Animals and infusions
Twenty-two neutered male, 15- to 18-month-old, healthy domestic shorthair cats (Charles River, L’Arbresle, France) were used following principles of laboratory animal care (Veterinary Office of Zürich, Switzerland, # 51/2007).
Cats were randomly divided into four groups. Groups I, II and III were infused over 10 days through a jugular catheter. Group I (six cats) received 50% glucose added to saline. Blood glucose was evaluated six to 12 times daily and the infusion rate was adjusted to target levels at 25–30 mmol/l. Group II (six cats) received lipids (Lipovenoes 10%; Fresenius-Kabi, Bad Homburg, Germany). The lipid composition is given in Electronic supplementary material (ESM) Table 1. Blood triacylglycerols were measured two or three times daily to target levels at 3–7 mmol/l. Group III (five cats) served as the control and was infused with saline. During the 10-day infusion, cats were housed in single cages. The remaining five cats (group IV) served as additional controls; they were not infused and were group-housed during the study. They allowed the investigation of the potential confounding effect of experiment-induced stress on glucose and lipid metabolism.
Plasma glucose was measured by a colorimetric hexokinase/glucose-6-phosphate dehydrogenase method, and triacylglycerols were measured by a colorimetric assay using glycerophosphate oxidase coupled to phenol and 4-aminophenazone (Roche, Vienna, Austria). Serum sodium and potassium were analysed with the ion-selective electrode method (Roche). Serum NEFA were measured using the NEFA-C kit (Wako, Richmond, PA, USA), plasma β-hydroxybutyrate with the 3-hydroxybutyrate dehydrogenase method (Randox, Crumlin, UK).
A cross-reacting porcine insulin radioimmunoassay (Linco, St Charles, MO, USA) was validated for cats (see ESM Validation of the porcine insulin radioimmunoassay for use in cats). Serum cortisol was measured by a competitive chemiluminescent immunoassay previously validated in cats (Bayer, Tarrytown, NY, USA) .
The acute-phase proteins of inflammation α1-acid glycoprotein and serum amyloid A were measured in plasma using a feline-specific radial immunodiffusion test and ELISA (Tridelta, Bray, Ireland).
Assessment of beta cell function
During the 10-day infusion in groups I–III overnight fasting blood samples were collected through the jugular catheter daily at 08:00 hours to measure glucose and insulin. An IVGTT was performed under anaesthesia in groups I–III 1 h after the 10-day infusion. Group IV was also admitted to the IVGTT. A glucose bolus of 1 g/kg was administered via the jugular catheter. Glucose and insulin were measured before the bolus and from 5 to 120 min thereafter. To estimate beta cell function the insulin secretion index was calculated (see ESM Insulin secretion in cats) . After IVGTT, cats were killed and the pancreas was excised.
Insulin content of the pancreas
Pancreatic insulin content was measured in a tissue specimen from the pancreas left lobe and 24 h later homogenised in 0.18 mol/l HCl in 70% ethanol. Results were normalised to the sample’s protein content measured by spectrophotometry (ND-1000, NanoDrop, Wilmington, DE, USA) at 280 nm.
Right and left lobes, and the body of the pancreas were examined. One aliquot of each was formalin-fixed for 24 h and paraffin-embedded. An additional aliquot collected from the left lobe was snap-frozen in liquid nitrogen and stored at −80°C.
Paraffin sections were stained with haematoxylin and eosin (H&E) for histomorphometry. For islet amyloid deposits, additional sections were stained with thioflavine-T and Congo red. For glycogen, frozen tissue sections were stained with periodic acid–Schiff’s reagent (PAS) with or without diastase digestion (1% α-amylase from Bacillus subtilis; Sigma-Aldrich, Buchs, Switzerland). A frozen section was stained with Oil Red O to detect islet accumulation of lipids.
Immunohistochemical staining procedures for insulin, amylin, NK6 homeobox 1 (NKX6.1), glucagon, cleaved caspase-3, antigen identified by monoclonal antibody Ki-67 (Ki67), proliferating nuclear antigen (PCNA) and myeloperoxidase (for neutrophils) were performed as described in ESM Histochemistry methods.
Insulin-, amylin- and glucagon-positive areas relative to the pancreatic area were calculated using ImageJ software (http://rsb.info.nih.gov/ij/) on 15 pictures collected from each cat at ×4 magnification. Large vessels, ducts and interlobular tissue were excluded from measurements.
The number of beta cells relative to the pancreatic area was calculated by counting NKX6.1-positive nuclei in sections that were double-stained with insulin and NKX6.1. Twenty pictures were collected from each cat at ×10 magnification using Image J.
Five-thousand cells were used to calculate the number of apoptotic islet cells, cleaved caspase-3-positive beta cells, Ki67-positive or PCNA-positive beta cells, and islet myeloperoxidase-immunostained neutrophils. Counts were not performed in small islets with the longer axis below 50 μm. Neutrophils were also counted in the exocrine pancreas in 50 microscopic fields at ×40 magnification, excluding those in large vessels.
Isolation of islets, RNA isolation, reverse transcription and quantitative analysis of mRNA
Islet isolation was performed as described by E. Zini and C. E. Reusch (unpublished results). Total RNA from islets was extracted using the RNeasy Mini Kit (Qiagen, Basel, Switzerland). Genomic DNA contamination was eliminated by including DNase treatments (DNase-Free DNase Set, Qiagen). RNA quality was assessed by gel electrophoresis. The complementary DNA (cDNA) was obtained from 1 μg samples of RNA (Omniscript RT-Kit, Qiagen) in the presence of 13 U of RNasin (Promega, Madison, WI, USA).
Islet cDNA was subjected to quantitative real-time PCR using feline-specific oligonucleotides established for insulin, Fas receptor, the caspase-8 inhibitor Flip (also known as Cflar), the chemokines Il8 and Mcp-1 (also known as Ccl2) and the housekeeping gene cyclophilin A (ESM Table 2). Published sets of primers and probes for feline Il6, Il1b, Tnfα (also known as Tnf) and the control gene Gapdh were also used (ESM Table 2) .
For SYBR-Green and probe-based assays, PCR were performed (E. Zini and C. E. Reusch, unpublished results). The cDNA samples were run in triplicate and transcripts were quantified using the relative standard curve method. Gene expression was normalised to cyclophilin A and Gapdh.
Data are expressed as mean ± SE. Data were analysed using GraphPad Prism 4.0 (GraphPad, San Diego, CA, USA). Results in glucose-, lipid- or saline-infused cats were compared using the Kruskal–Wallis and Dunn’s tests. Experimentally induced stress was assessed by comparing the results of group III (saline) and group IV (no infusion) using a Mann–Whitney test. Significance was set at p < 0.05. Pathology slides were evaluated in a blinded manner by two investigators (E. Zini, A. Perren or R.S. Heller).