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Original Paper

Nephron Physiol 2007;105:p42–p51 DOI: 10.1159/000098442

Insulin but Not Phlorizin Treatment Induces a Transient Increase in GLUT2 Gene Expression in the Kidney of Diabetic Rats

Helayne Soares Freitas a Beatriz D’Agord Schaan b Robinson Sabino da Silva a

Maristela Mitiko Okamoto a Maria Oliveira-Souza a Ubiratan Fabres Machado a

a Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo ,

and b Experimental Medicine Department, Institute of Cardiology of the State of Rio Grande do Sul/University

Foundation of Cardiology, Porto Alegre, Rio Grande do Sul , Brazil

mia, with opposite effects upon plasma insulin and urinary glucose, and both treatments decreased GLUT2 mRNA and protein (p ! 0.05). Conclusion: In kidney of diabetic rats, an initial and transient upregulation of GLUT2 was induced spe-cifically by insulin only. The 6-day normalization of GLUT2, however, was induced by both insulin and phlorizin treat-ment, which seems to be related to the plasma glucose low-ering. Copyright © 2007 S. Karger AG, Basel

Introduction

It is well known that renal glucose reabsorption is a coordinated process, which takes place in the epithelial cells of the proximal tubule, involving two classes of glu-cose transporters, the Na + -glucose transporters (SGLTs) and facilitative diffusion transporters (GLUTs) [1] . In the early S1 segment, where the bulk of filtered glucose is re-absorbed [2] , the low-affinity/high-capacity glucose transporters, SGLT2 and GLUT2, coexpress, respective-ly, in the luminal brush-border membrane (BBM) and in the basolateral membrane (BLM) [3] . Additionally, the presence of GLUT2 protein in the BBM of epithelial tu-

Key Words Diabetes � Insulin treatment � Phlorizin treatment � Glucose transporters � GLUT2

Abstract Background/Aims: Increases in the renal glucose transport-er gene expression are involved in renal tubule-glomerular diseases. Here we investigate the GLUT2 gene expression changes in the kidney of diabetic rats, by using insulin or phlorizin treatment. Methods: Rats were rendered diabetic and studied 20 days later: 4–12 h after one single injection of insulin or phlorizin, and 1–6 days after insulin or phlorizin injection twice a day, comparing with diabetic rats injected with placebo. GLUT2 was investigated by Northern and Western analysis. Results: In 20-day diabetic rats, acute treatment with insulin lowered the plasma glucose and in-creased the GLUT2 mRNA ( � 100%, p ! 0.001) without chang-es in the protein content, while phlorizin lowered the plasma glucose, but changed neither the GLUT2 mRNA nor the pro-tein expression. Twenty-four hours of insulin treatment in-creased both GLUT2 mRNA ( � 100%, p ! 0.001) and protein ( � 50%, p ! 0.01), but no effects of phlorizin were observed. After 6 days, insulin and phlorizin similarly reduced glyce-

Received: May 31, 2006 Accepted: October 13, 2006 Published online: January 9, 2007

Ubiratan Fabres Machado Department of Physiology and Biophysics, Institute of Biomedical Sciences University of Sao Paulo, Av. Prof. Lineu Prestes, 1524 05505-900, Sao Paulo (SP) (Brazil) Tel. +55 113 091 7494, Fax +55 113 091 7285, E-Mail ubiratan@icb.usp.br

© 2007 S. Karger AG, Basel 1660–2137/07/1053–0042$23.50/0

Accessible online at: www.karger.com/nep

GLUT2 Gene Expression and Diabetes Nephron Physiol 2007;105:p42–p51 p43

bular cells was already reported in diabetic rats, confirm-ing that facilitative glucose transport also occurs through-out the BBM, and pointing out the participation of GLUT2 in the glucose reabsorption across the tubule [4] .

Increases in the glucose transporter gene expression, which have been extensively reported in diabetes [2, 5–10] , are important to renal glucose reabsorption mainte-nance in this condition, since high blood and interstitial glucose concentrations may lower the outwardly directed glucose gradient, from tubule to blood [9] . Indeed, in-creased renal GLUT1 [11] and GLUT2 [6] gene expres-sion has been proposed to be involved in the develop-ment of diabetic nephropathy. Besides, mutations in the GLUT2 gene were described in the Fanconi-Bickel syn-drome [12–15] , in which glycogen accumulates in the proximal tubule and tubular nephropathy develops [16] , emphasizing the importance of this glucose transporter in the renal tubule-glomerular disease.

Glucose concentration has been proposed as an im-portant regulator of GLUT2 expression. However, this regulation seems to be tissue-specific. Supraphysiologic glucose concentration is known to enhance GLUT2 ex-pression in hepatocytes [17] , to suppress it in the human insulinoma cell line CM [18] , and to have no effect on rat pancreatic islets cultured for 4 or 6 weeks [19] . Besides, the modulation of GLUT2 gene expression in the kidney was firstly associated to changes in glucose filtration rate, as we and others have reported [6] , and recently we have proposed that insulin and glycemic control might inde-pendently regulate, in a time-dependent way, the GLUT2 gene expression of diabetic rats [20] . However, consider-ing that insulin treatment causes hyperinsulinemia and lowers glycemia and renal glucose filtration rate, the main effectors of the GLUT2 gene expression changes in the kidney are not clear yet.

The aim of the present study was to investigate the time course of GLUT2 gene expression changes in renal cortex of diabetic rats after insulin or phlorizin – an in-hibitor of SGLT that blocks the renal glucose reabsorp-tion – treatment. This strategy allowed us to show the individual effects of insulin or plasma and urinary glu-cose levels upon GLUT2 gene expression changes. We de-scribed here the GLUT2 gene expression in 20-day dia-betic rats subjected to either an acute (single injection) or a short-term (6 days) treatment with insulin or phlorizin. We demonstrated that an initial and transitory upregula-tion of GLUT2 is induced by insulin. However, 6-day normalization of GLUT2 gene expression is related to the plasma glucose lowering.

Methods

Animals and Experimental Protocols Male Wistar rats ( � 280 g BW), individually caged in an envi-

ronment maintained at controlled temperature (23 8 2 ° C), and lighting (from 6 a.m. to 6 p.m.), and allowed free access to water and standard rodent chow diet (Nuvilab CR-1, Nuvital, Curitiba, Brazil), were rendered diabetic by a single intravenous injection (penis vein) of alloxan (40 mg/kg BW). Nondiabetic control rats were injected with saline in the same volume. Plasma glucose and insulin as well as 24-hour urine glucose were evaluated. Twenty days after the alloxan injection, diabetic rats were compared to the nondiabetic rats, and submitted to the following protocols of study for further analysis:

Protocol 1 – Acute Effects of Insulin or Phlorizin Treatment. Twenty-day diabetic rats were evaluated 4 or 12 h after a single subcutaneous NPH insulin injection, 4 U (Biohulin, Biobrás, MG, Brazil) plus regular insulin, 2 U (Iolin, Biobrás), or subcutaneous phlorizin, 0.4 g kg � BW –1 (Sigma, Washington, D.C., USA). Phlo-rizin solution was 0.4 g � ml –1 of propylene glycol. Diabetic place-bo-treated rats were injected with the same volume of saline, in the insulin study, or propylene glycol, in the phlorizin study. The injections were performed at 8: 30 h a.m.

Protocol 2 – S hort-Term Effects of Insulin or Phlorizin Treat-ment. Twenty-day diabetic rats were evaluated after 1, 4 and 6 days of treatment with subcutaneous NPH insulin, 2 U in the morning (8: 30 a.m.) and 4 U in the afternoon (5: 30 p.m.), or subcutaneous phlorizin, 0.4 g/kg BW each 12 h (8: 30 a.m. and 8: 30 p.m.). Dia-betic placebo-treated rats were injected with the same volume of saline, in the insulin study, or propylene glycol, in the phlorizin study. Twenty-four-hour urine was collected during the 1st, 4th and 6th days of treatment. Animals were killed at 8: 30 a.m. For diabetes induction and final experimental procedures, animals were anesthetized with intraperitoneal injection of sodium pen-tobarbital (40 mg � kg –1 BW). Experimental procedures were ap-proved by the Ethical Committee for Animal Research of the In-stitute of Biomedical Sciences, of the University of Sao Paulo (pro-tocol #165/02).

Renal Sampling Renal out-cortices were sampled as previously described [20] .

Briefly, after clamping the aorta up to the renal arteries, and cut-ting the inferior vena cava up to the renal veins, the kidneys were perfused with Hanks buffer, to eliminate their intravascular blood content, and immediately removed. Renal out-cortex (slic-es � 1.5 mm thin) was carefully dissected and the tissue fragments were weighed and frozen in liquid nitrogen for further analysis procedures.

Protein and RNA Extraction The renal cortex samples were homogenized in 10 w/v buffer

(10 m M Tris-HCl, 1 m M EDTA and 250 m M sucrose, pH 7.4, containing 15 � g � ml –1 aprotinin, 5 � g � ml –1 leupeptin and 0.1 m M phenylmethanesulfonyl f luoride), using a Polytron set at 24,000 rpm for 30 s, and centrifuged at 3,000 g for 15 min. The supernatant, containing total cellular membrane proteins (TC), was centrifuged at 12,000 g for 20 min, to precipitate a plasma membrane rich fraction (PM), as previously characterized [20] . The protein concentration of the samples was determined by the Bradford method (Bio-Rad protein assay, Bio-Rad, USA). Com-

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Nephron Physiol 2007;105:p42–p51 p44

pared with the supernatant obtained after the first 3,000 g cen-trifugation, the PM fraction showed a 6.8-fold increase in 5 � nu-cleotidase activity (plasma membrane marker), and a 3.2-fold in-crease in alkaline phosphatase (BBM marker). Total RNA was extracted from 0.1 g renal cortex samples using Trizol reagent (Invitrogen Life Technologies, Carlsbad, Calif., USA) as described by the manufacturer’s instructions.

Western Blotting Immunoblotting was carried out as previously described

[6, 11, 20] . Briefly, equal amounts of plasma membrane protein (100 � g) were solubilized in sample buffer (0.5 M Tris-HCl, pH 7.5; 9% SDS; 15% glycerol; 0.05% bromophenol blue; 6% 2-mer-captoethanol), and resolved by SDS-PAGE (10%) electrophoresis. Proteins were electrotransferred to nitrocellulose membrane. The membrane was blocked with buffer containing 3% nonfat milk, incubated with anti-rat GLUT2 antiserum (1: 200 dilution), raised against the carboxyterminal decapeptide, for 3 h at 37 ° C, washed, and incubated with 1.5 � Ci � 50 ml –1 of PBS buffer pH 7.2 [ 125 I]protein-A (38 � Ci � � g –1 ) (Amersham Pharmacia Biotech, Amersham, UK) for 2 h at room temperature. After that, the membrane was washed and exposed to X-ray film for 5 to 7 days. The intensity of the blots was quantified by densitometry (Image Master 1D , Pharmacia Biotech, Uppsala, Sweden), and the pro-tein expression was normalized considering the placebo-treated diabetic rat values as 100%.

Northern Blotting Northern analysis was carried out as previously described [6,

20, 21] . In summary, 20 � g of total RNA were fractionated by 1% agarose-formaldehyde gel electrophoresis, transferred to nylon membranes, prehybridized in buffer with 100 � g � ml –1 salmon sperm DNA, and subsequently probed with a ( � - 32 P)dCTP-la-beled GLUT2 cDNA by random priming (Random Primers DNA Labeling System Kit, Invitrogen Life Technologies, Carlsbad, Calif., USA). The membranes were washed under high-stringency condition and exposed to hyperfilm at –70 ° C. After that, the membranes were stripped and rehybridized with a � -actin probe. The blots were analyzed by scanner densitometry as described for Western analysis, the relative intensity of the 3.0-kb GLUT2 mRNA was calculated as a ratio of the respective � -actin value, and expressed as GLUT2 mRNA. The GLUT2 mRNA expression was normalized considering the placebo-treated diabetic rat val-ues as 100%.

Analytical Procedures Plasma, obtained from blood sampled (tail vein) at the sacri-

fice of animals, and urine, obtained by 24-hour urine sampling, were assayed for glucose by the glucose-oxidase method (Anali-sa Diagnostica, Sao Paulo, Brazil). Insulin was also determined in plasma samples (RIE, Coat-a-Count, Diagnostic Products, Los Angeles, Calif., USA).

Data Analysis In each protocol, saline- or propylene glycol-treated rats re-

sults were pooled into placebo groups, since there were no differ-ences according to the period of treatment. All values were re-ported as mean 8 SEM. At least 4 different experiments of West-ern and Northern analysis were performed, evaluating at least 5 different animals in each group. Detailed data of sample size were

informed in the legends of figures and tables. The means were statistically analyzed by ANOVA, with Newman-Keuls (NK) post test. Two experimental groups were compared by Student’s t test when appropriate.

Results

As previously described [20] , the GLUT2 probe was seen to hybridize at � 3.0 and 3.9 kb, revealing the occur-rence of two transcripts, but only the � 3.0-kb GLUT2 mRNA content was modulated by diabetes, thus all re-sults presented here refer to this transcript. Despite the detection of two GLUT2 mRNA transcripts, the Western blotting analysis showed only one single � 59-kD band of GLUT2 protein.

Effects of Diabetes Compared to nondiabetic rats, 20-day diabetic rats

had lower body weights (325.8 8 10.1 vs. 274.8 8 15.7 g, n = 6 for each group, p ! 0.001). Plasma glucose was very high ( 6 20 m M ) in 2-, 10- and 20-day diabetic rats. Low plasma insulin levels were observed in 20-day diabetic rats (0.20 8 0.01 ng/ml), as compared to nondiabetic rats (0.91 8 0.19 ng/ml, n = 10). Glycosuria was 6 300 mg � day –1 since the second day after diabetes induction, whereas it was 3.5 8 0.8 mg � day –1 , n = 10, in nondiabet-ic rats. Additionally, 20 days of diabetes increased both the GLUT2 mRNA and protein by � 100% (p ! 0.05). These results are not shown in tables or figures, since they are similar to others reported previously [20] .

Acute Effects of Insulin or Phlorizin Treatment In protocol 1, 20-day diabetic rats were analyzed 4 or

12 h after a single injection of insulin, phlorizin or pla-cebo, as described in Methods. Since the results of each placebo group, saline- or propylene glycol-treated, re-spectively, for insulin or phlorizin, showed no differences between 4 and 12 h, they were pooled. Twenty-four-hour urinary glucose before the injections was similar in all groups, showing that the diabetic state achieved was sim-ilar among them.

Table 1 shows that the insulin injection induced a de-crease (p ! 0.001) in plasma glucose, which remained low for the next 12 h. However, 12 h after the injection, the mean plasma glucose was higher than the value observed after 4 h. Similarly, phlorizin injection induced a signifi-cant (p ! 0.001) decrease in plasma glucose after both 4 and 12 h. Clearly, the plasma glucose decrease in insulin-treated rats was a consequence of hyperinsulinemia ob-

GLUT2 Gene Expression and Diabetes Nephron Physiol 2007;105:p42–p51 p45

served. As expected, insulin levels of the phlorizin-treat-ed diabetic rats were very low.

Figure 1 shows the GLUT2 mRNA expression in insu-lin- and phlorizin-injected diabetic rats. Insulin induced a rapid and progressive increase in the GLUT2 mRNA content ( fig. 1 a). Four hours after the insulin injection the GLUT2 mRNA content was 50% higher (p ! 0.05) than that in placebo-treated rats, and 12 h later, this value was twice (p ! 0.001) that of the placebo-treated rats, showing a progressive elevation. Differently, phlorizin-treated rats, as compared to their placebo-treated rats ( fig. 1 b), showed no significant changes in the GLUT2 mRNA, suggesting that insulin was the cause of the rapid GLUT2 mRNA increase. Despite the changes observed in GLUT2 mRNA of the insulin-injected rats, the GLUT2 protein did not change in response to the acute insulin stimulus even 12 h later ( fig. 2 a). The GLUT2 protein content was also unchanged in phlorizin-treated rats, as expected ( fig. 2 b). To clarify whether insulin interferes with the intracellular traffic of GLUT2 protein, additional experi-ment was performed in 12-hour insulin-treated rats in which the protein content was measured in both PM and TC subcellular fractions. The TC GLUT2 content (total cellular) was 41.3 8 3.9 and 40.2 8 3.6 AU, respectively, in placebo- and insulin-treated rats.

Short-Term Effects of Insulin or Phlorizin Treatment In protocol 2, rats were made diabetic and 20 days lat-

er were submitted to insulin or phlorizin treatment twice

a day, as described in Methods. For each treatment, dif-ferent placebo-treated rats were studied, since the mo-ment and volume of the injections were different, as well as by the fact that placebo for insulin treatment was sa-line, whereas for phlorizin treatment was propylene gly-col. Since in each placebo treatment the results of 1, 4 and 6 days were similar concerning all parameters analyzed, their data were pooled in separate placebo groups, which did not differ between them (comparison not shown). As

Table 1. Plasma insulin and glucose of diabetic rats 4 or 12 h after an injection of insulin, phlorizin or placebo

Plasma insulin (ng/ml)

Plasma glucose (mM)

Insulin treatmentPlacebo (4 + 12 h) 0.2680.09 (14) 18.980.82 (10)4 h 18.483.60 (6)* 4.0080.45 (8)*12 h 6.482.71 (6)*, ** 13.881.00 (5)*, **Phlorizin treatmentPlacebo (4 + 12 h) 0.3080.11 (8) 21.1080.97 (10)4 h 0.2580.09 (7) 12.880.8 (7)*12 h 0.2880.11 (6) 13.180.8 (6)*

Placebo was saline for insulin treatment, or propylene glycol for phlorizin treatment. Data from 4- and 12-hour placebo-treat-ed rats were pooled. The numbers in parentheses following the actual data values represent the n for each measurement.

* p < 0.001 vs. respective placebo; ** p < 0.01 vs. 4 h, one-way ANOVA, Newman-Keuls post-test.

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Fig. 1. Acute effects of insulin ( a ) and phlorizin ( b ) on GLUT2 mRNA expression in renal out-cortex of diabetic rats studied 4 and 12 h after a single injection of insulin (NPH 4 U + regular 2 U, subcutaneously) or phlorizin (0.4 g/kg BW, subcutaneously). Placebo treatment was saline for insulin study and propylene gly-col for phlorizin study. 4- and 12-hour data of each placebo treat-ment were pooled, since there was no difference between them. Each panel shows, at the top, typical images of the � 3.0-GLUT2 mRNA and respective � -actin, at the bottom, relative content of the � 3.0-GLUT2 mRNA was normalized by the � -actin value, and expressed as percentage of the placebo group. Data are mean 8 SEM for 5–7 animals in each insulin- or phlorizin-treated group, and 10 animals in each placebo-treated group, evaluated in 4 different experiments. * p ! 0.05; *** p ! 0.001 vs. placebo; # p ! 0.05 vs. 12 h (ANOVA, NK post-test).

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Nephron Physiol 2007;105:p42–p51 p46

we pointed out in protocol 1, on the day before the begin-ning of the treatments, diabetic rats showed similar val-ues of 24-hour glycosuria, presenting a similar diabetic state (data not shown).

The body weight of each placebo-, insulin- or phlori-zin-treated group was similar at the beginning of the treatments (data not shown). Insulin treatment induced a rapid body weight gain ( table 2 ), and 4- and 6-day insu-lin-treated rats were 16 and 20% heavier than their pla-cebo-treated rats, respectively (p ! 0.05). On the other hand, phlorizin treatment did not induce any change in body weight gain ( table 2 ). This effect of insulin treat-ment in the body weight was clearly related to the ana-bolic effects of the hormone, since its plasma concentra-tions were very high (6.2–10.4 ng/ml).

Figure 3 a shows the plasma glucose concentration at sacrifice (8: 30 a.m.), before the insulin, phlorizin or pla-cebo injection. One day after the insulin treatment had begun, plasma glucose decreased by 20% (p ! 0.05), and in the 6-day insulin-treated rats the value was � 50% that of placebo-treated rats (p ! 0.01). Phlorizin treatment de-creased (p ! 0.001) the plasma glucose by � 50% after one day of treatment, in comparison with the placebo treat-ment ( fig. 3 c). This similar glycemic profile was accom-panied by completely opposite urinary volume and glu-cose concentration. Comparing with placebo-treated di-abetic rats, insulin-treated diabetic rats ( fig. 3 b) showed a progressive decrease in the 24-hour urinary glucose ex-cretion (p ! 0.001), which achieves � 95 mg � day –1 in 6-day-treated diabetic rats. Differently, the phlorizin-treat-ed diabetic rats, compared to their placebo-treated rats ( fig. 3 d), showed unchanged 24-hour urinary glucose ex-cretion at 1 and 4 days of treatment, and that was in-creased by 45% in the 6-day-treated diabetic rats (p !

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Fig. 2. Acute effects of insulin ( a ) and phlorizin ( b ) on GLUT2 protein expression in renal out-cortex of diabetic rats studied 4 and 12 h after a single injection of insulin (NPH 4 U + regular 2 U, subcutaneously) or phlorizin (0.4 g/kg BW, subcutaneously). Placebo treatment was saline for insulin study and propylene gly-col for phlorizin study. 4- and 12-hour data of each placebo treat-ment were pooled, since there was no difference between them. Each panel shows, at the top, typical images of the GLUT2 pro-tein, at the bottom, relative content of the GLUT2 protein, ex-pressed as percentage of the placebo group. Data are mean 8 SEM for 5–7 animals in each insulin- or phlorizin-treated group, and 10 animals in each placebo-treated group, evaluated in 4 different experiments. No significant differences were observed.

1 day 4 days 6 days 1 + 4 + 6 days

Insulin 279.189.9 (7) 312.3811.9 (8)* 321.2810.6 (5)*Placebo 269.0810.8 (10)Phlorizin 290.884.4 (5) 285.884.5 (8) 277.486.0 (7)Placebo 282.8810.1 (6)

Placebo was saline for insulin treatment, or propylene glycol for phlorizin treatment. Data from 1-, 4- and 6-day placebo-treated rats were pooled. The numbers in parenthe-ses following the actual data values represent the n for each measurement. * p < 0.05 vs. respective placebo and 1 day, one-way ANOVA, Newman-Keuls post-test.

Table 2. Body weight (g) of diabetic rats after 1, 4 or 6 days of treatment with insulin, phlorizin or placebo

GLUT2 Gene Expression and Diabetes Nephron Physiol 2007;105:p42–p51 p47

0.001). Although plasma glucose levels were shown to be decreased 24 h after phlorizin treatment, the urinary glu-cose excretion was not additionally increased, probably because the phlorizin effect had not yet been established along the 24-hour period.

The GLUT2 mRNA content in 1-day insulin-treated diabetic rats ( fig. 4 a), which was seen to be increased 12 h after the acute insulin injection, was still � 2-fold increased, as compared to the content of placebo-treated rats (p ! 0.001). However, the insulin treatment for more 3 days decreases the GLUT2 mRNA. Compared to pla-cebo-treated diabetic rats, the GLUT mRNA content was similar in the 4-day insulin-treated rats and � 40% lower in the 6-day insulin-treated rats (p = 0.0012 compared with placebo rats only, Student’s t test), finally reaching a value similar to that in nondiabetic rats. On the other hand, the GLUT2 mRNA in phlorizin-treated diabetic rats, compared to their placebo-treated rats ( fig. 4 b), was unchanged 1 day later, maintaining the absence of modulation observed in the acute experiment and was reduced (p ! 0.05) by � 30% in 4- and 6-day-treated rats, again reaching a value similar to that in nondiabet-ic rats.

Finally, the GLUT2 protein content, which remained unchanged during 12 h after a single insulin injection,

increased by 43% in 1-day insulin-treated diabetic rats (p ! 0.01 vs. placebo-rats), and progressively decreased to 75% in 6-day insulin-treated rats as compared to placebo-treated diabetic rats (p = 0.0223, Student’s t test), going back to a value close to that of non-diabetic rats ( fig. 5 a). Differently from the data observed in the protein modu-lation of insulin-treated diabetic rats, but similarly to the data observed in the mRNA of phlorizin-treated diabetic rats, the GLUT2 protein in phlorizin-treated animals ( fig. 5 b) was unchanged after 1 day of treatment, but de-creased (p ! 0.05) in 4- and 6-day-treated diabetic rats, as compared to their placebo-treated diabetic rats.

Discussion

The present study describes the time-course regulation of renal GLUT2 gene expression in insulin- and phlori-zin-treated diabetic rats, and unravels the in vivo mecha-nisms involved in the observed changes, since these treat-ments provided similar glycemic control, in spite of the opposite plasma insulin and urinary glucose levels. In-creased glucose reabsorption in the proximal renal tubule is a characteristic adaptive condition of uncontrolled dia-betes, which avoids high urinary losses, but contributes to

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Fig. 3. Plasma ( a, c ) and urinary glucose ( b, d ) of diabetic rats submitted to a short-term treatment with insulin (1, 4 and 6 days of NPH 2 U at 8: 30 a.m. and NPH 4 U at 5: 30 p.m., subcutaneously; a, b ) or phlo-rizin (1, 4 and 6 days of 0.4 g/kg BW, 8: 30 a.m. and 8: 30 p.m., subcutaneously; c, d ). Placebo treatment was saline for insulin study and propylene glycol for phlorizin study. 1-, 4- and 6-day data of each placebo treatment were pooled, since there was no difference among them. Plasma glucose is expressed in m M and urinary glucose in mg � day –1 . Data are mean 8 SEM for 5–7 animals in each insulin- or phlorizin-treated group, and 10 animals in each pla-cebo-treated group. * p ! 0.05, ** p ! 0.01, *** p ! 0.001 vs. placebo; ## p ! 0.01 vs. 1 day; §§§ p ! 0.001 vs. all groups (ANOVA, NK post-test).

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maintain hyperglycemia, and enhanced GLUT2 gene ex-pression plays a key role in this process [2] .

Two transcripts but only one protein has been reported in tissues that express GLUT2 such as pancreatic islets [22] , kidney and liver [20, 23] , similar to what we are now reporting. Fukumoto et al. [23] have demonstrated that

the 2.8- and 3.4-kb GLUT2 mRNA transcripts differ in the length of the 3 � untranslated region, thus explaining only one type of translated protein. In the present study, we have confirmed that both the 3.0-kb GLUT2 mRNA and protein contents were similarly increased in diabetic rats, suggesting a transcriptional upregulation of this gene.

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Fig. 4. Short-term effects of insulin ( a ) and phlorizin ( b ) on GLUT2 mRNA expression in renal out-cortex of diabetic rats studied after 1, 4 and 6 days of treatment with insulin (NPH 2 U at 8: 30 a.m. and NPH 4 U at 5: 30 p.m., subcutaneously) or phlo-rizin (0.4 g/kg BW, 8: 30 a.m. and 8: 30 p.m., subcutaneously). Pla-cebo treatment was saline for insulin study and propylene glycol for phlorizin study. 1-, 4- and 6-day data of each placebo treat-ment were pooled, since there was no difference among them. Each panel shows, at the top, typical images of the � 3.0-GLUT2 mRNA and respective � -actin, at the bottom, relative content of the � 3.0-GLUT2 mRNA was normalized by the � -actin value, and expressed as percentage of the placebo group. Data are mean 8 SEM for 5–7 animals in each insulin- or phlorizin-treated group, and 10 animals in each placebo-treated group, evaluated in 4 different experiments. * p ! 0.05, *** p ! 0.001 vs. placebo; # p ! 0.05, ### p ! 0.001 vs. 1 day (ANOVA, NK post test); f p = 0.0012 vs. placebo (Student’s t test).

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a

b

Fig. 5. Short-term effects of insulin ( a ) and phlorizin ( b ) on GLUT2 protein expression in renal out-cortex of diabetic rats studied after 1, 4 and 6 days of treatment with insulin (NPH 2 U at 8: 30 a.m. and NPH 4 U at 5: 30 p.m., subcutaneously) or phlo-rizin (0.4 g/kg BW, 8: 30 a.m. and 8: 30 p.m., subcutaneously). Pla-cebo treatment was saline for insulin study and propylene glycol for phlorizin study. 1-, 4- and 6-day data of each placebo treat-ment were pooled, since there was no difference among them. Each panel shows, at the top, typical images of the GLUT2 pro-tein, at the bottom, relative content of the GLUT2 protein, ex-pressed as percentage of the placebo group. Data are mean 8 SEM for 5–7 animals in each insulin- or phlorizin-treated group, and 10 animals in each placebo-treated group, evaluated in 4 different experiments. * p ! 0.05, ** p ! 0.01 vs. placebo; # p ! 0.05, ## p ! 0.01 vs. 1 day (ANOVA, NK post-test); f p = 0.0223 vs. placebo (Student’s t test).

GLUT2 Gene Expression and Diabetes Nephron Physiol 2007;105:p42–p51 p49

The reversal of diabetes-induced upregulation of GLUT2 gene expression by insulin treatment was recent-ly reported, and it was proposed to be due to the glycemic control and/or normal renal tubular glucose concentra-tion [20] . However, as expected, hyperinsulinemia was a consequence of insulin treatment, and the hormone could, per se, play as a modulator of the renal GLUT2 ex-pression. Thus, the main regulator of GLUT2 gene ex-pression was not clear yet.

Phlorizin is an agent that inhibits SGLTs co-transport-ers and suppresses renal glucose reabsorption, both in-creasing urinary excretion of glucose and reducing plas-ma glucose levels in diabetic rats [24] , in spite of low in-sulin levels. In 4-week-treated diabetic rats with T-1095 (a prodrug that should generate circulating phlorizin), it was already suggested that inhibition of renal glucose re-absorption could modulate GLUT2 expression in the kid-ney of diabetic rats [7] . However, that was a long-term treatment in which several other metabolic-hormonal mechanisms induced by the phlorizin precursor could be involved in the described GLUT2 gene regulation.

In this study we showed that both insulin and phlori-zin treatment for 6 days reversed the previously increased diabetes-induced GLUT2 gene expression, which re-turned to nondiabetic rats levels. Considering the oppo-site plasma insulin and urinary glucose levels induced by insulin and phlorizin treatments, this definitely reveals that plasma glucose and, consequently, its renal intersti-tial concentration are the main regulators of the GLUT2 mRNA and protein changes observed in the kidney of diabetic rats after 6 days of treatment.

Otherwise, insulin therapy induces a short-term ad-ditional upregulation of the GLUT2 gene expression [20] . This phenomenon is a transitory increase in the gene ex-pression, ranging from 2 h to 2 days, and would involve posttranscriptional regulation, once compared with the mRNA increase, the protein increase is retarded by at least 20 h [20] . Interestingly, the phlorizin treatment did not induce any acute change in both GLUT2 mRNA and protein, differently from what was observed in insulin-treated diabetic rats. Considering that both treatments were able to rapidly and significantly reduce plasma glu-cose, and that urinary glucose concentration was still very high after one day of insulin treatment, as it was ob-vious in phlorizin-treated rats, these results clearly showed that insulin is directly responsible for the transi-tory initial upregulation of GLUT2 expression in re-sponse to insulin treatment. This acute insulin effect of GLUT2 expression upregulation, in association with low-ered glycemic levels, is surprising, since studies in liver

and kidney have proposed that upregulation of GLUT2 mRNA is induced by high glucose concentration in few hours [8, 25] .

One important point is the initial discrepancy be-tween GLUT2 mRNA and protein content, which was already reported for GLUT1 and GLUT4 genes, and in-volves posttranscriptional modulation, such as reduced mRNA stability and/or efficiency of translation [26, 27] . Insulin is known to activate the PI3K-mTOR signalling transduction pathway, resulting in acute (i.e. occurring in minutes to hours) upregulation of protein synthesis, through modulation of multiple steps involved in medi-ating the initiation of mRNA translation [28] . In the pres-ent study, despite the increase of GLUT2 mRNA induced by insulin treatment in a few hours, the protein increas-ing was observed only after 1 day of treatment, and that seems to be a more retarded effect than the expected un-der high insulin stimulus. To clarify the initial discrep-ancy between GLUT2 mRNA and protein contents, fur-ther investigation should be performed, focusing on post-transcriptional regulation of this gene.

The transcriptional regulations observed may involve the action of several enhancer- and suppressor-transcrip-tional factors, which have already been reported as mod-ulators of GLUT2 gene expression [17, 29–33] . Some of them are strong candidates for the observed regulations, such as HNFs [29, 32] and SREBP [17] . The transcription factors HNF-1 � [32] and HNF-3 � /Foxa 3 [29] are activa-tors of GLUT2 transcription, acting in a synergistic way [34] . On the other hand, insulin and lowered glucose con-centration repress glucose-6-phosphatase (G-6-Pase) gene transcription, and that is related to HNF-1 � activity [35] . Thus, we speculate that the GLUT2 gene expression changes observed here could also be modulated by insu-lin and/or lowering plasma glucose-induced effects upon HNF-1 � .

Finally, high insulin was seen to acutely (in 4 h) in-crease GLUT2 gene expression in the liver by increased SREBP-1c activity induced: indirectly, by increasing glu-cose metabolism [17] , or directly, via the PI3-kinase path-way [36] . These mechanisms may be involved in the acute insulin-induced increase of GLUT2 mRNA observed in the present study.

In summary, we demonstrated that 6 days of insulin or phlorizin therapies in diabetic rats induced similar glycemic control improvement, although plasma insulin and urinary glucose effects are opposite in each treat-ment. Acutely, the insulin treatment induced an upregu-lation of the GLUT2 mRNA expression, additionally to the one diabetes had caused, which seems to involve a

Freitas /D’Agord Schaan /da Silva /Okamoto /Oliveira-Souza /Machado

Nephron Physiol 2007;105:p42–p51 p50

posttranscriptional regulation of the gene expression, re-straining a parallel increase of GLUT2 protein. This phe-nomenon seems to be related to the increased insulin concentration, since it was not observed in phlorizin-treated rats, which showed similar plasma and urinary glucose concentrations in this period, but no hyperinsu-linemia. Otherwise, after 6 days of treatment, both insu-lin and phlorizin downregulated the GLUT2 expression, which returned to nondiabetic levels, and definitely

pointed out the plasma/interstitium glucose concentra-tion as the key regulator of this gene.

Acknowledgments

The authors would like to thank Dr. Adauri Brezolin for care-ful English revision of the manuscript. This research was sup-ported by FAPESP [6/07384-4]. H.S.F. has a PhD grant from FAPESP (02/02017-3).

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