Pancreatic Hormones

Initially, crude pig-pancreatic lipase (PPL, 1 mg/mL) was dissolved in phosphate buffer (50 mM, pH 7) and then centrifuged at 12,000× g to remove insoluble components. To create an enzyme stock (0.1 mg/mL), the supernatant was diluted 10-fold with buffer. Prior research was used to evaluate the lipase inhibition potential [13 (link)]. A transparent 96-well microplate containing 100 µL of samples (ME, MEA, MH, SE, SEA, and SH) was combined with 20 µL of p-nitrophenyl butyrate (pNPB,10 mM in buffer) and incubated for 10 min at 37 °C. The outcome was compared to the reference drug orlistat, a well-known PPL inhibitor. At 405 nm, measurements were taken using a microplate reader. The unit of activity was calculated using the yield from the reaction rate of 1 mol of p-nitrophenol per minute at 37 °C. To measure the lipase inhibition activity, PPL activity was reduced in the test mixture by a specific amount. To ensure the validity of the study results, each sample was verified three times (in triplicate). The inhibitory data were obtained using the equation below.
$$\mathrm{Inhibition} \mathrm{of} \mathrm{Lipase} \mathrm{Activity} \left(\%\right)=100-\frac{\mathrm{B}-\mathrm{Bc}}{\mathrm{A}-\mathrm{Ac}}\times 100\%$$ A = activity without inhibitor; Ac—negative control without inhibitor; B—activity with inhibitor; Bc—negative control with inhibitor.

Animal experiments were conducted according to protocols approved by the Animal Care and Use Committee of the Institute of Molecular Genetics, Czech Academy of Sciences. All experiments were performed with littermates (males and females) cross-bred from two transgenic mouse lines: floxed Isl1 [Isl1^f/f; Isl1^tm2Sev, Stock No: 028501 Jackson Laboratory, [15 (link)]], and Neurod1-Cre [Tg(Neurod1-cre)1Able, Stock No: 028364 Jackson Laboratory, [32 (link)]]. Lines were maintained on C57BL/6 background. Neurod1-Cre mice do not have any detectable phenotype. Breeding scheme: Female mice Isl1^f/f were crossed with Isl1^f/+; Neurod1-Cre males, in which, Neurod1-cre knock-in allele was inherited paternally to minimize the potential influence of maternal genotype on the developing embryos. Isl1^f/+ or Isl1^f/f mice were used as the controls. The reporter tdTomato line (Ai14, B6.Cg-Gt(ROSA)26Sor^{tm14(CAG−tdTomato)Hze}, Stock No: 7914 Jackson Laboratory) was used. Genotyping was performed by PCR on tail DNA (Additional file 1: Table S1). Mice were kept under standard experimental conditions with a constant temperature (23–24 °C) and fed on soy-free feed (LASvendi, Germany). The females were housed individually during the gestation period and the litter size was recorded. Blood glucose levels were measured in animals by glucometer (COUNTOUR TS, Bayer); blood glucose levels maintained above 13.9 mmol/L are classified as diabetic. For total pancreatic insulin content, pancreases were excised, weighed, minced, and homogenized in acid–ethanol. A hormone concentration in extracts was measured by ELISA using Mouse Insulin ELISA kit (Mercodia, Sweden).

Hematoxylin abd eosin staining of each fixed 6‐μm pancreatic section was achieved as described previously [14 (link)]. After embedding in paraffin, each tissue fixed in 4% paraformaldehyde was cut into 6‐μm sections and applied to slides. For hematoxylin and eosin staining, tissues were subjected to rehydration, incubation in hematoxylin (2.5 min), rinsing with water, dipping in 0.5% HCl/70% ethanol (v/v), rinsing with water, immersion in 0.2% NaHCO₃, rinsing in water, dipping in 0.1% eosin for 20 s, rinsing briefly with water and, finally, dehydration and mounting.
For IHC evaluations, after embedding in paraffin, each tissue fixed in 4% paraformaldehyde was cut into 6‐μm sections and applied to slides. The sections were deparaffinized, rehydrated and then permeabilized in 0.2% Triton X‐100 for 5 min. Then, the permeabilized sections were blocked in PBS containing 10% inactivated fetal bovine serum for 90 min. Primary antibodies were provided in their required dilution in the same medium, applied on sections and then incubated overnight at 4 °C. After overnight incubation, each slide was incubated for 90 min with the appropriate secondary antibody after washing in PBS. Secondary antibodies were diluted in PBS containing 10% inactivated fetal bovine serum. Slides were viewed by fluorescence microscopy after washing in PBS and mounting with 4′,6‐diamidino‐2‐phenylindole [14 (link)].
The primary antibodies, including anti‐p‐extracellular signal‐regulated kinase (ERK)1/2 antibody sc‐81492 (dilution 1 : 1000), goat anti‐PAX4 antibody (ab101721) (dilution 1 : 1000), rabbit anti‐signal transducer and activator of transcription (STAT)5a antibody (ab30648) (dilution 1 : 1000), rabbit anti‐GAPDH antibody (ab181602) (dilution 1 : 1000), rabbit anti‐insulin antibody (ab63820) (dilution 1 : 500), mouse anti‐glucagon antibody sc‐514592 (dilution 1 : 500), mouse anti‐vimentin antibody sc‐6260 (dilution 1 : 500), rabbit recombinant anti‐Ki67 (ab197547) (dilution 1 : 500), rat anti‐CD3 antibody (ab11089) (dilution 1 : 500), mouse anti‐CD19 antibody (sc‐373897) (dilution 1 : 500), were used in western blotting and IHC assays. Also, all secondary antibodies were utilized at a concentration of 1 : 1000, including goat anti‐rat IgG H&L (ab6840), goat anti‐rabbit IgG H&L (ab6717), goat anti‐rabbit IgG H&L (ab72465), goat anti‐mouse IgG H&L (ab6785) and goat anti‐mouse IgG H&L (ab6787).

Based on the findings of a previous study [25] (link), lipase activity was determined by measuring the free fatty acid released after the enzyme – olive oil reaction. Briefly, 3 mL of olive oil was mixed with 2.5 mL of deionized water or the Miang extract, 1 mL of 100 mM sodium phosphate buffer pH 6.5, and 0.5 mL of Tween 80. The mixture was then vigorously mixed using a magnetic stirrer for 15 min to obtain an emulsion. The porcine pancreatic lipase (PPL) (100 U) was added to the emulsified mixture and incubated on a 150-rpm rotary shaker at 37 °C for 30 min. At the end of the incubation period, 3 mL of 95% ethanol was added prior to the mixture, which was then titrated with 50 mM NaOH using an automatic potentiometric titrator. The end point for the titration was set at pH 9.0. One unit of lipase activity was defined as the amount of enzyme that catalyzed the hydrolysis of triglycerides to release 1 microequivalent of fatty acids in 1 min under standard assay conditions. To determine the percentage of the inhibitory concentration of Miang against PPL, the PPL activity in the reaction was initially set up as 200 U. The PPL activity was assayed in the presence of various concentrations of the Miang extract as the inhibitor and compared to that without the presence of an inhibitor. The IC₅₀ value was determined from the regression curve and expressed as g/100 mL of Miang extract.