Hepatic SILAC proteomic data from PANDER transgenic model.

This article contains raw and processed data related to research published in "Quantitative Proteomic Profiling Reveals Hepatic Lipogenesis and Liver X Receptor Activation in the PANDER Transgenic Model" (M.G. Athanason, W.A. Ratliff, D. Chaput, C.B. MarElia, M.N. Kuehl, S.M., Jr. Stevens, B.R. Burkhardt (2016)) [1], and was generated by "spike-in" SILAC-based proteomic analysis of livers obtained from the PANcreatic-Derived factor (PANDER) transgenic mouse (PANTG) under various metabolic conditions [1]. The mass spectrometry output of the PANTG and wild-type B6SJLF mice liver tissue and resulting proteome search from MaxQuant 1.2.2.5 employing the Andromeda search algorithm against the UniprotKB reference database for Mus musculus has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with dataset identifiers PRIDE: PXD004171 and doi:10.6019/PXD004171. Protein ratio values representing PANTG/wild-type obtained by MaxQuant analysis were input into the Perseus processing suite to determine statistical significance using the Significance A outlier test (p<0.05). Differentially expressed proteins using this approach were input into Ingenuity Pathway Analysis to determined altered pathways and upstream regulators that were altered in PANTG mice.

Quantitative proteomic profiling reveals hepatic lipogenesis and liver X receptor activation in the PANDER transgenic model.

PANcreatic-DERived factor (PANDER) is a member of a superfamily of FAM3 proteins modulating glycemic levels by metabolic regulation of the liver and pancreas. The precise PANDER-induced hepatic signaling mechanism is still being elucidated and has been very complex due to the pleiotropic nature of this novel hormone. Our PANDER transgenic (PANTG) mouse displays a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased, a phenomena observed in type 2 diabetes. To examine the complex PANDER-induced mechanism of SHIR, we utilized quantitative mass spectrometry-based proteomic analysis using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) to reveal the global hepatic proteome differences within the PANTG under the metabolic states of fasting, fed and insulin-stimulated conditions. Proteomic analysis identified lipid metabolism as one of the top cellular functions differentially altered in all metabolic states. Differentially expressed proteins within the PANTG having a lipid metabolic role included ACC, ACLY, CD36, CYP7A1, FASN and SCD1. Central to the differentially expressed proteins involved in lipid metabolism was the predicted activation of the liver X receptor (LXR) pathway. Western analysis validated the increased hepatic expression of LXRα along with LXR-directed targets such as FASN and CYP7A1 within the PANTG liver. Furthermore, recombinant PANDER was capable of inducing LXR promoter activity in-vitro as determined by luciferase reporter assays. Taken together, PANDER strongly impacts hepatic lipid metabolism across metabolic states and may induce a SHIR phenotype via the LXR pathway.

Hepatic nutrient and hormonal regulation of the PANcreatic-DERived factor (PANDER) promoter.

PANcreatic-DERived factor (PANDER, FAM3B) has been shown to regulate glycemic levels via interactions with both pancreatic islets and the liver. Although PANDER is predominantly expressed from the endocrine pancreas, recent work has provided sufficient evidence that the liver may also be an additional tissue source of PANDER production. At physiological levels, PANDER is capable of disrupting insulin signaling and promoting increased hepatic glucose production. As shown in some animal models, strong expression of PANDER, induced by viral delivery within the liver, induces hepatic steatosis. However, no studies to date have explicitly characterized the transcriptional regulation of PANDER from the liver. Therefore, our investigation elucidated the nutrient and hormonal regulation of the hepatic PANDER promoter. Initial RNA-ligated rapid amplification of cDNA ends identified a novel transcription start site (TSS) approximately 26 bp upstream of the PANDER translational start codon not previously revealed in pancreatic ß-cell lines. Western evaluation of various murine tissues demonstrated robust expression in the liver and brain. Promoter analysis identified strong tissue-specific activity of the PANDER promoter in both human and murine liver-derived cell lines. The minimal element responsible for maximal promoter activity within hepatic cell lines was located between -293 and -3 of the identified TSS. PANDER promoter activity was inhibited by both insulin and palmitate, whereas glucose strongly increased expression. The minimal element was responsible for maximal glucose-responsive and basal activity. Co-transfection reporter assays, chromatin-immunoprecipitation (ChIP) and site-directed mutagenesis revealed that the carbohydrate-responsive element binding protein (ChREBP) increased PANDER promoter activity and interacted with the PANDER promoter. E-box 3 was shown to be critical for basal and glucose responsive expression. In summary, in-vitro and in-vivo glucose is a potent stimulator of the PANDER promoter within the liver and this response may be facilitated by ChREBP.

PANDER transgenic mice display fasting hyperglycemia and hepatic insulin resistance.

PANcreatic-DERived factor (PANDER, FAM3B) is a novel protein that is highly expressed within the endocrine pancreas and to a lesser degree in other tissues. Under glucose stimulation, PANDER is co-secreted with insulin from the ß-cell. Despite prior creation and characterization of acute hepatic PANDER animal models, the physiologic function remains to be elucidated from pancreas-secreted PANDER. To determine this, in this study, a transgenic mouse exclusively overexpressing PANDER from the endocrine pancreas was generated. PANDER was selectively expressed by the pancreatic-duodenal homeobox-1 (PDX1) promoter. The PANDER transgenic (PANTG) mice were metabolically and proteomically characterized to evaluate effects on glucose homeostasis, insulin sensitivity, and lipid metabolism. Fasting glucose, insulin and C-peptide levels were elevated in the PANTG compared with matched WT mice. Younger PANTG mice also displayed glucose intolerance in the absence of peripheral insulin sensitivity. Hyperinsulinemic-euglycemic clamp studies revealed that hepatic glucose production and insulin resistance were significantly increased in the PANTG with no difference in either glucose infusion rate or rate of disappearance. Fasting glucagon, corticosterones, resistin and leptin levels were also similar between PANTG and WT. Stable isotope labeling of amino acids in cell culture revealed increased gluconeogenic and lipogenic proteomic profiles within the liver of the PANTG with phosphoenol-pyruvate carboxykinase demonstrating a 3.5-fold increase in expression. This was matched with increased hepatic triglyceride content and decreased p-AMPK and p-acetyl coenzyme A carboxylase-1 signaling in the PANTG. Overall, our findings support a role of pancreatic ß-cell-secreted PANDER in the regulation of hepatic insulin and lipogenenic signaling with subsequent impact on overall glycemia.

Enhanced glucose tolerance in pancreatic-derived factor (PANDER) knockout C57BL/6 mice.

Pancreatic-derived factor (PANDER; also known as FAM3B) is a uniquely structured protein strongly expressed within and secreted from the endocrine pancreas. PANDER has been hypothesized to regulate fasting and fed glucose homeostasis, hepatic lipogenesis and insulin signaling, and to serve a potential role in the onset or progression of type 2 diabetes (T2D). Despite having potentially pivotal pleiotropic roles in glycemic regulation and T2D, there has been limited generation of stable animal models for the investigation of PANDER function, and there are no models on well-established genetic murine backgrounds for T2D. Our aim was to generate an enhanced murine model to further elucidate the biological function of PANDER. Therefore, a pure-bred PANDER knockout C57BL/6 (PANKO-C57) model was created and phenotypically characterized with respect to glycemic regulation and hepatic insulin signaling. The PANKO-C57 model exhibited an enhanced metabolic phenotype, particularly with regard to enhanced glucose tolerance. Male PANKO-C57 mice displayed decreased fasting plasma insulin and C-peptide levels, whereas leptin levels were increased as compared with matched C57BL/6J wild-type mice. Despite similar peripheral insulin sensitivity between both groups, hepatic insulin signaling was significantly increased during fasting conditions, as demonstrated by increased phosphorylation of hepatic PKB/Akt and AMPK, along with mature SREBP-1 expression. Insulin stimulation of PANKO-C57 mice resulted in increased hepatic triglyceride and glycogen content as compared with wild-type C57BL/6 mice. In summary, the PANKO-C57 mouse represents a suitable model for the investigation of PANDER in multiple metabolic states and provides an additional tool to elucidate the biological function and potential role in T2D.