**5.2 Animals, tissue preparation, light, and transmission electron microscopy**

Experiments were performed on 20–22-week-old male C57BL/6 J (RRID: IMSR\_ JAX:000664) mice purchased from Charles River (**Figure 1A**). Upon weaning, mice were fed with a standard rodent diet (CD, R70, Lantmännen, Stockholm, Sweden) with 72% of kcal from carbohydrates, 10% from fat, and 18% from protein until 12 weeks of age. From 12 to 20 weeks of age, a control group continued to be fed with CD while the second group was fed with WD (D12079B, Research diets inc., New Jersey, USA) containing 43% kcal from carbohydrates, 40% from fats and 17% from proteins (**Figure 1B**). Water was available *ad libitum*. Mice were housed in individually ventilated cages (Allentown LLC, USA) in groups of 1–4 animals per cage at 20–24°C, 45–65% relative humidity, and a 12-hour day-night lighting cycle. The bright part of the cycle was between 7 pm and 7 am. Mice were weighed before sacrifice.

Acute pancreas tissue slices from CD and WD mice were prepared as described previously [90–92]. For light microscopy (LM) and transmission electron microscopy (TEM), a small piece of the pancreatic splenic lobe was clamped using hemostatic forceps to avoid the leakage of agarose into this part of the pancreas. After removal from the body, small fragments of the pancreas were fixed in 2.45% glutaraldehyde and 2.45% paraformaldehyde in a 0.1M sodium cacodylate buffer (pH 7.4) at room temperature for 3 h, and at 4°C for 12 h. The tissue was washed in a 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature for 4 h and post-fixed with 2% OsO4 at room temperature for 2 h. After washing in a 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature for 3 hours, the tissue was dehydrated in a graded series of ethanol (50%, 70%, 90%, 96%, and 100%, each for 30 minutes at room temperature). The pieces of the tissue were embedded in TAAB embedding resin (Agar Scientific Ltd., Essex, England). For LM, semithin sections (2 μm) were stained with 0.5% toluidine blue in an aqueous solution. For TEM, ultrathin sections (70–75 nm) of the tissue were stained with uranyl acetate and lead citrate and analyzed with a Zeiss EM 902 transmission electron microscope.
