Hyperglycemia contributes insulin resistance in hepatic and adipose tissue but not skeletal muscle of ZDF rats

Masao Nawano1,2, Akira Oku2, Kiichiro Ueta2, Itsuro Umebayashi2, Tomomi Ishirahara2, Kenji Arakawa2, Akira Saito2, Motonobu Anai1, Masatoshi Kikuchi3, and Tomoichiro Asano1

1 Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113-0033; 2 Discovery Research Laboratory, Tanabe Seiyaku, Saitama 335-8505; and 3 Institute for Adult Disease, Asahi Life Foundation, Tokyo 160-0023, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine the contribution of hyperglycemia to the insulin resistance in various insulin-sensitive tissues of Zucker diabetic fatty (ZDF) rats, T-1095, an oral sodium-dependent glucose transporter (SGLT) inhibitor, was administered by being mixed into food. Long-term treatment with T-1095 lowered both fed and fasting blood glucose levels to near normal ranges. A hyperinsulinemic euglycemic clamp study that was performed after 4 wk of T-1095 treatment demonstrated partial recovery of the reduced glucose infusion rate (GIR) in the T-1095-treated group. In the livers of T-1095-treated ZDF rats, hepatic glucose production rate (HGP) and glucose utilization rate (GUR) showed marked recovery, with almost complete normalization of reduced glucokinase/glucose-6-phosphatase (G-6-Pase) activities ratio. In adipose tissues, decreased GUR was also shown to be significantly improved with a normalization of insulin-induced GLUT-4 translocation. In contrast, in skeletal muscles, the reduced GUR was not significantly improved in response to amelioration of hyperglycemia by T-1095 treatment. These results suggest that the contribution of hyperglycemia to insulin resistance in ZDF rats is very high in the liver and considerably elevated in adipose tissues, although it is very low in skeletal muscle.

glucose toxicity; Zucker diabetic fatty rat; sodium-dependent glucose transporter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN NON-INSULIN-DEPENDENT diabetes mellitus (NIDDM), insulin resistance is an initiating pathogenic mechanism, and when pancreatic beta -cells fail to secrete enough insulin to overcome insulin resistance, hyperglycemia becomes overt (38, 39, 40). However, hyperglycemia is not only a consequence of, but also an important factor in worsening both insulin resistance and insulin deficiency (4, 16, 38, 39, 40). Thus, in NIDDM patients whose glycemic control is poor, not only initiating factors such as obesity, high-fat diet, and insufficient exercise (23, 49), but also glucotoxicity, are regarded as factors contributing to the development of insulin resistance (12, 17). This study was designed to assess the contribution of hyperglycemia to insulin resistance in the liver, adipose tissue, and skeletal muscle in the Zucker diabetic fatty (ZDF) rat model.

ZDF rats are obese diabetic model animals that are polyphagic and hyperinsulinemic starting in the prehyperglycemic stage and then becoming hypoinsulinemic after establishment of hyperglycemia (42-44). This shift in disease phases is very similar to that seen in human NIDDM (2, 36). In this study, to remove the effect of hyperglycemia, a novel oral phlorizin derivative, T-1095, was administered to ZDF rats. An almost complete normalization of hyperglycemia was obtained at a high dosage of T-1095 without apparent changes in other factors, including overeating and obesity. By use of these T-1095-treated model animals, we investigated the extent to which hyperglycemia contributes to insulin resistance in the liver, adipose tissue, and skeletal muscle of ZDF rats. This is the first study to show clearly that the magnitude of the contribution of hyperglycemia to the development of insulin resistance differs among tissue types.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and analytical methods. T-1095 was synthesized at the Discovery Research Laboratory (Tanabe Seiyaku, Saitama, Japan). Plasma insulin levels were assayed by means of an ELISA kit (Seikagaku, Tokyo, Japan) with rat insulin as a standard. An RIA kit (insulin kit "Eiken," Eiken Chemical, Tokyo, Japan) with human insulin serving as the standard was used for the assay in the hyperinsulinemic euglycemic clamp study. Blood glucose levels were determined by means of commercially available kits based on the glucose oxidase method (New Blood Sugar Test, Boehringer Mannheim, Germany). Plasma glucose levels in the euglycemic clamp study and urinary glucose content were measured by a glucose analyzer (Apec, Danvers, MA). The hemoglobin AIc (Hb AIc) level was determined by an affinity column method (Glyc-Affin, Seikagaku). All other chemicals were standard high-purity materials obtained from commercial sources.

Animals. ZDF ( fa/fa) and lean control (fa/+) rats were purchased from Charles River Japan at 6 wk of age, housed in stainless wire cages, and given normal laboratory chow (CE-2, Clea Japan, Tokyo, Japan) and water ad libitum. The experiments were started at 9 wk of age. The animals were divided into experimental groups matched for both body wt and blood glucose level. All experiments that used animals were approved by the Animal Care and Use Committee at Tanabe Seiyaku.

For the single oral administration experiment, T-1095 was suspended in 0.1% hydrogenated caster oil polyethyleneglycol ether solution (Nikkol HCO-60, Nikko Chemical, Japan) and administered orally via stomach tube at a volume of 5 ml/kg. Blood samples were taken from the tail vein before, and 1, 2, 3, 5, 8, and 24 h after, drug administration to determine glucose levels. Urine samples were collected to measure glucose content by means of metabolic cages.

To achieve continuous administration, T-1095 was given as food admixtures. Both lean control and ZDF rats (9 wk old) were kept on a CE-2 diet containing 0.03 or 0.1% (wt/wt) T-1095. Doses were estimated from food intake and body wt. Blood and urine samples were collected as described above.

Hyperinsulinemic euglycemic clamp study. A hyperinsulinemic euglycemic clamp study was performed after 4 wk of continuous T-1095 administration. The animals were fasted for 16 h before this experiment to assure that the drug had been entirely eliminated, and insulin sensitivity was then investigated by use of the hyperinsulinemic euglycemic clamp technique (20, 25). In brief, rats were anesthetized with pentobarbital sodium (60 mg/kg ip), and catheters were inserted into both the right and left femoral veins for insulin and glucose infusion, respectively. A catheter in the left jugular vein was used for blood sampling and injection of [14C]glucose and deoxy-[2-3H]glucose. Regular human insulin (1.8 U · kg-1 · h-1, Humulin R, Eli Lilly, Indianapolis, IN) was infused intravenously, and whole blood glucose concentrations were determined at 5-min intervals. A 10% glucose solution was infused to maintain the blood glucose concentration at 120 mg/dl. When the blood glucose level reached a steady-state level, D-[U-14C] glucose (Amersham Life Science, Buckinghamshire, UK) was administered as an initial intravenous priming dose (4 µCi), and immediately after by continuous infusion at a rate of 0.2 µCi/min. After a stabilization period of >= 30 min, the bladder was emptied and urine sampling was started (i.e., time = 0 min). The steady-state glucose infusion rate (SSGIR) was determined as the mean of values obtained during a 0- to 60-min period. At 15 min, 2-deoxy-[1-3H]glucose (50 µCi, Amersham Life Science) was intravenously administered as a bolus injection. Additional blood samples were collected for the determination of plasma tracer concentrations and insulin levels at 17, 20, 25, 30, 35, 45, and 60 min, and at 0, 20, 40, and 60 min, respectively.

At completion of the glucose clamp study (i.e., 60 min), urine in the bladder was collected to evaluate the rate of urinary glucose loss (UGL) during the 60-min study. The liver, skeletal muscle (quadriceps femoris), white adipose tissue (epididymal fat), and brown adipose tissue (interscapular fat) were rapidly removed and frozen in liquid nitrogen. Twenty to 200 mg of tissues were weighed and dissolved in Soluen-350 (Packard Jpn, Tokyo, Japan) and the 3H activity was measured. The rate of glucose disappearance (Rd) was calculated by dividing the [14C]glucose infusion rate (dpm · min-1 · kg body wt-1) by the steady-state value of glucose-specific activity (dpm/mg). The whole body glucose utilization rate (GUR) and the hepatic glucose production rate (HGP) were calculated as follows: GUR = Rd - UGL, and HGP = Rd - SSGIR. The glucose utilization index (Rg'), an estimate of tissue glucose uptake, was calculated as described by James et al. (20).

Glucokinase activity assay. Hepatic glucokinase (GK) activity was measured by the spectrophotometric continuous assay method (10). Liver homogenates were prepared in ice-cold buffer (pH 7.5) containing 50 mM HEPES, 250 mM sucrose, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 2.5 mM dithioerythritol and centrifuged at 105,000 g for 60 min to sediment the microsomal fraction which was used for measuring glucose-6-phosphatase (G-6-Pase) activity. The enzyme activity of the supernatant (soluble fraction) was assayed in a buffer (pH 7.4) containing 50 mM HEPES, 7.5 mM MgCl2, 100 mM KCl, 5 mM ATP, 2.5 mM dithioerythritol, 10 mg/ml BSA, 0.5 mM NAD+, 4 U/ml glucose-6-phosphate dehydrogenase (L. mesenteroides) and 0.5 mM for hexokinase or 50, 25, 12.5, and 6.25 mM glucose for total phosphorylating activity. The hexokinase reaction was initiated by adding ATP, and the rate of NAD+ reduction was recorded at 340 nm. Total phosphorylating activity was determined as the difference of absorbance change in the presence and absence of ATP. GK activity was calculated as the difference between the total phosphorylating activity and hexokinase activities.

G-6-Pase activity assay. The microsomal fraction was resuspended in the homogenization buffer and diluted with ice-cold buffer (pH 6.5) containing 100 mM HEPES and 0.1 mM EDTA. The G-6-Pase reaction was initiated by adding 10, 5, 2.5, 1.25, and 0.625 mM glucose 6-phosphate at 30°C and stopped after 10 min with a 2.2-fold volume of the stop solution containing 3.7 mM ammonium molybdate and 240 mM SDS in 270 mM H2SO4. The absorption at 750 nm was monitored after color development by adding a 1/5 volume of 1.2 M ascorbic acid. Pi was used as a standard (26).

Preparation of isolated adipocytes from ZDF rats. ZDF and T-1095-treated ZDF rats were killed by decapitation after 5 wk of treatment. Epididymal fat pads were excised and digested with collagenase (1 mg/ml, Sigma, St. Louis, MO), and the isolated adipocytes were suspended in Krebs-Ringer bicarbonate (KRB) buffer supplemented with 30 mM HEPES, 1% BSA fraction V, 3 mM glucose, and 200 mM adenosine at pH 7.4 as described (9, 21). Adipocytes were prepared with a 25% adipocrit.

Subcellular fractionation and Western blotting. Isolated adipocytes were suspended in KRB buffer containing BSA (10 mg/ml, pH 7.4) and preincubated with or without insulin for 30 min at 37°C. After incubation, subcellular membrane fractions of adipocytes were prepared by differential centrifugation (9, 21). The homogenates were centrifuged at 3,000 g for 15 min to sediment the crude membrane fraction. The fat cakes were discarded, and the supernatant was centrifuged at 12,000 g for 15 min to sediment the plasma membrane (PM) fraction. The supernatant was centrifuged at 28,000 g for 15 min, and the resulting supernatant was centrifuged at 146,000 g for 75 min, yielding a particulate fraction termed low-density microsomes (LDM). Proteins (0.05 mg) in PM and LDM fractions were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 3% BSA in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T, vol/vol), and then incubated for 2 h at room temperature with antiserum to the COOH terminus of GLUT-4. After being washed with TBS-T, the bound immunoglobulins were probed with anti-rabbit IgG antibody. The membranes were immediately reacted with an enhanced chemiluminescence (ECL) reagent (Amersham Life Science), and the chemiluminescence level was quantified with a molecular imager GS-525 by use of Screen-HC.

Data analyses. The data are presented as means ± SE. Statistical analyses were performed by closed testing procedures. In brief, the untreated diabetic control group was initially compared with the lean group by unpaired Student's t-test. When the difference between these two groups was significant, a multiple comparison was performed with Dunnett's test to compare each T-1095-treated group with the diabetic control group. P values <0.05 were considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of a single administration of T-1095 on blood glucose and urinary glucose excretion. When administered orally, T-1095 is absorbed from the gut into the bloodstream and metabolized to an active form termed T-1095A. T-1095A suppresses the activity of SGLTs in the kidney, thereby inhibiting reabsorption of glucose (34, 46). Thus a dose-dependent increase in urinary glucose excretion was observed with the administration of T-1095 to ZDF rats (at 3 and 8 h after administration, as shown in Fig. 1A). Concurrently, T-1095 dose dependently lowered blood glucose levels in ZDF rats (Fig. 1B). The antihyperglycemic effect was apparent at a dose of 30 mg/kg, and the maximal dose of 100 mg/kg induced a sustained decrease in blood glucose levels for 24 h. In contrast, there was only a marginal effect on blood glucose levels in lean rats, even at a dosage of 100 mg/kg (Fig. 1C).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of single oral administration of T-1095 on urinary glucose excretion (A) and blood glucose (B, C) in Zucker diabetic fatty (ZDF) (A, B) and lean (C) rats. T-1095 was administered using an intragastic catheter, and changes in blood glucose and urinary glucose excretion were monitored for 24 h. Each value indicates mean ± SE (n = 6). ## P < 0.01 vs. lean control. * P < 0.05, ** P < 0.01 vs. corresponding control.

Effect of continuous treatment with T-1095 on hyperglycemia and physiological parameters. Continuous administration of T-1095 was shown to lower blood glucose levels significantly in ZDF rats at both low (0.03%) and high (0.1%) doses (Fig. 2A). In contrast, no such hypoglycemic effect of T-1095 was observed in lean rats (control lean rats, 86.3 ± 2.9 mg/dl, T-1095-treated lean rats, 86.1 ± 2.7 mg/dl). ZDF rats exhibited apparent hyperphagia and continued to weigh more than lean rats (Fig. 2B). T-1095 treatment did not affect the body wt increase of ZDF rats, despite slightly suppressing hyperphagia at 1 wk after the beginning of T-1095 administration (Fig. 2C).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of continuous oral administration of T-1095 on blood glucose level (A), body wt (B), and food consumption (C) in ZDF and lean rats. T-1095 was administered as dietary admixture. Each value indicates mean ± SE (n = 6). ## P < 0.01 vs. lean rat. * P < 0.05, ** P < 0.01 vs. ZDF control rat.

Marked glycosuria, polyuria, and polydipsia in ZDF rats were shown to progress with aging (Fig. 3). Continuous treatment with T-1095 dose dependently suppressed the age-related increases in urine volume (Fig. 3A), urinary glucose excretion (Fig. 3B), and water intake (Fig. 3C) in ZDF rats.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of continuous oral administration of T-1095 on urine volume (A), urinary glucose excretion (B), and water intake (C) in ZDF and lean rats. T-1095 was administered as dietary admixture. Each value indicates mean ± SE (n = 6). ## P < 0.01 vs. lean rat. * P < 0.05, ** P < 0.01 vs. ZDF control rat.

Table 1 summarizes the effects of T-1095 treatment on various parameters including fasting plasma glucose and insulin levels, Hb A1c, plasma triglyceride, free fatty acid, and total cholesterol levels of ZDF and lean control rats. The fasting blood glucose levels of lean, ZDF, and 0.03 and 0.1% T-1095-treated ZDF rats were 71.2, 196.8, 128.1, and 101.1 mg/dl, respectively. The plasma insulin level of ZDF rats was markedly higher than that of lean control rats at the pretreatment age of 9 wk. The high fasting plasma insulin levels of the ZDF rats were not altered significantly by T-1095 treatment.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Physiological parameters of nonfasting animals treated with or without T-1095 for 4 wk

Hb AIc, plasma triglyceride, free fatty acid, and total cholesterol levels of ZDF rats were apparently higher than those in lean control rats. Among these parameters, only Hb AIc was significantly reduced by T-1095 treatment. These results indicate that T-1095 reduces hyperglycemia without affecting either obesity or hyperlipidemia in ZDF rats (Table 1).

Effect of continuous T-1095 treatment on insulin sensitivity in hyperinsulinemic euglycemic clamp study. In vivo insulin sensitivity was quantitatively determined by the hyperinsulinemic euglycemic clamp study. During the clamp study, there were no significant differences in steady-state plasma glucose (SSPG), steady-state plasma insulin (SSPI), or urinary glucose loss (UGL) among lean, ZDF, and T-1095-treated ZDF rats (Table 2). When compared with lean control rats, there were decreases in steady-state GIR and GUR, and an increase in HGP in ZDF rats (Fig. 4). T-1095 treatment normalized GIR, GUR, and HGP in ZDF rats (Fig. 4). Rg' in skeletal muscle (quadriceps femoris), liver, white adipose tissue (epididymal fat pad), and brown adipose tissue were decreased in ZDF rats. T-1095 at a high dose elevated the Rg' levels significantly in liver and both types of adipose tissue (Fig. 5, A, C, and D). T-1095 produced no significant change in the Rg' of skeletal muscles (Fig. 5B).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Mean values of SSPG, SSPI and UGL during hyperinsulinemic euglycemic clamp test



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of continuous oral administration of T-1095 on glucose infusion (GIR) (A), glucose utilization (GUR) (B), and hepatic glucose production (HGP) rates (C) measured during hyperinsulinemic euglycemic clamp studies in ZDF and lean rats. Each value indicates mean ± SE (n = 6). ## P < 0.01 vs. lean rat. * P < 0.05, ** P < 0.01 vs. ZDF control rat.



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of continuous oral administration of T-1095 on glucose utilization index in liver (A), skeletal muscle (B), white adipose tissue (C), and brown adipose tissue (D) measured during hyperinsulinemic euglycemic clamp studies in ZDF and lean rats. Each value indicates mean ± SE (n = 6). ## P < 0.01 vs. lean rat. * P < 0.05 vs. ZDF control rat.

Effects of continuous T-1095 treatment on hepatic GK and G-6-Pase activities. To study changes in the key steps of hepatic glucose metabolism in ZDF rats, we measured the activities of both GK (Fig. 6A) and G-6-Pase (Fig. 6B). GK activity was decreased significantly to 46.0% of the control value, whereas G-6-Pase activity was increased to 152.3% in ZDF rats (Fig. 6). T-1095 treatment restored both GK and G-6-Pase activities in ZDF rats to those in lean control rats. There were no differences in Michaelis constant (Km) values for GK and G-6-Pase activities among groups.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of continuous oral administration of T-1095 on hepatic glucokinase (GK) (A), glucose-6-phosphatase (G-6-Pase) (B), and GK/G-6-Pase (C) activities in nonfasting ZDF and lean rats. Each value indicates mean ± SE (n = 6). # P < 0.05, ## P < 0.01 vs. lean rat. * P < 0.05, ** P < 0.01 vs. ZDF control rat.

Effect of continuous T-1095 treatment on insulin-induced GLUT-4 translocation in isolated adipocytes. Insulin stimulation of isolated adipocytes induced dramatic recruitment of GLUT-4 protein from LDM to PM. The GLUT-4 protein increase in the PM with insulin stimulation was markedly blunted in ZDF rats, and the T-1095 treatment apparently normalized this impairment (Fig. 7, A and B). Similarly, impaired insulin-induced reduction of GLUT-4 in LDM of ZDF rats was partially normalized with T-1095 treatment (Fig. 7, C and D). These results indicate that T-1095 treatment ameliorates impaired GLUT-4 translocation, which is in good agreement with increased Rg' levels in the hyperinsulinemic euglycemic clamp study.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of continuous oral administration of T-1095 on GLUT-4 protein contents in plasma membrane (A, B) and low-density microsomes (C, D) obtained from isolated adipocytes of ZDF and lean rats. Isolated adipocytes were stimulated with 10-6 M insulin for 15 min at 37°C. Each value indicates mean ± SE (n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

T-1095 is a derivative of phlorizin but, unlike phlorizin, it is absorbed efficiently from the gut into the bloodstream when administered orally. After entering the circulation, T-1095 is metabolized to an active form, termed T-1095A (33, 45), that suppresses the activity of renal SGLTs involved in glucose reabsorption in proximal tubules (22, 33, 34, 45). Thus orally administered, T-1095 exerts essentially the same effect as intravenously injected phlorizin, which cannnot be absorbed from the gut (8). However, taken as a food admixture, T-1095 efficiently suppresses postprandial blood glucose elevation, which results in a more efficient antihyperglycemic effect than phlorizin when given as a continuous venous infusion (45).

The ZDF rat is regarded as an insulin-resistant diabetic model exhibiting obesity and overeating because of an abnormality of the leptin system (18, 32, 35). In our experiments, the nonfasting blood glucose levels of ZDF rats averaged 250 ± 10 mg/dl at 9 wk of age, and a defect in insulin secretion had already been indicated in an oral glucose tolerance test at 11 wk (data not shown). Although the molecular mechanisms underlying insulin resistance remain unclear, the major cause of insulin resistance in ZDF rats has been considered to be obesity related (3, 18, 32, 35, 41-44, 48). Although the blood glucose level was normalized, the fasting insulin level was not reduced by T-1095 treatment. The ZDF strain of rat is a substrain of the Zucker fatty strain that is obese, hyperinsulinemic, and normoglycemic. Thus we speculate that these Zucker-strain rats are genetically hyperinsulinemic irrespective of blood glucose levels, and that correction of hyperglycemia does not, therefore, reduce hyperinsulinemia in ZDF rats.

In the present study, despite the degree of obesity being unchanged, the normalization of hyperglycemia with T-1095 treatment ameliorated insulin resistance significantly, as shown by a reduction in GIR in the hyperinsulinemic euglycemic clamp study. In fact, T-1095 treatment slightly improved total GUR. On the other hand, the elevated triglyceride (TG), free fatty acid (FFA), and total cholesterol (TCHO) values in ZDF rats were not altered by T-1095 treatment. We speculate that these levels are not related to abnormal leptin metabolism, because there is a functional leptin receptor defect in ZDF rats. In fact, plasma leptin levels in T-1095-treated ZDF rats were similar to those in untreated ZDF rats (data not shown). Thus it is very likely that the improved insulin sensitivity achieved with T-1095 treatment is attributable to removal of the effect of hyperglycemia (so-called glucotoxicity).

Of great interest in this study is the observation that the degree of insulin sensitivity improvement differs among tissues after blood glucose normalization. Insulin resistance in the liver was markedly suppressed by normalizing blood glucose levels. In contrast, although insulin resistance in adipose tissues was also improved significantly, this normalization was partial. Finally, insulin resistance in muscle was apparently not improved. These results show clearly that the contribution of hyperglycemia to insulin resistance differs in these three insulin-sensitive tissues.

First, T-1095 treatment profoundly reversed both reduced glucose uptake and elevated glucose production in the liver, as demonstrated by the hyperinsulinemic euglycemic clamp study. Although abrupt correction of hyperglycemia might have counterregulated HGP in ZDF rats during the glucose clamp study, elevations in fasting glucose level and the ratio of GK to G-6-Pase activities agree well with the result of the glucose clamp study. GK and G-6-Pase play important roles in blood glucose regulation. GK and G-6-Pase, both of which are known to be regulated by insulin, convert glucose to glucose 6-phosphate, and glucose 6-phosphate to glucose through glucose production, respectively (19, 30, 46, 47). In our experiment, the reduced GK/G-6-Pase activity ratio in ZDF rats was corrected by continuous T-1095 treatment. Hepatic GK activity and GK expression levels are reportedly increased in streptozotocin-treated (STZ) rats, and phlorizin treatment ameliorates the impaired GK activity (6, 27); therefore, it is likely that hyperglycemia altered GK and G-6-Pase activities, and that T-1095 treatment corrected the enzyme activities through amelioration of hyperglycemia.

Second, in adipose tissues, the normalization of insulin resistance with T-1095 treatment was demonstrated by the increased Rg' values in the glucose clamp study. It is well known that the rate-limiting step for glucose uptake into adipocytes is governed by the amount of GLUT-4 on the cell surface (11, 24, 28, 37). In fact, the translocation of GLUT-4 protein from LDM to PM was markedly impaired in adipose tissues of ZDF rats, and this impairment was shown to be ameliorated by T-1095 treatment. However, the degree of normalization was partial, such that factors worsening insulin sensitivity in ZDF fat tissues apparently persist. We speculate that the enlargement of adipocytes is likely to be one such factor, because it has been reported that larger adipocytes possess lower insulin sensitivity in glucose transport activity (29).

Finally, the reduced skeletal muscle Rg' of ZDF rats was not restored significantly by T-1095 treatment. A previous study demonstrated that impaired insulin sensitivity in STZ diabetic rats was improved by long-term correction of hyperglycemia with phlorizin (5). Thus, although in most nongenetic diabetic models (e.g., STZ rats) hyperglycemia is likely to be a major cause of the development of insulin resistance in peripheral tissues (5-7, 27, 30, 31, 38), glucotoxicity is not involved in the mechanism underlying insulin resistance in ZDF rat muscles (1, 51). Moreover, it was also reported that insulin resistance was not improved in muscle of ZDF rats treated with pioglitazone, an insulin sensitizer of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma ) agonist (13). On the contrary, it has been reported that the PPAR gamma -agonist improves muscle insulin resistance (51) and alpha -glucosidase inhibitor recovers muscle GLUT-4 contents in ZDF rats (15). In the present study, T-1095 reduced hyperglycemia without affecting plasma TG, FFA, and total cholesterol. These results suggest that insulin resistance in skeletal muscle is already developed by other mechanisms including lipotoxicity when hyperglycemia is established, and that hyperglycemia does not further increase insulin resistance, in ZDF rats. Hawkins et al. (16) have suggested that the increased availability of glucose and FFA affects insulin-induced glucose uptake via a common mechanism. In the present study, FFA levels were elevated in ZDF rats, whereas muscle TG contents were not determined. Therefore, although we speculate that the impaired insulin sensitivity in ZDF rat muscle may be related to an abnormality in lipid metabolism as well as the leptin system, further study is necessary to clarify this issue.

In summary, insulin resistance in hepatic and adipose tissues is induced by glucotoxicity resulting from long-term hyperglycemia in ZDF rats. In contrast, insulin resistance in skeletal muscles is not due to glucotoxicity. Taken together, these raise the possibility of the tissue dependence of glucotoxicity not only in other animal models but also in individual diabetic patients (14). However, although hyperglycemia is a consequence of insufficient insulin secretion and insulin resistance, these results suggest that normalization of hyperglycemia leads to an apparent improvement in insulin resistance. Thus hyperglycemia should be considered not only a consequence of but also a major factor in worsening insulin sensitivity. We now consider T-1095 to be a potentially novel therapeutic strategy for diabetes management, because it evidently reduces hyperglycemia-induced insulin resistance in liver and adipose tissues. Further study is required to elucidate the molecular mechanisms underlying insulin resistance in the skeletal muscle of ZDF rats.


    ACKNOWLEDGEMENTS

We thank Drs. Mamoru Matsumoto, Kenji Tsujihara, Takeshi Matsumoto (Tanabe Seiyaku, Japan) for reviewing the manuscript, Mitsuya Hongu for supplying T-1095, and Yasuo Kuronuma (Tanabe Seiyaku, Japan) for technical support.


    FOOTNOTES

This work was supported by a grant from Tanabe Medical Frontier Conference (TMFC, Tomoichiro Asano, Japan).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Saito, Discovery Research Laboratory, Tanabe Seiyaku, 2-2-50 Kawagishi, Toda-shi, Saitama 335-8505, Japan (E-mail: a-saito{at}tanabe.co.jp).

Received 24 June 1999; accepted in final form 15 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahmad, F., and B. J. Goldstein. Increased abundance of specific skeletal muscle protein-tyrosine phosphatases in a genetic model of insulin-resistant obesity and diabetes mellitus. Metabolism 44: 1175-1184, 1995[ISI][Medline].

2.   Ammon, H. P. Hyper- and hypoinsulinemia in type-2 diabetes: what may be wrong in the secretory mechanism of the B-cell. Exp. Clin. Endocrinol. Diabetes 105 (Suppl. 2): 43-47, 1991.

3.   Anai, M., M. Funaki, T. Ogihara, J. Terasaki, K. Inukai, H. Katagiri, Y. Fukushima, Y. Yazaki, M. Kikuchi, Y. Oka, and T. Asano. Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes 47: 13-23, 1998[Abstract].

4.   Barzilai, N., M. Hawkins, I. Angelov, M. Hu, and L. Rossetti. Glucosamine-induced inhibition of liver glucokinase impairs the ability of hyperglycemia to suppress endogenous glucose production. Diabetes 45: 1329-1335, 1996[Abstract].

5.   Blondel, O., D. Bailbe, and B. Portha. Insulin resistance in rats with non-insulin-dependent diabetes induced by neonatal (5 days) streptozotocin: evidence for reversal following phlorizin treatment. Metabolism 39: 787-793, 1990[ISI][Medline].

6.   Brichard, S. M., J. C. Henquin, and J. Girard. Phlorizin treatment of diabetic rats partially reverses the abnormal expression of genes involved in hepatic glucose metabolism. Diabetologia 36: 292-298, 1993[ISI][Medline].

7.   Burcelin, R., R. L. Printz, J. Kande, R. Assan, D. K. Granner, and J. Girard. Regulation of glucose transporter and hexokinase II expression in tissues of diabetic rats. Am. J. Physiol. Endocrinol. Metab. 265: E392-E401, 1993[Abstract/Free Full Text].

8.   Colombo, V., H. Lorenz-Meyer, and G. Semenza. Small intestinal phlorizin hydrolase: the "beta-glycosidase complex". Biochim. Biophys. Acta 327: 412-424, 1973[ISI][Medline].

9.   Cushman, S. W., L. J. Wardzala, I. A. Simpson, E. Karnieli, P. J. Hissin, T. J. Wheeler, P. C. Hinkle, and L. B. Salans. Insulin-induced translocation of intracellular glucose transporters in the isolated rat adipose cell. Federation Proc. 43: 2251-2255, 1984[ISI][Medline].

10.   Davidson, A. L., and W. J. Arion. Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity. Arch. Biochem. Biophys. 253: 156-67, 1987[ISI][Medline].

11.   Deems, R. O., J. L. Evans, R. W. Deacon, C. M. Honer, D. T. Chu, K. Burki, W. S. Fillers, D. K. Cohen, and D. A. Young. Expression of human GLUT-4 in mice results in increased insulin action. Diabetologia 37: 1097-1104, 1994[ISI][Medline].

12.   Del Prato, S., F. Leonetti, D. C. Simonson, P. Sheehan, M. Matsuda, and R. A. DeFronzo. Effect of sustained physiologic hyperinsulinaemia and hyperglycaemia on insulin secretion and insulin sensitivity in man. Diabetologia 37: 1025-35, 1994[ISI][Medline].

13.   Doebber, T. W., M. S. Wu, J. Ventre, and N. J. Rathway. Determination of the tissues responsible for insulin resistance in Zucker diabetic fatty rats as measured by the glucose clamp technique. Effect of pioglitazone. Diabetes 42, Suppl. 1: 66A, 1993..

14.   Edelman, S. V. Type II diabetes mellitus. Adv. Intern. Med. 43: 449-500, 1998[Medline].

15.   Friedman, J. E., J. E. de Venté, R. G. Peterson, and G. L. Dohm. Altered expression of muscle glucose transporter GLUT-4 in diabetic fatty Zucker rats (ZDF/Drt-fa). Am. J. Physiol. Endocrinol. Metab. 261: E782-E788, 1991[Abstract/Free Full Text].

16.   Hawkins, M., N. Barzilai, R. Liu, M. Hu, W. Chen, and L. Rossetti. Role of the glucosamine pathway in fat-induced insulin resistance. J. Clin. Invest. 99: 2173-2282, 1997[Abstract/Free Full Text].

17.   Henry, R. R. Glucose control and insulin resistance in non-insulin-dependent diabetes mellitus. Ann. Intern. Med. 124: 97-103, 1996[Abstract/Free Full Text].

18.   Hirose, H., Y. H. Lee, L. R. Inman, Y. Nagasawa, J. H. Johnson, and R. H. Unger. Defective fatty acid-mediated beta-cell compensation in Zucker diabetic fatty rats. Pathogenic implications for obesity-dependent diabetes. J. Biol. Chem. 271: 5633-5637, 1996[Abstract/Free Full Text].

19.   Holroyde, M. J., M. B. Allen, A. C. Storer, A. S. Warsy, J. M. Chesher, I. P. Trayer, A. Cornish-Bowden, and D. G. Walker. The purification in high yield and characterization of rat hepatic glucokinase. Biochem. J. 153: 363-373, 1976[ISI][Medline].

20.   James, D. E., K. M. Burleigh, and E. W. Kraegen. In vivo glucose metabolism in individual tissues of the rat. Interaction between epinephrine and insulin. J. Biol. Chem. 261: 6366-6374, 1986[Abstract/Free Full Text].

21.   James, D. E., and P. F. Pilch. Fractionation of endocytic vesicles and glucose-transporter-containing vesicles in rat adipocytes. Biochem. J. 256: 725-732, 1988[ISI][Medline].

22.   Kanai, Y., W. S. Lee, G. You, D. Brown, and M. A. Hediger. The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose. J. Clin. Invest. 93: 397-404, 1994[ISI][Medline].

23.   Knowler, W. C., K. M. Narayan, R. L. Hanson, R. G. Nelson, P. H. Bennett, J. Tuomilehto, B. Schersten, and D. J. Pettitt. Preventing non-insulin-dependent diabetes. Diabetes 44: 483-488, 1995[Abstract].

24.   Kozka, I. J., A. E. Clark, J. P. Reckless, S. W. Cushman, G. W. Gould, and G. D. Holman. The effects of insulin on the level and activity of the GLUT-4 present in human adipose cells. Diabetologia 38: 661-666, 1995[ISI][Medline].

25.   Kraegen, E. W., D. E. James, S. P. Bennett, and D. J. Chisholm. In vivo insulin sensitivity in the rat determined by euglycemic clamp. Am. J. Physiol. Endocrinol. Metab. 245: E1-E7, 1983[Abstract/Free Full Text].

26.   Lange, A. J., W. J. Arion, A. Burchell, and B. Burchell. Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride. J. Biol. Chem. 261: 101-7, 1986[Abstract/Free Full Text].

27.   Lavoie, L., D. Dimitrakoudis, A. Marette, B. Annabi, A. Klip, M. Vranic, and G. van de Werve. Opposite effects of hyperglycemia and insulin deficiency on liver glycogen synthase phosphatase activity in the diabetic rat. Diabetes 42: 363-366, 1993[Abstract].

28.   Lefebvre, A. M., M. Laville, N. Vega, J. P. Riou, L. van Gaal, J. Auwerx, and H. Vidal. Depot-specific differences in adipose tissue gene expression in lean and obese subjects. Diabetes 47: 98-103, 1998[Abstract].

29.   Macho, L., M. Fickova, E. Sebokova, A. Mitkova, and I. Klimes. Effect of dietary fish oil on 2-deoxy-D-3H glucose uptake in isolated adipocytes of rats fed various diets. Ann. NY Acad. Sci. 683: 237-243, 1993[Abstract].

30.   Massillon, D., N. Barzilai, W. Chen, M. Hu, and L. Rossetti. Glucose regulates in vivo glucose-6-phosphatase gene expression in the liver of diabetic rats. J. Biol. Chem. 271: 9871-9874, 1996[Abstract/Free Full Text].

31.   Napoli, R., M. F. Hirshman, and E. S. Horton. Mechanisms and time course of impaired skeletal muscle glucose transport activity in streptozocin diabetic rats. J. Clin. Invest. 96: 427-37, 1995[ISI][Medline].

32.   Ogawa, Y., H. Masuzaki, N. Isse, T. Okazaki, K. Mori, M. Shigemoto, N. Satoh, N. Tamura, K. Hosoda, and Y. Yoshimasa. Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats. J. Clin. Invest. 96: 1647-1652, 1995[ISI][Medline].

33.   Oku, A., K. Ueta, K. Arakawa, T. Ishihara, M. Nawano, Y. Kuronuma, M. Matsumoto, A. Saito, K. Tsujihara, M. Anai, T. Asano, Y. Kanai, and H. Endou. T-1095, an inhibitor of renal Na+-glucose cotransporters, may provide a novel approach to treating diabetes. Diabetes 48: 1794-1800, 1999[Abstract].

34.   Pajor, A. M., and E. M. Wright. Cloning and functional expression of a mammalian Na+/nucleoside cotransporter. A member of the SGLT family. J. Biol. Chem. 267: 3557-3560, 1992[Abstract/Free Full Text].

35.   Phillips, M. S., Q. Liu, H. A. Hammond, V. Dugan, P. J. Hey, C. J. Caskey, and J. F. Hess. Leptin receptor missense mutation in the fatty Zucker rat. Nat. Genet. 13: 18-19, 1996[ISI][Medline].

36.   Porte, D., Jr. Banting lecture 1990. Beta-cells in type II diabetes mellitus. Diabetes 40: 166-180, 1991[Abstract].

37.   Rea, S., and D. E. James. Moving GLUT-4: the biogenesis and trafficking of GLUT-4 storage vesicles. Diabetes 46: 1667-1677, 1997[Abstract].

38.   Rossetti, L., D. Smith, G. I. Shulman, D. Papachristou, and R. A. DeFronzo. Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J. Clin. Invest. 79: 1510-1515, 1987[ISI][Medline].

39.   Rossetti, L., G. I. Shulman, W. Zawalich, and R. A. DeFronzo. Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats. J. Clin. Invest. 80: 1037-1044, 1987[ISI][Medline].

40.   Rossetti, L., A. Giaccari, and R. A. DeFronzo. Glucose toxicity. Diabetes Care 13: 610-630, 1990[Abstract].

41.   Seufert, J., G. C. Weir, and J. F. Habener. Differential expression of the insulin gene transcriptional repressor CCAAT/enhancer-binding protein beta and transactivator islet duodenum homeobox-1 in rat pancreatic beta cells during the development of diabetes mellitus. J. Clin. Invest. 101: 2528-2539, 1998[Abstract/Free Full Text].

42.   Shimabukuro, M., Y. T. Zhou, M. Levi, and R. H. Unger. Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc. Natl. Acad. Sci. USA 95: 2498-2502, 1998[Abstract/Free Full Text].

43.   Sturis, J., W. L. Pugh, J. Tang, and K. S. Polonsky. Prevention of diabetes does not completely prevent insulin secretory defects in the ZDF rat. Am. J. Physiol. Endocrinol. Metab. 269: E786-E792, 1995[Abstract/Free Full Text].

44.   Tokuyama, Y., J. Sturis, A. M. DePaoli, J. Takeda, M. Stoffel, J. Tang, X. Sun, K. S. Polonsky, and G. I. Bell. Evolution of beta-cell dysfunction in the male Zucker diabetic fatty rat. Diabetes 44: 1447-1457, 1995[Abstract].

45.   Tsujihara, K., M. Hongu, K. Saito, M. Inamasu, K. Arakawa, A. Oku, and M. Matsumoto. Na(+)-glucose cotransporter inhibitors as antidiabetics. I. Synthesis and pharmacological properties of 4'-dehydroxyphlorizin derivatives based on a new concept. Chem. Pharm. Bull. (Tokyo) 44: 1174-1180, 1996[ISI][Medline].

46.   Wakelam, M. J., and D. G. Walker. The separate roles of glucose and insulin in the induction of glucokinase in hepatocytes isolated from neonatal rats. Biochem. J. 196: 383-390, 1981[ISI][Medline].

47.   Walker, D. G., and S. Rao. The role of glucokinase in the phosphorylation of glucose by rat liver. Biochem. J. 90: 360-368, 1964[ISI][Medline].

48.   Wang, M. Y., K. Koyama, M. Shimabukuro, D. Mangelsdorf, C. B. Newgard, and R. H. Unger. Overexpression of leptin receptors in pancreatic islets of Zucker diabetic fatty rats restores GLUT-2, glucokinase, and glucose-stimulated insulin secretion. Proc. Natl. Acad. Sci. USA 95: 11921-11926, 1998[Abstract/Free Full Text].

49.   Zachary, T., and M. D. Bloomgarden. American Diabetes Association Annual Meeting, 1998: insulin resistance, exercise, and obesity. Diabetes Care 22: 517-522, 1999[Free Full Text].

50.   Zierath, J. R., E. U. Frevert, J. W. Ryder, P. O. Berggren, and B. B. Kahn. Evidence against a direct effect of leptin on glucose transport in skeletal muscle and adipocytes. Diabetes 47: 1-4, 1998[Abstract].

51.   Zierath, J. R., J. W. Ryder, T. Doebber, J. Woods, M. Wu, J. Ventre, Z. Li, C. McCrary, J. Berger, B. Zhang, and D. E. Moller. Role of skeletal muscle in thiazolidinedione insulin sensitizer (PPAR gamma agonist) action. Endocrinology 139: 5034-5041, 1998[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 278(3):E535-E543
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society