**3. Results**

All animals in the intact control group showed stable plasma biochemical parameters which did not exceed the physiological norms during an observation period (glucose - 4,02 ± 0,1 mmol/L (Fig. 1), insulin - 1,65 ± 0,04 mU/ml., C-peptide - 4,65 ± 0,16 ng/ml.

Morphological study of the intact control group showed that the exocrine part of the pancreas has an alveolar-tubular structure which exhibited a division into lobules separated by interlobular connective tissue. Exocrinotcytes tended to be highly differentiated and they formed acini. Pancreatic islets in all parts of the pancreas were round or slightly oval in shape. They were single or were arranged in clusters around the intralobular excretory ducts. The total volume fraction of islet tissue in the gastric and splenic segments was almost twice as much as the volume fraction of islets of the intestinal segment (Table 3). The total area of nuclear β-endocrine cells in all segments of the pancreas had no statistically significant differences during all periods of observation. Insulin-positive cells were predominantly found in the central part of the pancreatic islets; however, glucagon-positive cells were more frequently encountered at the periphery. In this case there was a statistically significant increase (p <0.05) in β-cell area of the splenic segment compared with the intestinal and gastric segments of the pancreas (Fig. 2A). The maximum α-endocrine cell area was determined in the intestinal and gastric segments of the pancreas; however, this area was statistically significant lower (p <0.05) in the splenic segment (Table 3). There was a statistically significant (p ≤ 0.05) direct relationship between the total volume fraction of pancreatic islets and the area of βendocrine cells depending on their location. The amount of islets and β-endocrine cells tended to increase in the following segments of the pancreas: intestinal →gastric → splenic.

228 Apoptosis and Medicine

antibodies.

**3. Results** 

(Allred DC, et al. 1998).

Immunohistochemistry was performed according to manufacturers' protocols using ABC (Novocastra, UK), «UltraVision» (Lab Vision, UK) and «EnVision» (Dako Cytomation, Denmark) antibody detection systems and a chromogen, diaminobenzidine under the protocol on high temperature antigen unmasking technique using «*Pascal*» mini autoclave (Dako Cytomation, Denmark) (Kumar G.L. et al. 2009). The reliability of the obtained results was defined using both positive and negative control antigens, as well as negative control

Immunohistochemical reaction was based on visual evaluation, taking into account the intensity of color, or on determination of the specific amount of positively stained cells

Photo images were captured with an «AxioScope» microscope (Carl Zeiss, Germany) and a «PowerShot» digital camera (Canon, Japan). Morphometric analysis was performed using "VideoTestMorfo-4" software (Russia). In the course of the study we determined the relationship between the total α-and β- endocrine cell area and the total area of the islet (S,%), between the volume fraction (ML, %) of islets and exocrine glands, as well as the area of β-cell nuclei (S, µm2). We also measured apoptotic index (AI), i.e. the relative amount of

The research results were processed using basic statistical analysis techniques as well as "Video TestMorfo-4», Excel Microsoft Office (Microsoft, USA) and STATISTICA 6.0 software (StatSoft Inc., USA). The analysis of the parameters for normally-distributed values was performed using Student's t test. Nonparametric statistics was calculated using Mann-Whitney test. To compare qualitative variables, the chi-square test and the Fisher exact test

All animals in the intact control group showed stable plasma biochemical parameters which did not exceed the physiological norms during an observation period (glucose - 4,02 ± 0,1

Morphological study of the intact control group showed that the exocrine part of the pancreas has an alveolar-tubular structure which exhibited a division into lobules separated by interlobular connective tissue. Exocrinotcytes tended to be highly differentiated and they formed acini. Pancreatic islets in all parts of the pancreas were round or slightly oval in shape. They were single or were arranged in clusters around the intralobular excretory ducts. The total volume fraction of islet tissue in the gastric and splenic segments was almost twice as much as the volume fraction of islets of the intestinal segment (Table 3). The total area of nuclear β-endocrine cells in all segments of the pancreas had no statistically significant differences during all periods of observation. Insulin-positive cells were predominantly found in the central part of the pancreatic islets; however, glucagon-positive cells were more frequently encountered at the periphery. In this case there was a statistically significant increase (p <0.05) in β-cell area of the splenic segment compared with the intestinal and gastric

β-endocrine cells with apoptotic structural and immunohistochemical changes.

were used. Differences were considered significant if error probability was p <0.05.

mmol/L (Fig. 1), insulin - 1,65 ± 0,04 mU/ml., C-peptide - 4,65 ± 0,16 ng/ml.

**Figure 1.** Blood glucose dynamics (mmol/L) in rats with different models of experimental diabetes. \* - Significant changes compared with the intact control group.



Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 231

and destructive changes were revealed in the central part of the islets of Langerhans. Immunohistochemical reaction in the central part of the pancreatic islets revealed insulinpositive cells with marked expression of insulin. A slight increase in the size of β-cell nuclei was reported. Compared with the intact control group, the total area of islet cells in all segments of the pancreas was slightly reduced. The total glucagon-positive cell area did not

At day 7 the islets were swollen, hyperemic and collapsed. There was no statistically significant reduction in the total volume fraction of islets in all segments of the pancreas. In the central part of the islets we observed moderate necrobiotic changes associated with a significant decrease (p <0.05) in the total β-cell area predominantly in the gastric and splenic segments when compared with the intact control group (Table 5). There was a statistically significant increase (p <0.05) in the area of α-endocrine cells in the gastric and splenic segments of the pancreas. β-cell necrosis was accompanied by moderate leukocyte infiltration of the stroma and acinar cells with only few leukocytes in the islets. The area of nuclear β-islet cells in all segments of the pancreas increased by 4.5 µm2; however, no statistically significant changes

Edema significantly reduced by day 14; however, blood vessels of the acinar tissue and capillaries of the islets of Langerhans were still slightly hyperemic. There were focal sclerotic changes in some islets. Compared with the control group, there were not any statistically significant changes in the islet volume fraction in all segments of the pancreas. Insulinpositive cells tended to be located in a random pattern; however, glucagon-positive cells were predominantly found at the periphery. The total β-endocrine cell area in the gastric and splenic segments, as compared with the intact control group, was significantly decreased (p <0.05), while the total α-cell area was increased (p <0.05) (Table 5) (Fig.2B).

At 28 day edema, hyperemia of blood vessels and inflammatory infiltration were replaced by focal sclerotic changes of the acinar tissue and pancreatic islets. As before, a slight decrease in the islet volume fraction in all segments of the pancreas was reported. Also, in all segments of the pancreas the total area of β-cells was slightly increased, compared with day 14 of the experiment, but was significantly lower (p <0.05) as compared with the animals in the intact control group. The volume fraction of α-endocrine cells slightly decreased but was statistically greater (p <0.05) than in the intact control group (Table 5). The average area of nuclei was slightly decreased as compared with the intact control group

**Intestinal Gastric Splenic** 

3 intact 6,8±2,5 12,6±6,0 13,8±7,3 diabetes 6,7±2,1 11,8±5,4 13,9±10,2 7 intact 6,7±2,2 12,0±4,5 12,9±2,1 diabetes 4,5±3,1 11,2±3,2 11,1±5,2

There was moderate hypertrophy of β-cell nuclei in all segments of the pancreas.

**Indicator Day of experiment Segment of the pancreas**

change as compared with the control group (Table 5).

were identified when compared with the intact control group.

and with day 14 of the experiment.

Volume fraction of islets,%

\* - Significant difference in the intestinal segment of the pancreas (p <0.05),

\*\* - Significant difference in the gastric segment of the pancreas (p <0.05).

**Table 3.** Morphometric indicators of pancreatic islets in the intact control group (M ± m).

Bax protein expression was negative in all islet cells. A weak positive cytoplasmic staining of islet cells in the central part of the islet for Bcl-2 protein was reported. In the nuclei of individual β-endocrine cells ambiguous or weak expression of p53 protein was determined. Most of β-islet cells had a positive nuclear staining for MDM2 protein. In some β-endocrine cells cytoplasmic expression of caspase 3 and TRAIL was poorly defined or ambiguous (Table 4) (Fig.3A, 4A). Apoptotic index was low (Table 5).


**Table 4.** Immunohistochemical characteristics of β-cells

#### **Alloxan-induced diabetes**

In alloxan-induced diabetes simulation model a significant increase in blood glucose of rats compared with the intact control was reported (Fig. 1).

At day 3 of the experiment a well pronounced interstitial edema associated with hyperemia of blood vessels and capillaries in the acinar tissue of pancreatic islets was determined histologically. Compared with the intact control group, the total volume fraction of the pancreatic islets was not significantly reduced. However, there was a slight decrease in the area of β-cells in all segments of the pancreas (Table 5). The cells with moderate dystrophic and destructive changes were revealed in the central part of the islets of Langerhans. Immunohistochemical reaction in the central part of the pancreatic islets revealed insulinpositive cells with marked expression of insulin. A slight increase in the size of β-cell nuclei was reported. Compared with the intact control group, the total area of islet cells in all segments of the pancreas was slightly reduced. The total glucagon-positive cell area did not change as compared with the control group (Table 5).

230 Apoptosis and Medicine

Total area of βcell nuclei, µm2

**Group Day of** 

**Alloxan-induced diabetes** 

Intact control

Intact control **experiment** 

**Table 4.** Immunohistochemical characteristics of β-cells

compared with the intact control was reported (Fig. 1).

**Indicator Day of experiment Segment of the pancreas** 

**Table 3.** Morphometric indicators of pancreatic islets in the intact control group (M ± m).

Bax protein expression was negative in all islet cells. A weak positive cytoplasmic staining of islet cells in the central part of the islet for Bcl-2 protein was reported. In the nuclei of individual β-endocrine cells ambiguous or weak expression of p53 protein was determined. Most of β-islet cells had a positive nuclear staining for MDM2 protein. In some β-endocrine cells cytoplasmic expression of caspase 3 and TRAIL was poorly defined or ambiguous

**Intensity of expression**

**Amount of cells**

In alloxan-induced diabetes simulation model a significant increase in blood glucose of rats

At day 3 of the experiment a well pronounced interstitial edema associated with hyperemia of blood vessels and capillaries in the acinar tissue of pancreatic islets was determined histologically. Compared with the intact control group, the total volume fraction of the pancreatic islets was not significantly reduced. However, there was a slight decrease in the area of β-cells in all segments of the pancreas (Table 5). The cells with moderate dystrophic

\* - Significant difference in the intestinal segment of the pancreas (p <0.05), \*\* - Significant difference in the gastric segment of the pancreas (p <0.05).

(Table 4) (Fig.3A, 4A). Apoptotic index was low (Table 5).

**p53 Caspase-3** 

**Intestinal Gastric Splenic** 

3 24,2±1,2 24,4±1,0 23,9±0,2 7 24,3±1,3 23,8±1,2 24,7±1,0 14 24,3±1,2 24,8±1,3 24,6±1,3 28 24,4±1,0 24,3±1,2 24,7±2,0

**Primary antibodies** 

**2** 

**MDM2 NOS-3** 

**NFkB** 

**TRAIL Bax Bcl-**

3 - + + - + ++ - ++ 7 - + + - + ++ - ++ 14 - + + - + ++ - ++ 28 - + + - + ++ - ++

3 - + + - + +++ - ++ 7 - + + - + +++ - ++ 14 - + + - + +++ - ++ 28 - + + - + +++ - ++ At day 7 the islets were swollen, hyperemic and collapsed. There was no statistically significant reduction in the total volume fraction of islets in all segments of the pancreas. In the central part of the islets we observed moderate necrobiotic changes associated with a significant decrease (p <0.05) in the total β-cell area predominantly in the gastric and splenic segments when compared with the intact control group (Table 5). There was a statistically significant increase (p <0.05) in the area of α-endocrine cells in the gastric and splenic segments of the pancreas. β-cell necrosis was accompanied by moderate leukocyte infiltration of the stroma and acinar cells with only few leukocytes in the islets. The area of nuclear β-islet cells in all segments of the pancreas increased by 4.5 µm2; however, no statistically significant changes were identified when compared with the intact control group.

Edema significantly reduced by day 14; however, blood vessels of the acinar tissue and capillaries of the islets of Langerhans were still slightly hyperemic. There were focal sclerotic changes in some islets. Compared with the control group, there were not any statistically significant changes in the islet volume fraction in all segments of the pancreas. Insulinpositive cells tended to be located in a random pattern; however, glucagon-positive cells were predominantly found at the periphery. The total β-endocrine cell area in the gastric and splenic segments, as compared with the intact control group, was significantly decreased (p <0.05), while the total α-cell area was increased (p <0.05) (Table 5) (Fig.2B). There was moderate hypertrophy of β-cell nuclei in all segments of the pancreas.

At 28 day edema, hyperemia of blood vessels and inflammatory infiltration were replaced by focal sclerotic changes of the acinar tissue and pancreatic islets. As before, a slight decrease in the islet volume fraction in all segments of the pancreas was reported. Also, in all segments of the pancreas the total area of β-cells was slightly increased, compared with day 14 of the experiment, but was significantly lower (p <0.05) as compared with the animals in the intact control group. The volume fraction of α-endocrine cells slightly decreased but was statistically greater (p <0.05) than in the intact control group (Table 5). The average area of nuclei was slightly decreased as compared with the intact control group and with day 14 of the experiment.



Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 233

**Primary antibodies** 

**<sup>2</sup>MDM2 NOS-**

**3** 

**NFkB** 

**<sup>3</sup>TRAIL Bax Bcl-**

3 - + + - + ++ - ++ 7 - + + - + ++ - ++ 14 - + + - + ++ - ++ 28 - + + - + ++ - ++

7 - ++ - ++ - + - + 14 - ++ - ++ - + - + 28 - + - + - ++ - ++

3 - + + - + +++ - ++ 7 - + + - + +++ - ++ 14 - + + - + +++ - ++ 28 - + + - + +++ - ++

7 - ++ - ++ - ++ - + 14 - ++ - ++ - ++ - + 28 - + - + - ++ - +

amount of cells with NF-kB-and MDM2-positive stained nuclei was reported. At day 7 there was a significant (p <0.05) increase in the amount of apoptotic β-cells, compared with day 3 of the experiment, which was followed by a significant (p <0.05) decrease by day 14 (Table 12). At day 28 the amount of cells and the intensity of expression of apoptotic markers significantly decreased and anti-apoptotic proteins were expressed in large quantities in the endocrine cells of pancreatic islets. Apoptotic index was significantly decreased as compared with day 7; however, it was still statistically more significant than in the intact

**Intensity of expression**

Diabetes 3 - + - + - ++ - ++

**Amount of cells**

Diabetes 3 - + - + - + - +

Within the period from day 7 to day 14 we observed cytoplasmic staining for caspase-3 and Bax proteins accompanied by decreased amount of cells with NF-kB-and MDM2-positive stained nuclei (Fig. 4B). At day 7 there was a significant (p <0.05) increase in the amount of apoptotic β-cells compared with day 3 of the experiment, which was followed by a

Thus, in alloxan-induced diabetes we observe a decrease in the total β-cell area from day 7 to day 28 of observation which is accompanied by a simultaneous increase in the total α-cell area. These changes occur in all segments of the pancreas; however, they are predominantly obvious and are statistically significant in the gastric and splenic segments. Along with

**Table 6.** Immunohistochemical characteristics of β-endocrine cells in alloxan-induced diabetes

significant (p <0.05) decrease by day 14 (Table 12).

**p53 Caspase-**

control group (Table 6).

**Group Day of** 

Intact control

Intact control **experiment** 

\* - Significant difference compared with the intact control group (p <0.05).

**Table 5.** Morphometric indicators of pancreatic islets in rats with alloxan-induced diabetes (M ± m)

Immunohistochemical study which, was performed at day 3 and day 28, showed negative staining of β-endocrine cells for p53, TRAIL, endothelial NO-synthase and Bcl-2 proteins (Fig. 3B) in all segments of the pancreas. At day 3 we observed mild cytoplasmic staining for anti-caspase-3 and anti-Bax-antibody in some endocrine cells. Compared with the intact control group, apoptotic index was significantly increased (p <0.05) during all observation periods. The expression of NF-kB and MDM2 proteins tended to decrease. At 7-14 day moderate cytoplasmic staining for caspase-3 and Bax proteins accompanied by decreased amount of cells with NF-kB-and MDM2-positive stained nuclei was reported. At day 7 there was a significant (p <0.05) increase in the amount of apoptotic β-cells, compared with day 3 of the experiment, which was followed by a significant (p <0.05) decrease by day 14 (Table 12). At day 28 the amount of cells and the intensity of expression of apoptotic markers significantly decreased and anti-apoptotic proteins were expressed in large quantities in the endocrine cells of pancreatic islets. Apoptotic index was significantly decreased as compared with day 7; however, it was still statistically more significant than in the intact control group (Table 6).

232 Apoptosis and Medicine

Total area of β-cells, %

Total area fraction of α-cells,%

Total area of β-cell nuclei, µm2

\* - Significant difference compared with the intact control group (p <0.05).

**Indicator Day of experiment Segment of the pancreas**

**Intestinal Gastric Splenic** 

14 intact 6,9±3,1 12,1±5,2 14,0±2,9 diabetes 4,2±1,4 8,4±1,6 9,3±2,1 28 intact 6,8±2,1 11,9±3,7 13,7±3,2 diabetes 3,1±1,1 7,6±2,3 9,5±2,4

3 intact 51,3±9,2 64,6±9,1 78,2±6,1 diabetes 50,4±8,5 65,9±8,2 69,8±4,9 7 intact 53,8±7,6 63,4±7,0 75,4±7,7 diabetes 42,2±2,1 **45,5±1,4\* 58,1±1,4\***  14 intact 52,1±5,5 64,5±8,7 79,4±4,6 diabetes 40,4±2,4 **39,7±2,2\* 57,4±2,0\***  28 intact 51,1±7,5 65,2±6,0 77,4±4,4 diabetes **32,3±1,3\* 40,2±1,4\* 49,7±2,1\*** 

3 intact 37,4±5,2 31,2±3,5 22,2±3,4 diabetes 36,5±4,8 30,8±2,5 25,7±2,4 7 intact 39,3±4,3 30,4±4,0 21,3±3,0 diabetes 41,1±4,1 **49,3±2,1\* 35,4±2,2\***  14 intact 38,5±3,5 32,4±3,2 19,7±2,3 diabetes 32,2±1,5 **47,4±2,0\* 32,2±2,1\***  28 intact 30,0±4,4 31,4±3,9 18,9±3,2 diabetes **30,1±2,1\* 42,3±3,1\* 30,1±4,1\*** 

3 intact 24,2±1,2 24,4±1,0 23,9±0,2 diabetes 27,8±5,9 27,5±5,5 26,6±4,6 7 intact 24,3±1,3 23,8±1,2 24,7±1,0 diabetes 29,5±4,3 28,4±3,2 29,6±4,3 14 intact 24,3±1,2 24,8±1,3 24,6±1,3 diabetes 30,3±3,2 29,6±2,3 30,4±3,4 28 intact 23,9±1,2 24,2±1,2 24,6±1,2 diabetes 28,5±5,1 27,4±3,4 28,5±4,0

**Table 5.** Morphometric indicators of pancreatic islets in rats with alloxan-induced diabetes (M ± m)

Immunohistochemical study which, was performed at day 3 and day 28, showed negative staining of β-endocrine cells for p53, TRAIL, endothelial NO-synthase and Bcl-2 proteins (Fig. 3B) in all segments of the pancreas. At day 3 we observed mild cytoplasmic staining for anti-caspase-3 and anti-Bax-antibody in some endocrine cells. Compared with the intact control group, apoptotic index was significantly increased (p <0.05) during all observation periods. The expression of NF-kB and MDM2 proteins tended to decrease. At 7-14 day moderate cytoplasmic staining for caspase-3 and Bax proteins accompanied by decreased


**Table 6.** Immunohistochemical characteristics of β-endocrine cells in alloxan-induced diabetes

Within the period from day 7 to day 14 we observed cytoplasmic staining for caspase-3 and Bax proteins accompanied by decreased amount of cells with NF-kB-and MDM2-positive stained nuclei (Fig. 4B). At day 7 there was a significant (p <0.05) increase in the amount of apoptotic β-cells compared with day 3 of the experiment, which was followed by a significant (p <0.05) decrease by day 14 (Table 12).

Thus, in alloxan-induced diabetes we observe a decrease in the total β-cell area from day 7 to day 28 of observation which is accompanied by a simultaneous increase in the total α-cell area. These changes occur in all segments of the pancreas; however, they are predominantly obvious and are statistically significant in the gastric and splenic segments. Along with

pronounced necrotic changes in the cells of the insular apparatus of the pancreas, there is also an increase in apoptogenic activity of β-endocrine cells due to the damage to mitochondrial membranes (increased expression of Bax and inhibition of Bcl-2 proteins), which might be caused by impaired intracellular calcium homeostasis and activation of the mitochondrial apoptotic pathway with subsequent activation of caspase cascade without the involvement of p53 protein (Table 12).

Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 235

**Segment of the pancreas Intestinal Gastric Splenic** 

**Primary antibodies** 

**<sup>2</sup>MDM2 NOS-**

**3** 

**NFkB** 

**<sup>3</sup>TRAIL Bax Bcl-**

28 - + + - + ++ - ++

28 - ++ + ++ - + + +

28 - + + - + +++ - ++

28 - + + + - ++ + ++

28 intact 40,0±4,4 31,4±3,9 18,9±3,2 diabetes 40,9±4,8 42,7±4,8 **30,3±4,7\*** 

7 intact 24,3±1,3 23,8±1,2 24,7±1,0 diabetes 23,8±3,1 25,7±2,5 **29,5±1,8\***  28 intact 23,9±1,2 24,2±1,2 24,6±1,2 diabetes 25,3±1,2 25,9±1,0 **29,3±1,1\*** 

**Table 7.** Morphometric indicators of pancreatic islets in rats with streptozotocin-induced diabetes (M ± m)

Immunohistochemical reaction, which was performed at day 7, revealed that most endocrine cells exhibited marked expression of proapoptotic proteins, such as caspase-3, TRAIL and Bax (Fig. 3C, 4C). Activation of apoptosis was accompanied by inhibited expression of anti-apoptotic Bcl-2, MDM2, nuclear factor NF-kB proteins (Table 8). There was a statistically significant increase in the amount of cells with morphological features of

By day 28 there was a decrease in the amount of β-cells as well as in the intensity of expression of caspase 3 in the surviving cells (Table 12). We did not observe any significant changes in expression of either mitochondrial proapoptotic Bax protein or membrane TRAIL (Table 8). Compared with the intact control group, there was a statistically significant increase in apoptotic index. With regard to day 7 of observation, a statistically significant decrease in apoptotic index was stated (Table 8). Moderate expression of endothelial NOsynthase in the capillaries of pancreatic islets was found during all observation periods

**p53 Каспаза-**

**Intensity of expression** Intact control 7 - + + - + ++ - ++

Diabetes 7 - +++ + ++ - + + +

**Amount of cells** Intact control 7 - + + - + +++ - ++

Diabetes 7 - ++ + + - + + +

**Table 8.** Immunohistochemical characteristics of β-endocrine cells in streptozotocin-induced diabetes

**Indicator Day of** 

Total area of β-cell nuclei,

µm2

(Table 8).

**Characteristic Day of** 

**experiment** 

**experiment** 

\* - Significant difference compared with the intact control group (p <0.05).

apoptosis when compared with the intact control group (Table 8).

### **Streptozotocin-induced diabetes**

In streptozotocin-induced diabetes a statistically significant elevation of plasma glucose levels, as compared with those in the intact control animals, was reported (Fig. 1).

Histological investigation which was performed at day 7 showed pronounced hyperemia of blood vessels associated with lymphocytic infiltration and marked edema of the interlobular connective tissue. There were not any statistically significant differences in the total volume fraction of islets either with regard to the intact control group, or to different segments of the pancreas. We revealed marked necrotic changes in endocrine cells both in the central part and at the periphery of the islets (Fig. 2C). We also stated a statistically significant decrease (p <0.05) in the area of β-cells in all segments of the pancreas. However, a significant increase (p <0.05) in the volume fraction of α-cells exclusively in the gastric and splenic segments of the pancreas was reported. There was marked hypertrophy of β-islet cell nuclei (p <0.05) predominantly in the splenic segment of the pancreas (Table 7).

By day 28 moderate hyperemia of blood capillaries of pancreatic islets and accumulation of lymphocytes at some sites were observed. Focal necrotic changes were reported in some islets, whereas other islets underwent sclerotic changes. No significant changes in the islet volume fraction were observed. The total β-islet cell area was still significantly lower (p <0.05) compared with the intact control group during the same period of observation. The total volume fraction of α-endocrine cells was significantly increased (p <0.05) exclusively in the splenic segment of the pancreas. The nuclei of β-cells were hypertrophic and hypertrophy was statistically significant (p <0.05) in the cells of the splenic segment of the pancreas (Table 7).



\* - Significant difference compared with the intact control group (p <0.05).

234 Apoptosis and Medicine

involvement of p53 protein (Table 12).

**Streptozotocin-induced diabetes** 

**Indicator Day of** 

Volume fraction of islets,%

Total area of β-cells,%

Total area of α-cells,%

**experiment** 

pronounced necrotic changes in the cells of the insular apparatus of the pancreas, there is also an increase in apoptogenic activity of β-endocrine cells due to the damage to mitochondrial membranes (increased expression of Bax and inhibition of Bcl-2 proteins), which might be caused by impaired intracellular calcium homeostasis and activation of the mitochondrial apoptotic pathway with subsequent activation of caspase cascade without the

In streptozotocin-induced diabetes a statistically significant elevation of plasma glucose

Histological investigation which was performed at day 7 showed pronounced hyperemia of blood vessels associated with lymphocytic infiltration and marked edema of the interlobular connective tissue. There were not any statistically significant differences in the total volume fraction of islets either with regard to the intact control group, or to different segments of the pancreas. We revealed marked necrotic changes in endocrine cells both in the central part and at the periphery of the islets (Fig. 2C). We also stated a statistically significant decrease (p <0.05) in the area of β-cells in all segments of the pancreas. However, a significant increase (p <0.05) in the volume fraction of α-cells exclusively in the gastric and splenic segments of the pancreas was reported. There was marked hypertrophy of β-islet cell nuclei

By day 28 moderate hyperemia of blood capillaries of pancreatic islets and accumulation of lymphocytes at some sites were observed. Focal necrotic changes were reported in some islets, whereas other islets underwent sclerotic changes. No significant changes in the islet volume fraction were observed. The total β-islet cell area was still significantly lower (p <0.05) compared with the intact control group during the same period of observation. The total volume fraction of α-endocrine cells was significantly increased (p <0.05) exclusively in the splenic segment of the pancreas. The nuclei of β-cells were hypertrophic and hypertrophy was statistically significant (p <0.05) in the cells of the splenic segment of the pancreas (Table 7).

> 7 intact 6,7±2,2 12,0±4,5 12,9±2,1 diabetes 4,7±3,1 10,2±6,7 11,8±1,5 28 intact 6,8±2,1 11,9±3,7 13,7±3,2 diabetes 3,2±2,1 8,2±3,1 9,7±2,3

7 intact 53,8±7,6 63,4±7,0 75,4±8,7 diabetes **24,3±2,1\* 39,2±3,2\* 40,1±2,0\***  28 intact 51,1±7,5 65,2±6,0 77,4±4,4 diabetes **29,8±5,5\* 43,2±5,4\* 47,7±8,2\*** 

7 intact 39,3±4,3 30,4±4,0 21,3±3,0 diabetes 44,2±4,5 **42,3±2,2\* 29,8±2,1\*** 

**Segment of the pancreas Intestinal Gastric Splenic** 

levels, as compared with those in the intact control animals, was reported (Fig. 1).

(p <0.05) predominantly in the splenic segment of the pancreas (Table 7).

**Table 7.** Morphometric indicators of pancreatic islets in rats with streptozotocin-induced diabetes (M ± m)

Immunohistochemical reaction, which was performed at day 7, revealed that most endocrine cells exhibited marked expression of proapoptotic proteins, such as caspase-3, TRAIL and Bax (Fig. 3C, 4C). Activation of apoptosis was accompanied by inhibited expression of anti-apoptotic Bcl-2, MDM2, nuclear factor NF-kB proteins (Table 8). There was a statistically significant increase in the amount of cells with morphological features of apoptosis when compared with the intact control group (Table 8).

By day 28 there was a decrease in the amount of β-cells as well as in the intensity of expression of caspase 3 in the surviving cells (Table 12). We did not observe any significant changes in expression of either mitochondrial proapoptotic Bax protein or membrane TRAIL (Table 8). Compared with the intact control group, there was a statistically significant increase in apoptotic index. With regard to day 7 of observation, a statistically significant decrease in apoptotic index was stated (Table 8). Moderate expression of endothelial NOsynthase in the capillaries of pancreatic islets was found during all observation periods (Table 8).


**Table 8.** Immunohistochemical characteristics of β-endocrine cells in streptozotocin-induced diabetes

Thus, the development of streptozotocin-induced diabetes is associated not only with hyperglycemia, but also with marked necrotic changes in endocrine cells of pancreatic islets, decreased β-islet cell area accompanied by hyperplasia of their nuclei, increased αendocrine cell area, as well as increased apoptotic index (Table 12). High expression levels of endothelial NO-synthase in the capillaries of pancreatic islets are indicative of endothelial dysfunction.

Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 237

**Section of the pancreas Intestinal Gastric Splenic** 

7 intact 53,8±7,6 63,4±7,0 75,4±8,7 diabetes **29,8±5,5\* 43,2±5,4\* 47,7±8,2\***  14 intact 52,1±5,5 64,5±8,7 79,4±4,6 diabetes **25,4±3,2\* 40,1±3,2\* 42,3±1,2\***  28 intact 51,1±7,5 65,2±6,0 77,4±4,4 diabetes **30,2±4,5\* 44,3±2,3\* 40,1±3,2\*** 

7 intact 39,3±4,3 30,4±4,0 21,3±3,0 diabetes 45,2±1,2 40,4±5,1 **56,7±4,6\***  14 intact 38,5±3,5 32,4±3,2 19,7±2,3 diabetes 44,1±2,1 42,3±4,2 **56,4±3,4\***  28 intact 40,0±4,4 31,4±3,9 18,9±3,2 diabetes 33,9±3,2 **49,5±4,4\* 50,9±3,2\*** 

7 intact 24,3±1,3 23,8±1,2 24,7±1,0 diabetes 25,8±1,1 25,2±2,3 27,5±2,2 14 intact 24,3±1,2 24,8±1,3 24,6±1,3 diabetes 25,6±2,3 24,6±2,3 26,4±2,1 28 intact 23,9±1,2 24,2±1,2 24,6±1,2 diabetes 24,9±2,0 25,1±1,2 25,8±2,2

**Table 9.** Morphometric indicators of pancreatic islets in rats with insulin-dependent diabetes (M ± m)

Immunohistochemical study during all observation periods showed absence or doubtful expression of p53, Bax, MDM2 as well as Bcl-2 proteins in certain β-islet cells. Within the period from day 7 to day 14 we observed increased expression of TRAIL proapoptotic protein and caspase 3 protein (compared with the intact control group and at day 14 compared with day 7 of the experiment) (Table 10, 12, Fig. 3D, 4D) in most β-cells accompanied by a slight reduction by day 28. Simultaneously, apoptotic index during all observation periods was significantly increased compared with the intact control group (Table 5). The expression of NO-synthase in the endothelium of the pancreatic islet

**Intensity of expression**

**Primary antibodies** 

**kB** 

**p53 Caspase-3 TRAIL Bax Bcl-2 MDM2 NOS-3 NF-**

7 - + + - + ++ - ++ 14 - + + - + ++ - ++ 28 - + + - + ++ - ++

**Indicator Day of** 

Total area of β-cells,%

Total area of α-cells,%

Total area of β-cell nuclei,

µm2

**Group** 

Intact control **experiment** 

\* - Significant difference compared with the intact control group (p <0.05).

capillaries during all observation periods enhanced.

**Day of experiment, days** 

#### **Immune-dependent diabetes**

Persistent hyperglycemia and a reduction in plasma insulin by 67.8% were reported in immune-dependent diabetes (Fig. 1), as compared with the intact control group.

Microscopic examination, which was performed at day 7, found that the total volume fraction of the islets decreased when compared with the intact control group. The islets had an irregular shape due to marked necrotic changes in the endocrine cells, hyperemia of the capillaries, as well as mild lymphocytic infiltration. There were single insulin-positive cells or they were arranged in small clusters in the central part of the islets around the capillaries. We observed a significant decrease (p <0.05) in the total β-cell area in all segments of the pancreas as compared with the intact controls (Table 9). The total α-endocrine cell area was significantly increased exclusively in the splenic segment of the pancreas.

At day 14 some symptoms of insulitis, i.e. inflammation of insular cells, persisted. Compared with the intact control group, the area covered by β-endocrine cells in all segments of the pancreas tended to become smaller (p <0.05) due to marked necrotic changes in β-endocrine cells (Fig. 2D). However, we observed moderate hypertrophy of the nuclei of β-islet cells as well as an increase in the area covered by α-endocrine cells in the splenic segment of the pancreas (Table 9). Some pancreatic islets were reported to undergo sclerotic changes.

By day 28, as before, a statistically significant reduction (p <0.05) in the area covered by βislet cells in all segments of the pancreas, as compared with the intact control group, was observed. Inflammatory infiltration, swelling and hyperemia of blood vessels tended to reduce, too. However, the amount of islets which developed sclerotic changes increased and β-endocrine cells had moderately hypertrophic nuclei. There was an increase in the area covered by α-endocrine cells in the gastric and splenic segments of the pancreas (Table 9).



\* - Significant difference compared with the intact control group (p <0.05).

236 Apoptosis and Medicine

dysfunction.

sclerotic changes.

Volume fraction of islets,%

**Indicator Day of** 

**experiment** 

**Immune-dependent diabetes** 

Thus, the development of streptozotocin-induced diabetes is associated not only with hyperglycemia, but also with marked necrotic changes in endocrine cells of pancreatic islets, decreased β-islet cell area accompanied by hyperplasia of their nuclei, increased αendocrine cell area, as well as increased apoptotic index (Table 12). High expression levels of endothelial NO-synthase in the capillaries of pancreatic islets are indicative of endothelial

Persistent hyperglycemia and a reduction in plasma insulin by 67.8% were reported in

Microscopic examination, which was performed at day 7, found that the total volume fraction of the islets decreased when compared with the intact control group. The islets had an irregular shape due to marked necrotic changes in the endocrine cells, hyperemia of the capillaries, as well as mild lymphocytic infiltration. There were single insulin-positive cells or they were arranged in small clusters in the central part of the islets around the capillaries. We observed a significant decrease (p <0.05) in the total β-cell area in all segments of the pancreas as compared with the intact controls (Table 9). The total α-endocrine cell area was

At day 14 some symptoms of insulitis, i.e. inflammation of insular cells, persisted. Compared with the intact control group, the area covered by β-endocrine cells in all segments of the pancreas tended to become smaller (p <0.05) due to marked necrotic changes in β-endocrine cells (Fig. 2D). However, we observed moderate hypertrophy of the nuclei of β-islet cells as well as an increase in the area covered by α-endocrine cells in the splenic segment of the pancreas (Table 9). Some pancreatic islets were reported to undergo

By day 28, as before, a statistically significant reduction (p <0.05) in the area covered by βislet cells in all segments of the pancreas, as compared with the intact control group, was observed. Inflammatory infiltration, swelling and hyperemia of blood vessels tended to reduce, too. However, the amount of islets which developed sclerotic changes increased and β-endocrine cells had moderately hypertrophic nuclei. There was an increase in the area covered by α-endocrine cells in the gastric and splenic segments of the pancreas (Table 9).

> 7 intact 6,7±2,2 12,0±4,5 12,9±2,1 diabetes 4,7±3,1 10,2±6,7 11,8±1,5 14 intact 6,9±3,1 12,1±5,2 14,0±2,9 diabetes 4,2±1,2 8,9±2,0 9,2±2,2 28 intact 6,8±2,1 11,9±3,7 13,7±3,2 diabetes 4,4±1,0 7,4±1,3 8,7±3,4

**Section of the pancreas Intestinal Gastric Splenic** 

immune-dependent diabetes (Fig. 1), as compared with the intact control group.

significantly increased exclusively in the splenic segment of the pancreas.

**Table 9.** Morphometric indicators of pancreatic islets in rats with insulin-dependent diabetes (M ± m)

Immunohistochemical study during all observation periods showed absence or doubtful expression of p53, Bax, MDM2 as well as Bcl-2 proteins in certain β-islet cells. Within the period from day 7 to day 14 we observed increased expression of TRAIL proapoptotic protein and caspase 3 protein (compared with the intact control group and at day 14 compared with day 7 of the experiment) (Table 10, 12, Fig. 3D, 4D) in most β-cells accompanied by a slight reduction by day 28. Simultaneously, apoptotic index during all observation periods was significantly increased compared with the intact control group (Table 5). The expression of NO-synthase in the endothelium of the pancreatic islet capillaries during all observation periods enhanced.



Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 239

**Intestinal Gastric Splenic** 

sclerotic changes. Compared with the intact control group, there was a statistically significant reduction in the area of β-cells in the splenic segment. Immunopositive material was unevenly distributed both in the insular cells and within the cytoplasm of an insular cell. Hypertrophy of the nuclei of islet cells was reported in all segments of the pancreas.

At day 14 a mosaic pattern of cell damage was still noted. Both histologically intact islets and islets revealing sclerotic changes were defined. Their amount tended to decrease in all segments of the pancreas. Simultaneously, the total β-cell area decreased in the gastric and splenic segments of the pancreas. Hypertrophy of the nuclei of β-cells persisted in all

By day 28 a statistically significant reduction in β-cell area in the gastric segment of the pancreas was reported, as compared with the control group. Hypertrophy of the nuclei of β-

> 3 intact 6,8±2,5 12,6±6,0 13,8±7,3 diabetes 3,8±1,0 8,3±1,0 5,4±3,2 7 intact 6,7±2,2 12,0±4,5 12,9±2,1 diabetes 4,2±1,2 7,8±1,4 6,7±2,5 14 intact 6,8±2,1 11,9±3,7 13,7±3,2 diabetes 5,4±1,3 6,9±1,2 8,9±4,2 28 intact 6,6±4,2 12,8±4,5 14,3±5,2 diabetes 5,3±1,1 7,9±1,0 11,3±2,1

> 3 intact 51,3±9,2 64,6±9,1 78,2±6,1 diabetes 43,4±4,9 52,4±3,4 65,7±3,4 7 intact 53,8±7,6 63,4±7,0 75,4±7,7 diabetes 44,6±4,2 53,2±6,1 **57,7±3,3\***  14 intact 51,1±7,5 65,2±6,0 77,4±4,4 diabetes 37,4±5,6 **47,6±4,3\* 56,2±4,2\***  28 intact 54,3±5,4 66,2±3,6 77,3±4,3 diabetes 41,2±2,3 **50,2±3,1\*** 67,8±2,6

> 3 intact 24,2±1,2 24,4±1,0 23,9±0,2 diabetes 26,4±1,1 26,8±1,0 **26,5±0,9\***  7 intact 24,3±1,3 23,8±1,2 24,7±1,0 diabetes **29,8±1,0\* 29,6±1,2\* 30,1±0,6\***  14 intact 23,9±1,2 24,2±1,2 24,6±1,2 diabetes **29,1±1,3\* 29,6±1,0\* 30,0±1,0\***  28 intact 24,4±1,0 24,3±1,2 24,7±2,0 diabetes **28,7±1,2\* 28,7±1,1\* 29,7±1,2\***

**Table 11.** Morphometric indicators of pancreatic islets in rats with experimental streptozotocin-

**Indicator Day of experiment Segment of the pancreas**

segments of the pancreas (Fig. 2E).

cells persisted (Table 11).

Total volume fraction of islets,%

Total area of β-cells,%

Total area of β-cell nuclei,

\* - Significant difference compared with the intact control (p <0.05).

nicotinamide-induced diabetes (M ± m)

µm2

**Table 10.** Expression of pro-and β-anti-apoptotic proteins in endocrine cells of insulin-dependent diabetic rats

Thus, it was found that immuno-dependent diabetes is characterized by the following changes: hyperglycemia, reduced area of β-islet cells, inflammatory infiltration of islets, marked β-necrotic changes in endocrine cells, focal sclerosis of the islets of Langerhans.

## **Streptozotocin-nicotinamide-induced diabetes**

The results of the 28-day streptozotocin-nicotinamide-induced diabetes study indicated that plasma C-peptide concentration in the control animals with diabetes increased by 36.8% (p<0,05), as compared with intact rats. Blood glucose levels in rats with strep-tozototsinnicotinamide-induced diabetes during the entire experiment was significantly superior to glycemia in intact animals (Fig. 1).

Histologically, the maximum reduction in the volume fraction of islets was found at day 3 with a tendency to increase by day 28 in all segments of the pancreas.

At day 3 the pancreatic tissue appeared unevenly swollen and hyperemic. There were minor destructive changes of endocrine cells in pancreatic islets. The proportion of β-islet cells in all segments of the pancreas markedly decreased. At the same time no significant changes in the area of β-cells in islets were observed. The islets showed uneven accumulation of insulin-positive cells. Along with the cells which exhibited a marked accumulation of insulin, endocrine cells showing uneven distribution of immunopositive material in the cytoplasm with moderate or weak staining were singled out. Endocrine cells in the splenic segment of the pancreas were characterized by moderate hypertrophy of the nuclei and it was statistically significant, as compared to the intact control (Table 11).

By day 7 swelling hyperemia decreased and a microscopic mosaic image was observed. Both histologically intact and impaired islets of Langerhans were found. In a number of islets we could observe focal necrotic changes of endocrine cells, while others exhibited focal proliferation of connective tissue, and sometimes poorly defined intralobular and periductal sclerotic changes. Compared with the intact control group, there was a statistically significant reduction in the area of β-cells in the splenic segment. Immunopositive material was unevenly distributed both in the insular cells and within the cytoplasm of an insular cell. Hypertrophy of the nuclei of islet cells was reported in all segments of the pancreas.

238 Apoptosis and Medicine

**Day of experiment, days** 

**Streptozotocin-nicotinamide-induced diabetes** 

glycemia in intact animals (Fig. 1).

**Primary antibodies** 

**kB** 

**p53 Caspase-3 TRAIL Bax Bcl-2 MDM2 NOS-3 NF-**

14 - +++ ++ ++ - + ++ + 28 - ++ + + - ++ ++ +

7 - + + - + +++ - ++ 14 - + + - + +++ - ++ 28 - + + - + +++ - ++

14 - ++ + ++ - ++ + + 28 - ++ + + - ++ + +

Diabetes 7 - +++ +++ ++ - + ++ +

**Amount of cells**

Diabetes 7 - ++ + ++ - ++ + +

**Table 10.** Expression of pro-and β-anti-apoptotic proteins in endocrine cells of insulin-dependent

Thus, it was found that immuno-dependent diabetes is characterized by the following changes: hyperglycemia, reduced area of β-islet cells, inflammatory infiltration of islets, marked β-necrotic changes in endocrine cells, focal sclerosis of the islets of Langerhans.

The results of the 28-day streptozotocin-nicotinamide-induced diabetes study indicated that plasma C-peptide concentration in the control animals with diabetes increased by 36.8% (p<0,05), as compared with intact rats. Blood glucose levels in rats with strep-tozototsinnicotinamide-induced diabetes during the entire experiment was significantly superior to

Histologically, the maximum reduction in the volume fraction of islets was found at day 3

At day 3 the pancreatic tissue appeared unevenly swollen and hyperemic. There were minor destructive changes of endocrine cells in pancreatic islets. The proportion of β-islet cells in all segments of the pancreas markedly decreased. At the same time no significant changes in the area of β-cells in islets were observed. The islets showed uneven accumulation of insulin-positive cells. Along with the cells which exhibited a marked accumulation of insulin, endocrine cells showing uneven distribution of immunopositive material in the cytoplasm with moderate or weak staining were singled out. Endocrine cells in the splenic segment of the pancreas were characterized by moderate hypertrophy of the nuclei and it

By day 7 swelling hyperemia decreased and a microscopic mosaic image was observed. Both histologically intact and impaired islets of Langerhans were found. In a number of islets we could observe focal necrotic changes of endocrine cells, while others exhibited focal proliferation of connective tissue, and sometimes poorly defined intralobular and periductal

with a tendency to increase by day 28 in all segments of the pancreas.

was statistically significant, as compared to the intact control (Table 11).

**Group** 

Intact control

diabetic rats

At day 14 a mosaic pattern of cell damage was still noted. Both histologically intact islets and islets revealing sclerotic changes were defined. Their amount tended to decrease in all segments of the pancreas. Simultaneously, the total β-cell area decreased in the gastric and splenic segments of the pancreas. Hypertrophy of the nuclei of β-cells persisted in all segments of the pancreas (Fig. 2E).

By day 28 a statistically significant reduction in β-cell area in the gastric segment of the pancreas was reported, as compared with the control group. Hypertrophy of the nuclei of βcells persisted (Table 11).


\* - Significant difference compared with the intact control (p <0.05).

**Table 11.** Morphometric indicators of pancreatic islets in rats with experimental streptozotocinnicotinamide-induced diabetes (M ± m)


\* - Significant difference compared with the intact control (p <0.05) **^** - significant difference compared with day 3 of experiment (p <0.05); # - significant difference compared with day 7 of experiment (p <0.05).

**Table 12.** Apoptotic index of β-cells (%)

Thus, hyperglycemia as well as reduced plasma C-peptide concentrations are common to experimental streptozotocin-nicotinamide-induced diabetes. Along with biochemical changes, it displays some characteristic morphological changes, including a statistically significant decrease in insular cell area associated with hypertrophy of β-cell nuclei, mosaic islet damage resulting in the destruction of endocrine cells as well as sclerotic changes of pancreatic islets during long-term observation period.

**A.** Intact control **B.** Alloxan-induced diabetes

Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 241

**E.** Streptozotocin-nicotinamide-induced diabetes

**A.** Intact control **B.** Alloxan-induced diabetes

**C.** Streptozotocin-induced diabetes **D.** Immune-dependent diabetes

**Figure 2.** Reduction in the amount of cells under different experimental diabetes conditions, day 14 (Streptozotocin-induced diabetes, day 7), splenic segment of the pancreas. Primary antibody against

insulin, DAB staining. Magnification: х400

**C.** Streptozotocin-induced diabetes **D.** Immune-dependent diabetes

Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 241

**E.** Streptozotocin-nicotinamide-induced diabetes

**Figure 2.** Reduction in the amount of cells under different experimental diabetes conditions, day 14 (Streptozotocin-induced diabetes, day 7), splenic segment of the pancreas. Primary antibody against insulin, DAB staining. Magnification: х400

240 Apoptosis and Medicine

**Intact control** 

**Table 12.** Apoptotic index of β-cells (%)

**Alloxaninduced diabetes** 

pancreatic islets during long-term observation period.

experiment (p <0.05); # - significant difference compared with day 7 of experiment (p <0.05).

**Streptozotocininduced diabetes** 

3 4,1±0,2 **10,2±1,2\*** 3,9±0,1 - 5,1±1,8 7 3,9±0,1 **17,4±0,9\* ^** 4,0±1,1 **28,6±1,5\*** 5,4±2,1 14 4,2±1,0 **14,9±1,0\* ^ 32,3±4,2\* 35,2±2,9\*#** 4,8±1,9 28 4,0±1,1 **12,5±0,6\* # 21,3±2,2\* # 30,3±2,7\*** 5,0±4,1 \* - Significant difference compared with the intact control (p <0.05) **^** - significant difference compared with day 3 of

Thus, hyperglycemia as well as reduced plasma C-peptide concentrations are common to experimental streptozotocin-nicotinamide-induced diabetes. Along with biochemical changes, it displays some characteristic morphological changes, including a statistically significant decrease in insular cell area associated with hypertrophy of β-cell nuclei, mosaic islet damage resulting in the destruction of endocrine cells as well as sclerotic changes of

**A.** Intact control **B.** Alloxan-induced diabetes

**C.** Streptozotocin-induced diabetes **D.** Immune-dependent diabetes

**Immunedependent diabetes** 

**Streptozotocinnicotinamideinduced diabetes** 

**Day experiment** 

**A.** Intact control **B.** Alloxan-induced diabetes

**C.** Streptozotocin-induced diabetes **D.** Immune-dependent diabetes

**E.** Streptozotocin-nicotinamide-induced diabetes

**Figure 3.** TRAIL expression in pancreatic islet cells under different experimental diabetes conditions, day 14 (Streptozotocin-induced diabetes, day 7), splenic segment of the pancreas. TRAIL primary antibody, DAB staining. Magnification: х400.

**C.** Streptozotocin-induced diabetes **D.** Immune-dependent diabetes

Pancreatic Islet Beta-Cell Apoptosis in Experimental Diabetes Mellitus 243

**E.** Streptozotocin-nicotinamide-induced diabetes

It is known that type I diabetes develops when pancreatic β-cells are damaged due to certain inflammatory, autoimmune and other pathological processes. Selective organ-specific tissue destruction of the insulin-producing pancreatic β-cells is associated with insulin deficiency resulting in impairment of glucose homeostasis. A large body of experimental evidence emphasizes the key role of apoptosis in the pathogenesis of diabetes mellitus (Kim B.M. et al. 2001; Severgina E.S. 2002; Butler A.E. et al. 2003; Bertalli E. et al. 2005; Rees D.A., et al. 2005; Srinivasan K., et al. 2007; Islam S., et al. 2009; Pisarev V.B. et al. 2009). Toxic effects of certain chemicals (e.g. alloxan, streptozotocin, etc.) used to induce diabetes specifically in pancreatic β-cells, manifest themselves by alkylation of DNA and formation of toxic compounds, such as superoxide anion, peroxynitrite, and nitric oxide. Damage to DNA and intracellular structures causes necrosis and activates apoptosis of pancreatic β-cells (Daisy

Selective toxicity of streptozotocin can be explained by destruction of antiradical protective system and pancreatic β-cell DNA fragmentation. Numerous experiments show that the principal cause of streptozotocin-induced β-cell death is alkylation of DNA. Exposure of cells to streptozotocin results in the formation of toxic compounds, such as superoxide anion, peroxynitrite, and nitric oxide (NO). However, the contribution of NO to the cytotoxic activity remains controversial, because low concentrations of NO in the cells inhibit the inducible forms of NO-synthase, thus reducing DNA fragmentation (Szkudelski

Receptor- or mitochondrial-mediated pathway may trigger programmed pancreatic β-cell death (Szkudelski T. 2001). Induction of β-cell apoptosis can occur through a TRAILmediated mechanism. DNA damage can activate p53 gene which regulates the expression of

**Figure 4.** Apoptotic cells under different experimental diabetes conditions, day 14 (Streptozotocininduced diabetes, day 7), splenic segment of the pancreas. Caspase 3 primary antibody, DAB staining.

Magnification: х400.

**4. Discussion** 

Mythili M., et al. 2004).

T. 2001; Lenzen S. 2008).

**E.** Streptozotocin-nicotinamide-induced diabetes

**Figure 4.** Apoptotic cells under different experimental diabetes conditions, day 14 (Streptozotocininduced diabetes, day 7), splenic segment of the pancreas. Caspase 3 primary antibody, DAB staining. Magnification: х400.
