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

Abstract

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.

Keywords: FAM3B; Glucose tolerance; Glycemic regulation; Hepatic insulin signaling; Knockout model; Pancreatic-derived factor.

Fig. 1.

Long- and short-term fasting glycemic measurements of PANKO-C57 mice. Mice of both genders were 4 months of age. (A) Long-term fasting (overnight, 16 hours) blood glucose measurements of male PANKO-C57 (KO) and WT mice (n=12–15). (B) Short-term fasting (4 hours) blood glucose measurements of males (n=8–11). Values are expressed as means±s.e.m. (C) Long-term fasting blood glucose measurements of female PANKO-C57 and WT mice (n=7–12). (D) Short-term fasting (4 hours) blood glucose measurements of females (n=6–10). *P<0.05, **P<0.01, Student’s t-test.

Fig. 2.

Enhanced glucose tolerance in male PANKO-C57 mice. All mice were 4 months of age. (A) Intraperitoneal (i.p.) glucose tolerance tests (GTTs) were performed following overnight fast (~16 hours) on male PANKO-C57 (KO) and WT mice through injection of glucose at 2 g/kg of body weight and measurement of plasma glucose concentration at the indicated time points (n=8–12). (B) Intraperitoneal insulin tolerance tests (ITTs) were performed on male PANKO-C57 and WT mice following a 4-hour fast by injection of insulin at 1 unit/kg of body weight and subsequent measurement of plasma glucose concentration at all indicated time points. Results are presented as the percentage of the baseline glucose concentration measured at time point 0 (n=8–11). (C) GTTs performed on female PANKO-C57 and WT mice as described above (n=7–12). (D) ITTs performed on female PANKO-C57 and WT mice as described above (n=7–11). Values are expressed as the mean±s.e.m. *P<0.05, **P<0.01 as determined by using two-way ANOVA.

Fig. 3.

Increased body weight of male and female PANKO-C57 mice. Body weight measurements were recorded at similar times from 8 to 23 weeks of age following an overnight fast. (A) Male PANKO-C57 (KO) and WT mice weight over time (n=18–27). (B) Female PANKO-C57 and WT mice weight over time (n=13–22). Values are expressed as the means±s.e.m. *P<0.05, **P<0.01, ***P<0.001 as determined by using two-way ANOVA.

Fig. 4.

Hormonal evaluation of male PANKO-C57 during fasting. The plasma levels of (A) insulin, (B) glucagon and (C) leptin were measured during fasting at 2 and 5 months of age in male PANKO-C57 (KO) and WT mice using the Mouse Metabolic Hormone Panel (Millipore) as measured by the MAGPIX^® Luminex system (n=4–6). (D) Overnight fasting levels of C-peptide from mice aged 2 months using the MAGPIX^® Luminex system as above (n=4). (E) Corticosterone levels were measured from plasma collected during fasting conditions at 2 and 5 months of age using the Corticosterone EIA Kit (Enzo Life Sciences) (n=3). (F) Plasma insulin levels during the course of GTTs (n=3–5). Values are expressed as means±s.e.m. *P<0.05, **P<0.01, ***P<0.001 as determined by using Student’s t-test for A–E and two-way ANOVA in F. IP, intraperitoneal.

Fig. 5.

Hepatic glycogen, triglyceride and gluconeogenic content and expression in PANKO-C57. (A) Hepatic glycogen content (in μg/μl of tissue lysate) measurement of male PANKO-C57 (KO) and WT mice at 4–5 months of age following insulin stimulation (2 U/kg of body weight) or an overnight fast using the Glycogen Assay Kit (Abcam^®) (n=3 per group). (B) Hepatic triglyceride content evaluation of 4-to 5-month-old male PANKO-C57 and WT mice as described in A, using 100 mg of liver tissue and the Triglyceride Quantification Kit (Abcam^®) (n=3). (C) Serum triglycerides from serum after fasting of PANKO-C57 and WT male mice were analyzed longitudinally at 2 and 5 months of age using the Triglyceride Quantification Kit (Abcam^®) (n=5). Values are expressed as the mean+s.e.m. *P<0.05, **P<0.01 as determined by using Student’s t-test.

Fig. 6.

Western blot analysis of PANKO-C57 hepatic signaling. (A) Western blot analysis for the protein levels of phosphorylated PI3K (p-PI3K; at residue Tyr 508), total and phosphorylated Akt (p-Akt; at residue Thr 308), mature and precursor (pre) SREBP-1, total and phosphorylated AMPKα (p-AMPKα; at residue Thr 172) in lysates from the livers of male PANKO-C57 and WT mice that had been subject to overnight fast. GAPDH served as loading control. The first three lanes correspond to PANKO-C57 mice and the final three lanes correspond to age- and gender-matched C57BL/6J WT mice. *Statistical significance of P<0.05 as determined by measuring using ImageJ as detailed in B–E. (B) Densitometry analysis of hepatic protein levels of phosphorylated PI3K, (C) Akt, (E) AMPK and (D) levels of mature SREBP-1 (mSREBP-1). The levels were determined and normalized to the total protein levels of the respective protein followed by normalization of the total protein to GAPDH (n=3). Values are expressed as the mean+s.e.m. *P<0.05 as determined by using unpaired Student’s t-test.