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Effect of dapagliflozin alone and in combination with insulin in a rat model of type 1 diabetes

Dapagliﬂozin is a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor; it reduces glucose reabsorption via the kidney and increases the glucose excretion in urine. This inhibitor functions through a unique insulin-independent mechanism, and is therefore a potential new approach for the treatment of hyperglycemia in patients with diabetes. In this study, we evaluated the effectiveness of the SGLT2 inhibitor, dapagliflozin, by using a rat model of type 1 diabetes. Type 1 diabetes was induced by a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ). The STZ-induced rats showed marked hyperglycemia and other metabolic abnormalities. We clarified the hypoglycemic effect of the combination treatment of dapagliflozin with a low dose of insulin compared with dapagliflozin alone and insulin alone in 3-week and 8-week studies. Our results showed that dapagliflozin in combination with a low dose of insulin significantly lowered hyperglycemia, hypercholesterolemia, and hypertriglyceridemia. Furthermore, the antioxidant status and body weight were improved. In contrast, treatment with dapagliflozin alone did not improve the blood glucose levels, lipid profile, antioxidant status, or body weight. These findings suggested that in type 1 diabetes, dapagliflozin was effective in combination with a low dose of insulin; however, the administration of dapagliflozin alone did not achieve a significant effect.


148
In a preliminary study in which 2 U/rat of insulin alone was administered to diabetic rats, 149 one of the diabetic rats was able to normalize blood glucose (132 mg/dl) and seven rats were 150 unable to lower their blood glucose level (569 ± 40.9 mg/dl). As shown in Fig. 1, the animals in 151 the untreated diabetic group in the 8-week study developed severe hyperglycemia. Although the 152 oral administration of dapagliflozin alone did not significantly decrease the blood glucose levels, 153 the levels were significantly decreased in the high-dose insulin and the combination-treated 154 groups. In the 3-week study (data shown in the Fig. S1), the blood glucose levels were similar to 155 the first 3 weeks of the 8-week study. 156 In the 8-week study, the body weight of animals in the untreated diabetic group and the 157 dapagliflozin-treated group was not significantly increased compared with the normal growth 158 observed in the control group (Fig. 2). In contrast, the body weight in groups treated with insulin 159 alone and in combination with dapagliflozin was increased from the second week of treatment, in 160 a comparable manner the control group. In the 3-week study (data shown in the Fig. S2), the 161 changes in body weight were similar to those in the first 3 weeks of the 8-week study. 162 In the 3-week study, the plasma total cholesterol levels were significantly increased in the 163 untreated diabetic group compared with the control group. In the dapagliflozin-treated and the 164 insulin-treated groups, the cholesterol levels decreased, but these changes were not significant. 165 However, the combination treatment significantly decreased the plasma cholesterol. In the 166 8-week study, the total cholesterol levels were increased significantly in the untreated diabetic 167 group. Although the oral administration of dapagliflozin alone did not significantly decrease the 168 plasma cholesterol levels, it was significantly reduced in diabetic rats treated with insulin alone 169 or in combination with dapagliflozin (Table 1).

170
In the 3-week study, the plasma triglyceride levels were significantly increased in the 171 untreated diabetic group and the dapagliflozin-treated group compared with the control group. decreased triglyceride levels. In the 8-week study, the untreated diabetic group exhibited 174 increases in plasma triglyceride levels, but these were not significant. The diabetic group treated 175 with dapagliflozin showed significant increase in triglyceride levels. In the insulin-treated group, 176 the triglyceride levels decreased, but these changes were not significant. In contrast, the 177 triglyceride levels were significantly decreased in diabetic rats that received the combination 178 treatment (Table 1).

179
In the 3-week study, the liver GSH levels significantly decreased in the untreated diabetic 180 group and the dapagliflozin-treated group compared with the control group. The insulin-treated 181 group also exhibited a significant decrease in GSH compared with the control group, but a 182 significant increase compared with the diabetic untreated group. The GSH levels were 183 significantly increased in the combination-treated group. In the 8-week study, GSH levels were 184 significantly decreased in both the untreated diabetic group and the dapagliflozin-treated diabetic 185 group; however, the insulin-and the combination-treated groups showed a significant increase 186 (Table 1).

187
In the 3-week study, the SOD activities in the liver were significantly decreased in the 188 untreated diabetic group and three of treated groups. In the 8-week study, the liver SOD 189 activities were decreased in the untreated diabetic group and the dapagliflozin-treated diabetic 190 group, but were not significantly different to the control group. However, the insulin-treated 191 group and the combination-treated group showed a significant increase compared with the 192 untreated diabetic group and the dapagliflozin-treated diabetic group (Table 1). 193 In the pancreas, the control animals showed several normal islets of Langerhans, which showed that treatment with 1.5-2.5 U of insulin in combination with dapagliflozin significantly 250 decreased the blood glucose level, to a similar level as treatment with 3-5 U of insulin alone.

251
These results were comparable with those in a previous study [23], that showed that, in proper glycemic control in these animals (Fig. 2).  Insulin not only plays an important role in glucose metabolism, but also in lipid metabolism.  Glutathione, a major endogenous antioxidant, ameliorates free radical-mediated damage. 289 Studies have shown that the tissue GSH concentrations in STZ-induced diabetic rats were 290 significantly lower than those in the control rats [10]. The decreased GSH levels in diabetic rats 291 may be due to its increased consumption that is required to relieve oxidative stress [4]. This is 292 consistent with our results that GSH levels were significantly decreased in the diabetic untreated 293 group and the dapagliflozin-treated group. In addition, the depletion of hepatic GSH was 294 corrected with insulin treatment; this finding was consistent with those of previous results [20].
In the combination-treated group, the GSH levels were similar to the insulin-treated group ( [18]. In this study, the SOD activity was low in the diabetic untreated group and the three treated 302 groups. The SOD activity was significantly improved after 8 weeks of treatment with insulin 303 alone and insulin in combination with dapagliflozin (Table 1). Our results suggest that the 304 combination of dapagliflozin with a low dose of insulin significantly improved the antioxidant 305 status in diabetic rats, with an effect that was similar to a full dose of insulin alone. This 306 improvement may be due to the antihyperglycemic effect of the treatment, which reduces the 307 oxidative stress burden.

308
In the pancreas of the untreated diabetic rats and dapagliflozin-treated rats, the islets of 309 Langerhans were noticeably smaller than those of the control rats. However, insulin treatment 310 and the combined insulin and dapagliflozin treatment improved the size of the islets of 311 Langerhans (Fig. 3D or E). Our results were consistent with those of a previous study, in which 312 at 8 weeks after STZ injection, the islet size was significantly decreased compared with the 313 control rats, and insulin treatment improved the islet size in diabetic rats [26]. However, there 314 were swollen cells in the center and small, densely packed cells in the periphery of normal-sized 315 islets in the rats treated with insulin or the combination of insulin and dapagliflozin (Fig. 3E). 316 These findings implied that insulin secretion has not functionally improved in normal-sized islets 317 over the 8-week experiment. However, blood glucose levels were controlled for up to 8 weeks by 318 the same dose of insulin and dapagliflozin (Fig. 1) and hypoglycemia was not seen in any treated rats. Therefore, further studies are needed to confirm the effect of the combination treatment of 320 insulin and dapagliflozin on the islets of Langerhans.

321
The liver of the untreated diabetic rats and dapagliflozin-treated rats showed glycogen 322 depletion in the hepatocytes. These changes were confirmed by the PAS reaction, which is 323 specific to glycogen. The decrease in hepatic glycogen content in diabetes is due to the lack of 324 insulin in the diabetic state, which results in the inactivation of the glycogen synthase systems 325 [36]. However, in the insulin-and combination-treated groups, this glycogen depletion was 326 reversed, and was most likely caused by the re-activation of the glycogen synthase systems in a 327 normal insulin-controlled state (Fig. 4).

328
The kidneys of the untreated diabetic rats and the dapagliflozin-treated rats showed 329 glycogen deposits in the distal convoluted tubules. Our results are consistent with those of a 330 previous study [33] that observed that prolonged hyperglycemia was the only driving force for 331 glycogen accumulation in the renal tubules. As diabetes is prolonged, more glycogen 332 accumulates and spreads into the renal tubules. However, it is not yet clear whether glycogen 333 accumulation in the renal tubules is an inevitable change in the diabetic condition in humans, as 334 an early phase in pathogenesis that will contribute to the end-stage diabetic nephropathy that is 335 always associated with the end-stage nephropathy, or if it has a role in inducing a pathway that 336 leads to the pathophysiological changes observed in diabetic nephropathy [3,32]. These renal 337 changes were reversed in the insulin-and combination-treated groups, as shown by the glycogen 338 deposits in the liver tissues (Fig. 4). 339 Our results suggested that in type 1 DM, the combination treatment of dapagliflozin with 340 a low dose of insulin was able to correct hyperglycemia, which, in turn, improved the lipid 341 profile and antioxidant status. These effects may help to reduce the risk of diabetic complications 342 and hypoglycemia. Furthermore, the effect of this combination treatment appears to be more 343 potent than that of dapagliflozin treatment alone; however, further investigations are required. In conclusion, the combination of dapagliflozin with a low dose of insulin is a potential treatment 345 for patients with type 1 DM. 346 42. Yamamoto, K., Uchida, S., Kitano, K., Fukuhara, N., Okumura-Kitajima, L., Gunji, E.,  Weekly blood glucose measurements to evaluate the hypoglycemic effect of treatment using dapagliflozin (0.1 mg/kg) alone, insulin (3-5U/rat) alone, or a combination of dapagliflozin (0.1 mg/kg) and insulin (1.5-2.5U/rat) in the 8-week study. Values are expressed as mean ± standard deviation.