isolated from epididymal fat pads in nondiabetic and diabetic male Wistar rats (180–220 g), as previously reported (12,13). After a preincubation period of 45 min at 37°C, each vial, containing 400 µl of cell suspension in Krebs-Ringer bicarbonate buffer and the drugs being tested, received an isotopic dilution of 2-DG in a final concentration of 0.1 mmol/l, equivalent to ~1,300,000 dpm/vial. 2-DG transport assays were performed as previously reported (12,13). Isolated fat cells from control or treated rats were disrupted for total membrane preparation by hypo-osmotic lysis in a 20 mmol/l HES buffer and an antiprotease cocktail, as previously reported (12). Protein concentrations were determined by the Bradford method (18), with {gamma}-globulin as a standard.

Amine oxidase activity assays.
The radiochemical determination of amine oxidase activity was performed basically as described by Fowler and Tipton (19), with slight modifications (13).

Electrophoresis and immunoblot analysis.
SDS-PAGE was performed on membrane proteins following the method of Laemmli (20). Proteins were transferred to etorphine hydrochloride (Immobilon) in buffer consisting of 20% methanol, 200 mmol/l glycine, and 25 mmol/l Tris (pH 8.3). After transfer, the filters were blocked with 5% nonfat milk in PBS for 1 h at 37°C and then incubated with polyclonal antibodies raised against GLUT-4 or ß1-integrin. The immune complex was detected by enhanced chemiluminescence Western blot, in the linear response range.

Analytical methods.
The glucose concentration in plasma or urine was determined by the glucose oxidase method (MPR-3 glucose/GOD-PAP Method; Boehringer) in glucose-tolerance experiments and with a rapid glucose analyzer (Accutrend Sensor Comfort; Roche) in chronic treatments. The plasma immunoreactive insulin concentration was determined with a sensitive rat insulin radioimmunoassay kit (Linco Research, St. Charles, MO).

Calculations and statistical analysis.
Insulin and glucose responses during the glucose tolerance tests were calculated as the incremental plasma values integrated over a 45-min period in control animals and 150-min period in diabetic animals after the injection of glucose. Areas under the curve for insulin and glucose responses were calculated using the Graph Prism program (GraphPad Software). Data are presented as means ± SE; an unpaired Student’s t test was used to compare two groups. When experimental series involved more than two groups, statistical analysis was done by one-way analysis of variance and further post hoc Dunnett’s or Tukey’s t tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Acute administration of benzylamine and vanadate enhanced glucose tolerance in nondiabetic rats.
We have previously reported that in vitro substrates of SSAO, such as benzylamine or tyramine, in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in 3T3-L1 and rat adipocytes (12,13). In this study, we examined whether the combination of SSAO substrates and vanadate exhibit insulin-like effects in vivo.

Control anesthetized nondiabetic rats were injected via the tail vein with vehicle (PBS) or 7 µmol/kg benzylamine and/or 20 µmol/kg sodium orthovanadate. Glucose load (0.8 g/kg body wt by saphenous vein) was carried out 30 min after administration of benzylamine, vanadate, or both. Acute intravenous administration of vanadate did not alter glucose tolerance (Fig. 1A), and acute administration of benzylamine only caused reduced glycemia values 5 min after glucose injection, but the integrated glucose area under the curve remained unaltered (Figs. 1B and D). In the same conditions, administration of benzylamine plus vanadate reduced glycemia at different times after glucose injection (Fig. 1C) and increased glucose tolerance by 35% (Fig. 1D). No changes in glucose concentrations in urine were detected between the control and benzylamine + vanadate groups (0.38 ± 0.07 vs. 0.42 ± 0.09 mmol/l, respectively) during the glucose tolerance test (data not shown).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 1. Effect of acute administration of benzylamine and vanadate on glucose tolerance in control rats. Glucose was injected in the saphenous vein (0.8 g/kg body wt) 30 min after intravenous administration of vehicle (A, B, and C; {blacklozenge}), 20 µmol/kg vanadate (A; {circ}), 7 µmol/kg benzylamine (B; {triangleup}), or vanadate plus benzylamine (C; {square}). Results are expressed as increments in plasma glucose. Plasma glucose concentrations at time zero were 5.5 ± 0.2, 5.1 ± 0.4, 5.4 ± 0.2, and 4.6 ± 0.3 µmol/l in control, vanadate, benzylamine, and benzylamine plus vanadate groups, respectively. D: Integrated increase in glycemia. Data are means ± SE of six to nine observations presented in A, B, and C and are expressed as a percentage of the control (PBS) group. Last bar corresponds to the increase in glycemia from rats pretreated with semicarbazide (SCZ; 5 mg/kg) for 3 days and receiving benzylamine (Benz) plus vanadate (V) just before the glucose tolerance test. *Different from vehicle control at P < 0.05.

 
As expected, plasma insulin was maximal 5 min after glucose injection (Table 1), and returned to basal levels 30 min later (data not shown). The acute administration of benzylamine, vanadate, or both had no effect on plasma insulin levels (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Effect of acute administration of benzylamine and vanadate on plasma insulin concentrations in control rats

 
Semicarbazide treatment prevented the effects of benzylamine and vanadate on glucose tolerance in nondiabetic rats.
To determine whether the effect of benzylamine plus vanadate on glucose tolerance was a consequence of the SSAO activity, rats previously treated for 3 days with the SSAO inhibitor semicarbazide (5 mg · kg-1 · day-1 i.p.) were subjected to a glucose tolerance test. Semicarbazide treatment caused >90% inhibition of SSAO activity in adipose tissue extracts (data not shown). In keeping with the inhibition of SSAO activity, isolated rat adipocytes from semicarbazide-treated rats did not respond to the combination of 0.1 mmol/l benzylamine plus 0.1 mmol/l vanadate by stimulating glucose transport (data not shown).

Acute administration of benzylamine (7 µmol/kg body wt) with vanadate (20 µmol/kg body wt) in semicarbazide-treated rats had no effect on glucose tolerance (Fig. 1D). In these conditions, plasma insulin levels were normal (data not shown).

Acute administration of benzylamine plus vanadate enhanced glucose tolerance in STZ-induced diabetic rats.
Next, we tested the in vitro and in vivo effects of the combination of benzylamine and vanadate in STZ-induced diabetic rats. Adipose tissue from diabetic rats shows normal SSAO activity (21; G. Enrique-Tarancón, unpublished observations); however, we evaluated whether adipose cells from diabetic rats respond to SSAO substrates and vanadate by stimulating glucose transport.

As expected from previous reports (22,23), isolated adipocytes from diabetic rats showed a reduced maximal stimulation of glucose transport in response to insulin (Table 2). Interestingly, the combination of 0.1 mmol/l benzylamine and 0.1 mmol/l vanadate stimulated glucose transport to a similar extent in adipocytes from diabetic and nondiabetic rats (Table 2). Moreover, in diabetic rats, this stimulation was comparable to that of insulin, although it reached only 70–80% of insulin’s effect in control animals (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2 Effect of benzylamine and vanadate on glucose transport in isolated adipocytes from control or STZ-induced diabetic rats, as measured by 2-DG uptake (nmol · 100 mg-1 lipid · 5 min-1)

 
Nonanesthetized diabetic rats were injected via the tail vein with vehicle or 7 µmol/kg body wt benzylamine and/or 20 µmol/kg sodium orthovanadate. An oral glucose tolerance test (2 g/kg body wt) was carried out 15 min after drug injection (Fig. 2). The acute intravenous administration of benzylamine plus vanadate reduced glycemia (Fig. 2A) and enhanced glucose tolerance in the STZ-induced diabetic rats (42% increase), but benzylamine or vanadate alone did not alter glucose tolerance (Fig. 2B). The effect of benzylamine plus vanadate on glucose tolerance in vivo was independent of changes in plasma insulin concentrations (Table 3). In addition, the improvement in glucose tolerance induced by benzylamine plus vanadate was abolished in semicarbazide-treated diabetic rats, indicating that intact SSAO activity was required to improve glucose tolerance (Fig. 2B). No changes in glycosuria were detected in control or benzylamine + vanadate groups (515 ± 55 vs. 447 ± 43 mmol/l, respectively) during the tolerance test.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 2. Effect of the acute administration of benzylamine plus vanadate on oral glucose tolerance test in STZ-induced diabetic rats. A: Relative changes in glycemia with an oral glucose load (2 g/kg) starting 15 min after intravenous injection of vehicle ({blacklozenge}) or 20 µmol/kg vanadate + 7 µmol/kg benzylamine ({square}). Results are expressed as increments in plasma glucose. B: Integrated increase in glycemia. Concentrations of plasma glucose at time zero were 21.1 ± 1.2, 23.1 ± 2.3, 23.2 ± 3.9, 21.2 ± 1.3, and 26.2 ± 1.1 mmol/l in diabetic rats untreated (PBS) or treated with vanadate (V), benzylamine (Benz), Benz + V, and Benz + V + semicarbazide (SCZ), respectively. Data are means ± SE of 4–15 observations expressed as percentage, with 100% corresponding to the PBS group. *A significant difference versus PBS group at P < 0.05.

 

View this table:
[in this window]
[in a new window]
  		TABLE 3 Effect of the acute administration of benzylamine and vanadate on plasma insulin concentrations in diabetic rats

 
Chronic administration of benzylamine plus vanadate reduced hyperglycemia in diabetic rats.
Based on the finding of potent insulin-like effects of acute administration of benzylamine plus vanadate, we tested chronic administration of these compounds in diabetic rats. To this end, STZ-induced diabetic rats were implanted subcutaneously with osmotic minipumps releasing benzylamine (84 µmol · kg-1 · day-1) or were sham-operated. Preliminary studies have indicated that benzylamine is stable for 2 weeks in implanted osmotic minipumps, based on its capacity to further stimulate glucose transport in isolated adipocytes after this period (data not shown). Another group of diabetic animals was subjected to daily intraperitoneal injection of vanadate (50 µmol/kg body wt) for 2 weeks, as compared with a group of diabetic rate that received both benzylamine and vanadate. Although treatment with vanadate caused a moderate reduction of glycemia, only the combination of benzylamine plus vanadate normalized glycemia after 1 week of treatment (Fig. 3A). Administration of benzylamine plus vanadate for 2 weeks did not alter SSAO activity in extracts from adipose cells (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3. Chronic treatment with benzylamine and vanadate ameliorates glycemia in STZ-induced diabetic rats. A: Diabetic rats received daily intraperitoneal injection of vanadate (50 µmol/kg) alone ({circ}) or in combination with benzylamine (84 µmol · kg-1 · day-1) ({square}) delivered by osmotic minipumps. Control animals ({blacklozenge}) were sham-operated diabetic rats that received a daily injection of the vehicle (PBS). Data are means ± SE of 4–12 observations. *Significantly different from sham-operated group from day 6 at P < 0.05. B: Rats received daily intraperitoneal injections of vanadate at 25 and 50 µmol/kg body wt for the 1st and 2nd weeks, respectively. Glycemia of diabetic rats untreated (PBS; {blacklozenge}) or treated with vanadate ({circ}) or benzylamine plus vanadate ({square}) is presented. Data are means ± SE of three to six observations per group. *Significantly different (P < 0.05) versus PBS group from day 11.

 
In other studies, diabetic rats were daily injected intraperitoneally with 25 µmol/kg vanadate for the 1st week and 50 µmol/kg vanadate for the 2nd week. These rats, as well as diabetic rats treated with 84 µmol/kg benzylamine alone, remained hyperglycemic (Fig. 3B; data not shown). However, the combination of benzylamine plus vanadate reduced hyperglycemia in diabetic rats from day 10 of treatment (Fig. 3B).

The chronic treatment with benzylamine plus vanadate caused a substantial decrease in food and water consumption to normal levels, and a 45% increase in the weight of epididymal adipose tissue (Table 4). All these variations occurred in the absence of changes in body weight. The effects on food and water intake were not detected when benzylamine or vanadate was administered alone (Table 4).


View this table:
[in this window]
[in a new window]
  		TABLE 4 Effect of chronic treatment with benzylamine and vanadate on body weight, epididymal adipose tissue weight, food and water intake, and plasma insulin

 
The normalization of glycemia caused by chronic treatment with both benzylamine and vanadate took place in the absence of changes in plasma insulin concentrations (Table 4).

Chronic administration of benzylamine plus vanadate stimulated glucose transport and GLUT4 expression in adipocytes from diabetic rats.
To determine whether the antidiabetic effects caused by chronic treatment with benzylamine plus vanadate were a consequence of stimulation of peripheral glucose utilization, we analyzed glucose transport and glucose transporter expression in adipose cells obtained from rats treated for 2 weeks with different combinations of benzylamine and/or vanadate. Treatment for 2 weeks with benzylamine plus vanadate stimulated basal glucose transport in isolated adipocytes and normalized insulin-stimulated glucose transport (Fig. 4A). These effects were not detected in adipose cells obtained from rats treated with benzylamine or vanadate alone. Indeed, in diabetic rats there was a negative correlation between glycemia at the end of the experiment and basal glucose transport in adipose cells (Fig. 4B).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. Chronic treatment with benzylamine plus vanadate stimulates glucose transport in adipocytes from diabetic rats. A: STZ-induced diabetic rats were implanted subcutaneously with an osmotic pump containing benzylamine (84 µmol · kg-1 body wt · day-1) or sham-operated. Rats received daily intraperitoneal injections of vanadate at 25 and 50 µmol/kg body wt for the 1st and 2nd weeks, respectively. After 2 weeks of treatment, isolated adipocytes from nondiabetic rats ({blacksquare}) and untreated (), vanadate-treated (), benzylamine-treated (), and benzylamine plus vanadate-treated diabetic rats ({square}) were obtained from epididymal adipose tissue. Adipose cells were incubated for 30 min in basal conditions or in the presence of 100 nmol/l insulin. Subsequently, 2-DG transport was measured for 5 min (expressed as nmol · 5 min-1 · 100 mg-1 lipid). Data are means ± SE of four to five observations per group. *P < 0.05. B: Correlation between basal glucose transport in isolated rat adipocytes and plasma glycemia of the rats (r = 0.77, P < 0.01). Data from rats treated with PBS ({blacklozenge}), vanadate ({circ}), benzylamine ({triangleup}), and benzylamine plus vanadate ({square}) are shown.

 
Treatment with benzylamine plus vanadate increased GLUT4 expression (by sevenfold) more than vanadate alone (Fig. 5). In addition, there was a correlation between the amount of GLUT4 and basal glucose transport in adipocytes (data not shown). No changes in GLUT4 expression were detected in soleus or extensor digitorum longus muscles in response to chronic treatment with benzylamine plus vanadate (data not shown).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 5. Chronic treatment with benzylamine plus vanadate stimulates GLUT4 glucose transporter expression in adipocytes from diabetic rats. STZ-induced diabetic rats were implanted subcutaneously with an osmotic pump containing benzylamine (84 µmol · kg-1 body wt · day-1) or were sham-operated ({blacklozenge}). Rats received daily intraperitoneal injections of vanadate at 25 and 50 µmol/kg body wt for the 1st and 2nd weeks, respectively. After 2 weeks of treatment, isolated adipocytes from untreated diabetic rats and diabetic rats treated with vanadate, benzylamine, and benzylamine plus vanadate were obtained from epididymal adipose tissue. Total adipocyte membranes were obtained and subjected to SDS-PAGE and further immunoblotting using specific antibodies directed against GLUT4 or ß1-integrin. A: Representative autoradiogram. B: GLUT4 abundance corrected by ß1-integrin. Data are means ± SE from six independent experiments. *Significant differences with the PBS group at P < 0.05; {dagger}significant difference with the vanadate-treated group at P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have demonstrated that many SSAO substrates, in combination with low vanadate concentrations, stimulate glucose transport in rat and 3T3-L1 adipose cells (14). The stimulation of glucose transport is dependent on SSAO activity and hydrogen peroxide production rather than on aldehyde production (13). The enhanced glucose transport induced by SSAO substrates plus vanadate is characterized by the recruitment of GLUT4 glucose transporters to the cell surface, as demonstrated by subcellular fractionation and plasma membrane lawn techniques (12,13). As to the nature of the mechanisms involved, SSAO substrates plus vanadate markedly stimulate tyrosine phosphorylation of IRS-1 and -3 as well as PI 3-kinase activity (14). On the basis of these data, we proposed that SSAO substrates and vanadate synergistically stimulate one or several tyrosine protein kinases or inhibit protein tyrosine phosphatases, leading to activation of an intracellular pathway similar to that triggered by insulin (14).

In keeping with these observations in vitro, we found that acute administration of an SSAO substrate, benzylamine, together with a low dosage of vanadate, enhances glucose tolerance in nondiabetic and diabetic rats. This effect did not alter the profile of plasma insulin concentrations after glucose challenge, was not associated with alterations in renal glucose reabsorption, and required SSAO activity. These data indicate that the combination of benzylamine and vanadate stimulates glucose disposal after a glucose challenge in vivo.

Because the benzylamine plus vanadate combinations stimulated glucose transport in isolated rat adipocytes from nondiabetic and diabetic rats, we suggest that adipose tissue participates in this response. In addition, given the low concentrations of insulin in diabetic rats, we favor the view that benzylamine and vanadate have insulin-like effects in adipose tissue.

Interestingly, the chronic administration of benzylamine plus vanadate reduced glycemia in STZ-induced diabetic rats, whereas in some experimental protocols, this combination normalized glycemia. As to the time-dependence of these effects, a moderate reduction of glycemia was already noted at day 5–6 after the onset of treatment, which is similar to the time-dependence shown by vanadate or peroxovanadate treatments (24,25). This antihyperglycemic effect of benzylamine plus vanadate was also accompanied by normalization in food and water intake and, as reported for vanadate alone (26), was not a consequence of any increase in plasma insulin.

The chronic administration of benzylamine plus vanadate stimulated glucose uptake in adipose cells. Thus, cells obtained after combined benzylamine and vanadate treatment showed enhanced rates of basal and insulin-stimulated glucose transport and GLUT4 expression. These effects were observed only when both compounds were given in combination but not when they were given separately. The enhanced basal glucose transport may have been a result of the acute effects of benzylamine and vanadate in promoting GLUT4 recruitment to the cell surface or the presence of a larger GLUT4 population. However, the enhanced glucose uptake by adipose cells and the normalization of glycemia may also have been consequences of enhanced insulin sensitivity.

Our data indicate that the combination of benzylamine and vanadate was required for both acute and chronic effects; as previously observed in vitro (12,13), benzylamine or vanadate alone had little or no effect. Studies of adipose cells in vitro have suggested that hydrogen peroxide production is crucial for triggering the stimulation of glucose transport and GLUT4 recruitment to the cell surface, as catalase blocks the effects (12,13). Given that peroxovanadate is a potent insulin-like agent (27,28,29,30,31), it may be formed either inside or outside the cell and may be responsible for the effects of the combination of vanadate and SSAO substrates. Based on the fact that peroxovanadate inhibition of protein tyrosine phosphatases (PTPs) is irreversible, whereas the effect of vanadate is reversible (32), we have indirectly evaluated the generation of peroxovanadate by assessing PTP activity in the presence of 1 mmol/l EDTA and using extracts from adipose cells obtained from diabetic rats chronically treated with different compounds. Chronic vanadate treatment inhibited PTP activity, a result that is in keeping with prior observations (33); the administration of vanadate plus benzylamine also inhibited PTP activity, which was comparable to the effects of vanadate alone (data not shown). These data indicate that there is no correlation between total PTP activity and activation of basal glucose transport in adipose cells or reduction of hyperglycemia in diabetic rats treated with vanadate or benzylamine plus vanadate. Thus, although peroxovanadate may explain the effects of benzylamine plus vanadate, our data do not justify any conclusion on whether peroxovanadate is the only signal generated in adipose cells in response to benzylamine plus vanadate treatment.

Restoration of adipose glucose transport activity may be insufficient to normalize glucose levels in diabetic rats after benzylamine plus vanadate treatment. In adipose cells, benzylamine plus vanadate may generate molecules that have insulin-like effects in other insulin-sensitive tissues. In addition, amelioration of adipose tissue metabolism subsequent to benzylamine plus vanadate treatment, via molecules or hormones released from adipose cells, such as leptin, tumor necrosis factor-{alpha}, or resistin (34,35,36), may have a profound impact on muscle metabolism. It is important to determine whether skeletal muscle increases glucose disposal in response to benzylamine and vanadate.


   ACKNOWLEDGMENTS
 
This study was supported by research grants from the Dirección General de Investigación Científica y Técnica (PM98/0197), Grant 1999SGR 00039 from Generalitat de Catalunya, Fondo de Investigaciones Sanitarias (00/2101), European Commission (Quality of Life, QLG-CT-1999-00295), "Accords INSERM/CSIC," "Actions Integrées Franco-Espagnoles PICASSO," COST B17 Action, and Fundació Marató de TV3 (300720). A.A. is a recipient of a predoctoral fellowship from the Universitat de Barcelona. L.M. is a recipient from a Marie Curie postdoctoral fellowship from the European Union.

We thank Robin Rycroft for his editorial support, Judith Garcia for technical assistance, Mar Grasa and Luc Penicaud for scientific advice, and Roser Casamitjana for help in insulin determinations.


   FOOTNOTES
 
Address correspondence and reprint requests to Antonio Zorzano, Ph.D., Departament de Bioquìmica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain. E-mail: azorzano{at}porthos.bio.ub.es.

Received for publication 25 September 2000 and accepted in revised form 30 May 2001.

L.M. and A.A. contributed equally to the study.

2-DG, 2-deoxy-D-[1,2-3H]glucose; IRS, insulin receptor substrate; PBS, phosphate-buffered saline; PI, phosphatidylinositol; PTP, protein tyrosine phosphatase; SSAO, semicarbazide-sensitive amine oxidase; STZ, streptozotocin; VAP-1, vascular adhesion protein-1.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lyles GA: Substrate-specificity of mammalian tissue-bound semicarbazide-sensitive amine oxidase. Prog Brain Res 106:293–303, 1995[Medline]
  2. Salmi M, Jalkanen S: A 90-kilodalton endothelial cell molecule mediating lymphocyte binding in humans. Science 257:1407–1409, 1992[Abstract/Free Full Text]
  3. Smith DJ, Salmi M, Bono P, Hellman J, Leu T, Jalkanen S: Cloning of vascular adhesion protein 1 reveals a novel multifunctional adhesion molecule. J Exp Med 188:17–27, 1998[Abstract/Free Full Text]
  4. Bono P, Salmi M, Smith DJ, Jalkanen S: Cloning and characterization of mouse vascular adhesion protein-1 reveals a novel molecule with enzymatic activity. J Immunol 160:5563–5571, 1998[Abstract/Free Full Text]
  5. Morris NJ, Ducret A, Aebersold R, Ross SA, Keller SR, Lienhard GE: Membrane amine oxidase cloning and identification as a major protein in the adipocyte plasma membrane. J Biol Chem272:9388–9392, 1997
  6. Barrand MA, Callingham BA: Monoamine oxidase activities in brown adipose tissue of the rat: some properties and subcellular distribution. Biochem Pharmacol 31:2177–2184, 1982[Medline]
  7. Raimondi L, Pirisino R, Ignesti G, Capecchi S, Banchelli G, Buffoni F: Semicarbazide-sensitive amine oxidase activity (SSAO) of rat epididymal white adipose tissue. Biochem Pharmacol 41:467–470, 1991[Medline]
  8. Moldes M, Fève B, Pairault J: Molecular cloning of a major mRNA species in murine 3T3 adipocyte lineage: differentiation-dependent expression, regulation, and identification as semicarbazide-sensitive amine oxidase. J Biol Chem 274:9515–9523, 1999[Abstract/Free Full Text]
  9. Bono P, Jalkanen S, Salmi M: Mouse vascular adhesion protein 1 is a sialoglycoprotein with enzymatic activity and is induced in diabetic insulitis. Am J Pathol 155:1613–1624, 1999[Abstract/Free Full Text]
  10. Raimondi L, Pirisino R, Banchelli G, Ignesti G, Conforti L, Buffoni F: Cultured preadipocytes produce a semicarbazide-sensitive amine oxidase (SSAO) activity. J Neural Transm 32 (Suppl):331–336, 1990
  11. Hayes GR, Lockwood DH: Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide. Proc Natl Acad Sci U S A84:8115–8119, 1987
  12. Enrique-Tarancón G, Marti L, Morin N, Lizcano JM, Unzeta M, Sevilla L, Camps M, Palacín M, Testar X, Carpéné C, Zorzano A: Role of semicarbazide-sensitive amine oxidase on glucose transport and GLUT4 recruitment to the cell surface in adipose cells. J Biol Chem 273:8025–8032, 1998[Abstract/Free Full Text]
  13. Marti L, Morin N, Enrique-Tarancón G, Prévot D, Lafontan M, Testar X, Zorzano A, Carpéné C: Tyramine and vanadate synergistically stimulate glucose transport in rat adipocytes by amine oxidase-dependent generation of hydrogen peroxide. J Pharmacol Exp Ther 285:342–349, 1998[Abstract/Free Full Text]
  14. Enrique-Tarancon G, Castan I, Morin N, Marti L, Abella A, Camps M, Casamitjana R, Palacin M, Testar X, Degerman E, Carpene C, Zorzano A: Substrates of semicarbazide-sensitive amine oxidase cooperate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells. Biochem J 350:171–180, 2000
  15. Gumà A, Mora C, Santalucía T, Viñals F, Testar X, Palacín M, Zorzano A: System A transport activity is stimulated in skeletal muscle in response to diabetes. FEBS Lett 310:51–54, 1992[Medline]
  16. Laury MC, Takao F, Bailbe D, Penicaud L, Portha B, Picon L, Ktorza A: Differential effects of prolonged hyperglycemia on in vivo and in vitro insulin secretion in rats. Endocrinology 128:2526–2533, 1991[Abstract]
  17. Wang X, Rondu F, Lamouri A, Dokhan R, Marc S, Touboul E, Pfeiffer B, Manechez D, Renard P, Guardiola-Lemaitre B, Godfroid JJ, Ktorza A, Penicaud L: Effect of S-21663 (PMS 812), an imidazoline derivative, on glucose tolerance and insulin secretion in a rat model of type II diabetes. J Pharmacol Exp Ther278:82–89, 1996
  18. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254, 1976[Medline]
  19. Fowler CJ, Tipton KF: Concentration dependence of the oxidation of tyramine by the two forms of rat liver mitochondrial monoamine oxidase. Biochem Pharmacol 30:3329–3332, 1981[Medline]
  20. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685, 1970[Medline]
  21. Conforti L, Pirisino R, Ignesti G, Banchelli G, Raimondi L: Semicarbazide-sensitive amine oxidase activity in white adipose tissue of the insulin-deficient rat. J Pharm Pharmacol47:420–424, 1995
  22. Kasuga M, Akanuma Y, Iwamoto Y, Kosaka K: Insulin binding and glucose metabolism in adipocytes of streptozotocin-diabetic rats. Am J Physiol 235:E175-E182, 1978
  23. Kobayashi M, Olefsky JM: Effects of streptozotocin-induced diabetes on insulin binding, glucose transport, and intracellular glucose metabolism in isolated rat adipocytes. Diabetes 28:87–95, 1979[Medline]
  24. Meyerovitch J, Farfel Z, Sack J, Shechter Y: Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats: characterization and mode of action. J Biol Chem 262:6658–6662, 1987[Abstract/Free Full Text]
  25. Yale JF, Lachance D, Bevan AP, Vigeant C, Shaver A, Posner BI: Hypoglycemic effects of peroxovanadium compounds in Sprague-Dawley and diabetic BB rats. Diabetes 44:1274–1279, 1995[Abstract]
  26. Brichard SM, Ongemba LN, Girard J, Henquin JC: Tissue-specific correction of lipogenic enzyme gene expression in diabetic rats given vanadate. Diabetologia 37:1065–1072, 1994[Medline]
  27. Kadota S, Fantus IG, Deragon G, Guyda HJ, Hersh B, Posner BI: Peroxide(s) of vanadium: a novel and potent insulin-mimetic agent which activates the insulin receptor kinase. Biochem Biophys Res Commun 147:259–266, 1987[Medline]
  28. Fantus IG, Kadota S, Deragon G, Foster B, Posner BI: Pervanadate (peroxide[s] of vanadate) mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase. Biochemistry 28:8864–8871, 1989[Medline]
  29. Posner BI, Faure R, Burgess JW, Bevan AP, Lachance D, Zhang-Sun G, Fantus IG, Ng JB, Hall DA, Soo Lum B, Shaver A: Peroxovanadium compounds: a new class of potent phosphotyrosine phosphatase inhibitors which are insulin mimetics. J Biol Chem 269:4596–4604, 1994[Abstract/Free Full Text]
  30. Lönnroth P, Eriksson JW, Posner BI, Smith U: Peroxovanadate but not vanadate exerts insulin-like effects in human adipocytes. Diabetologia 36:113–116, 1993[Medline]
  31. Shisheva A, Shechter Y: Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes: dissociation from vanadate effect. Endocrinology 133:1562–1568, 1993[Abstract]
  32. Huyer G, Liu S, Kelly J, Moffat J, Payette P, Kennedy B, Tsaprailis G, Gresser MJ, Ramachandran C: Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate. J Biol Chem 272:843–851, 1997[Abstract/Free Full Text]
  33. Begum N, Sussman KE, Draznin B: Differential effects of diabetes on adipocyte and liver phosphotyrosine and phosphoserine phosphatase activities. Diabetes 40:1620–1629, 1991[Abstract]
  34. Hotamisligil GS, Shargill NS, Spiegelman BM: Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259:87–91, 1993[Abstract/Free Full Text]
  35. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM: Positional cloning of the mouse gene and its human homologue. Nature 372:425–432, 1994[Medline]
  36. Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA: The hormone resistin links obesity to diabetes. Nature 409:307–312, 2001[Medline]

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 DiabetesHome page
S. Garcia-Vicente, F. Yraola, L. Marti, E. Gonzalez-Munoz, M. J. Garcia-Barrado, C. Canto, A. Abella, S. Bour, R. Artuch, C. Sierra, et al.
Oral Insulin-Mimetic Compounds That Act Independently of Insulin
Diabetes, February 1, 2007; 56(2): 486 - 493.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. C. Iglesias-Osma, M. J. Garcia-Barrado, V. Visentin, M. F. Pastor-Mansilla, S. Bour, D. Prevot, P. Valet, J. Moratinos, and C. Carpene
Benzylamine Exhibits Insulin-Like Effects on Glucose Disposal, Glucose Transport, and Fat Cell Lipolysis in Rabbits and Diabetic Mice
J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1020 - 1028.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
P. H. Yu, M. Wang, H. Fan, Y. Deng, and D. Gubisne-Haberle
Involvement of SSAO-mediated deamination in adipose glucose transport and weight gain in obese diabetic KKAy mice
Am J Physiol Endocrinol Metab, April 1, 2004; 286(4): E634 - E641.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. Tardif, N. Julien, J.-L. Chiasson, and L. Coderre
Stimulation of glucose uptake by chronic vanadate pretreatment in cardiomyocytes requires PI 3-kinase and p38 MAPK activation
Am J Physiol Endocrinol Metab, June 1, 2003; 284(6): E1055 - E1064.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
A. Abella, L. Marti, M. Camps, M. Claret, J. Fernandez-Alvarez, R. Gomis, A. Guma, N. Viguerie, C. Carpene, M. Palacin, et al.
Semicarbazide-Sensitive Amine Oxidase/Vascular Adhesion Protein-1 Activity Exerts an Antidiabetic Action in Goto-Kakizaki Rats
Diabetes, April 1, 2003; 52(4): 1004 - 1013.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
N. Mercier, M. Moldes, K. E. Hadri, and B. Feve
Regulation of Semicarbazide-Sensitive Amine Oxidase Expression by Tumor Necrosis Factor-alpha in Adipocytes: Functional Consequences on Glucose Transport
J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1197 - 1208.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
N. Morin, V. Visentin, D. Calise, L. Marti, A. Zorzano, X. Testar, P. Valet, Y. Fischer, and C. Carpene
Tyramine Stimulates Glucose Uptake in Insulin-Sensitive Tissues in Vitro and in Vivo via Its Oxidation by Amine Oxidases
J. Pharmacol. Exp. Ther., December 1, 2002; 303(3): 1238 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
M. Salmi, C. Stolen, P. Jousilahti, G. G. Yegutkin, P. Tapanainen, T. Janatuinen, M. Knip, S. Jalkanen, and V. Salomaa
Insulin-Regulated Increase of Soluble Vascular Adhesion Protein-1 in Diabetes
Am. J. Pathol., December 1, 2002; 161(6): 2255 - 2262.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Marti, L.
Right arrow Articles by Zorzano, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marti, L.
Right arrow Articles by Zorzano, A.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS