Glucagon

One of the most important features of an evidence map is the cataloging of the large number and variety of outcomes reported in the published literature. This step typically occurs after data extraction since the scope of an evidence-map database is large. Thus, it is often difficult to pre-define all outcome categories of interest. The research team worked with the stakeholder panel to classify outcomes into clinically and biologically meaningful outcome categories that could be used in evidence-map analyses. The research team recorded outcomes reported in each publication and took the first attempt in identifying clinically and biologically relevant groups. Standardized coding was then developed for each outcome category. Feedback was sought from the stakeholder panel, and the outcome categories and coding were modified based on the final consensus of the stakeholder panel. Table 2 shows the final list of outcomes for each outcome category that are reported in the studies included in the LCS evidence-map database. Specifically, outcomes related to appetite or satiety ratings such as hunger score and desire to eat were often rated by a visual analog scale (VAS) and were classified under the ‘Appetite’ category. Outcomes focused on neurological measurements and sensing signals by the brain were classified under the ‘Energy Sensing’ category. Body weight, body composition and changes in weight-related outcomes were classified under the ‘Body Weight or Composition’ category. The ‘Dietary Intake’ category included outcomes such as energy intake, dietary intake, food intake and carbohydrate intake, and finally the ‘Glycemic’ category included glucose, insulin and gastric hormones. Our stakeholder panel did not identify additional outcomes that were not reported in the literature. Both outcome categories and full outcome lists were included in the evidence-map database, which can be used in future analyses with current or new outcome category coding.Table 2

Outcomes of interest by outcome groups in the LCS evidence-map database

Outcome groups	Outcomes of interest
Appetite	Appetite ratings using a visual analog scale (VAS), hunger, desire to eat, fullness, prospective consumption, thirst, motivational and behavioral factors reported through questionnaire
Energy sensing by brain	Neurological measurements (fMRI, EEG), sensory rating (sweetness, intensity, pleasantness, sensory specific satiation), taste, perception and preference, taste reaction time
Body weight or body composition	Body weight, body composition, BMI, waist circumferences, weight or BMI changes
Dietary intake	Energy intake, dietary intake, food intake, carbohydrate intake, sugar intake, salt intake, water intake
Glycemic	Glucose, Hemoglobin A1c (HbA1c), insulin concentration, insulin sensitivity, hypoglycemia, glucagon, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), peptide tyrosine tyrosine (PYY), cholecystokinin (CCK), enterostatin, ghrelin, leptin, somatostatin, oxyntomodulin

For vibratome sections, dissected tissues were fixed in 4% PFA, embedded in 4% agarose gel and sectioned at 80 µm on a Leica VT1000S vibratome. All company names and catalog numbers of primary and secondary antibodies, and their dilutions used in this study, are in Additional file 1: Table S3. The nuclei were counterstained with Hoechst 33342. Image acquisition was completed using the Zeiss LSM 880 NLO scanning confocal microscope, with ZEN lite software. The number of glucagon (GCG) and insulin (INS) expressing cells, and Ki67⁺ cells were counted in one vibratome section of Isl1CKO and control embryos or mice (n = 5 pancreases per genotype and for each age) with the largest pancreatic footprint per individual using the Cell Counter plugin of Image J (NIH). The number of INS and pHH3 expressing cells were counted in vibratome sections of Isl1CKO and control pancreases at E17.5 (n = 3 pancreases per genotype) and P0 (n = 4 pancreases per genotype). The number of NEUROD1⁺/ISL1⁺ cells at E10.5 were counted in the whole mount of the dorsal pancreas (n = 5 pancreases per genotype) using the Cell Counter plugin of Image J (NIH). For the evaluation of glucagon delaminating cells at E11.5 were quantified using the thresholding tool Image J (NIH) and expressed as a percentage of the total GCG⁺ area to PDX1⁺ area (n = 9 control pancreases; n = 8 Isl1CKO pancreases).

Fig. S1 shows that Prdm16 is transiently expressed in the premalignant lesions. Fig. S2 shows that Prdm16KO mice display normal insulin and glucagon expression and distribution as well as normal blood glucose levels. Fig. S3 provides additional data demonstrating that inactivation of Prdm16 accelerates KrasG12D-driven PDAC. Fig. S4 displays that Prdm16 is required for IPMN-to-PDAC progression and that Prdm16 deletion led to the accumulation of cells with high vimentin expression. In addition, Fig. S4 shows the effects of deleting PRDM16 on the proliferation of PANC-1 and BxPC-3 cell lines. Fig. S5 further expands upon the notion that concomitant inactivation of Prdm16 and Smad4 mimics the phenotype of complete TGF-β signaling inactivation through TβRII ablation.