**4. Application of TEM in pancreas physiology**

Glucose-induced insulin secretion is based on oxidative metabolism, thus mitochondrial function is of crucial importance for beta cells [74]. Mutations in mitochondrial

#### *Application of Transmission Electron Microscopy to Detect Changes in Pancreas Physiology DOI: http://dx.doi.org/10.5772/intechopen.104807*

DNA result in reduced insulin secretion and hyperglycemia [75]. The genetic background is not the only factor affecting mitochondrial function. The diabetic environment, with chronic hyperglycemia, can also affect mitochondria [76]. Such a milieu could be responsible for the progression of T2DM and the reduced capacity of insulin release in these patients. Mitochondria appear round-shaped and hypertrophic in endocrine cells from T2DM islets [76–78]. Besides mitochondria, the structure of other cellular organelles is also altered [79]. The volume density of the endoplasmic reticulum is typically increased, and autophagic vacuoles are present [78, 80, 81]. TEM is also a very useful technique that enables us to identify apoptotic cells [82].

Several studies showed that TEM is suitable to detect morphological characteristics of the diabetic pancreas, but only a few studies have reported results in quantitative terms. Due to the large variability and plasticity of cells and their organelles, especially mitochondria, qualitative evaluations are not sufficient [83]. Furthermore, quantitative morphometry is needed to evaluate subtle structural changes early during the progression of the metabolic syndrome that may precede overt functional changes occurring during the development of T2DM.

Since mouse models of T2DM exhibit comparable developmental features and can provide significant insight into the mechanisms of T2DM development in humans [84], they are widely used in these studies. From several animal models, C57Bl/6 J mice have been the most susceptible to the development of the metabolic syndrome and diet-induced diabetes [85–89]. We performed the morphological and morphometric evaluation of pancreatic endocrine and exocrine tissue obtained from C57Bl/6J mice fed with control diet (CD) or western diet (WD). During 8 weeks on WD, these mice developed obesity, hyperglycemia, and hyperinsulinemia (data not shown). Pancreatic tissue samples were taken in parallel with the preparation of acute pancreas tissue slices intended for electrophysiological studies, confocal calcium imaging, and expression analyses. Although the functional response to glucose in terms of intracellular calcium concentration changes and sensitivity of the exocytotic machinery to calcium were well preserved in WD mice (data not shown), TEM revealed severe morphological changes in the endocrine and exocrine part of the pancreas. In the following section, we demonstrate how to use image segmentation methods and ultrastructural morphometry to analyze and quantify structural changes in the rough endoplasmic reticulum, mitochondria, and exocytotic vesicles and vacuolization in the endocrine and endocrine part of the pancreas from control animals and animals fed with WD. These ultrastructural lesions presented below can be detected early during the progression of the metabolic syndrome and precede cell apoptosis, necrosis, fat infiltration, and overt functional changes. Therefore, TEM enables a more direct assessment of the functional characteristics of the pancreatic tissue and is therefore a suitable and probably the crucial method for detecting alterations associated with early pancreas dysfunction.

### **5. Sample preparation and analysis**

#### **5.1 Ethical statement**

The study protocol was approved by the Administration for Food Safety, Veterinary Sector and Plant Protection, Ministry of agriculture, Forestry and Food Republic of Slovenia (approval number: U34401–12/2015/3). The study was conducted in strict accordance with all national and European recommendations pertaining to care and work with laboratory animals, and every effort was made to minimize animal suffering.
