PASK gene expression is regulated by glucose in human pancreatic islets of Langerhans and is lowered in type 2 diabetes
We have previously shown that Pask mRNA and protein levels are increased by high glucose in rat islets and clonal MIN6 beta cells . Here, we first determined whether islet PASK expression may also be regulated by glucose or by diabetes in humans. Measured in human islets of Langerhans by quantitative PCR, PASK mRNA was elevated after 24 h culture at 11 mmol/l glucose (2.7 ± 0.2-fold vs culture at 3 mmol/l glucose [means ± SE]; Fig. 1a). Measured after culture at 11 mmol/l glucose, PASK gene expression was significantly lower in islets from patients with type 2 diabetes than in control islets (Fig. 1b).
mRNA expression data obtained using whole islets may be confounded by variable degrees of contamination with other pancreatic cell types, as well as by other factors. Therefore, we sought to further support the above results by using laser capture microdissection and array analysis to quantify mRNA levels selectively in beta cell-enriched samples . A clear tendency for decreased PASK expression in islets in type 2 diabetic pancreases vs controls was also observed using this approach (ESM Fig. 1). In contrast, consistent changes in levels of the AMPK-alpha1 and -alpha2 subunit (encoded by PRKAA1 and PRKAA2, respectively) were not observed with array data on samples obtained by laser capture microdissection. However, with quantitative PCR done on the same samples with enough RNA (10 vs 10), a small but statistically significant decrease in PRKAA1 expression was apparent (18%; p < 0.019), using RPL32 as an internal control.
Pask is expressed in murine pancreatic beta and alpha cells
In the mouse, Pask mRNA has previously been shown to be most abundant in testes as well as in haemopoietic tissues including thymus and spleen . Levels in other tissues, including islets, are much lower  (G. A. Rutter and G. Sun, unpublished observations). Indeed, the expression of Pask in adult mouse islets and its induction by glucose have recently been questioned, since expression in these cells of the lacZ gene, expressed in mice null for Pask alleles under the Pask promoter, was barely detectable by beta-Gal staining . By contrast, we have previously detected Pask mRNA in mouse islets and MIN6 cells , data recently confirmed by others . Examined here in islets from mice on a mixed C57BL/6/sv129 background cultured at 11 mmol/l glucose, massive parallel sequencing (RNAseq) confirmed the presence of Pask mRNA at low but detectable levels, lying in the lower 30th percentile of all mRNAs at approximately 0.2 ± 0.3% (n = 3 female mice) of β-actin mRNA levels (ESM Fig. 2).
To determine in which cell types Pask was expressed in mouse islets, we next compared the expression of Pask mRNA in highly purified primary mouse alpha and beta cells (Fig. 1c). These were obtained by fluorescence-activated cell sorting of transgenic mice selectively expressing the fluorescent protein, Venus, in the alpha cell [34, 35] and by real-time quantitative PCR analysis. This approach confirmed the presence of Pask mRNA in both cell types and indicated that levels in purified alpha cells (1.45 ± 0.46% of beta-actin mRNA) tended to be higher than in beta cells (0.54 ± 0.044%; n = 3 preparations; p = 0.07 by two-tailed Student’s t test).
Pask−/− mice display normal glucose tolerance but impaired plasma glucagon concentration
Given the greater expression of Pask in alpha than in beta cells and the dysregulation of glucagon secretion observed in type 2 diabetes, as reviewed by others , we next analysed the potential contribution of this enzyme to the regulation of glucagon secretion using Pask
−/− mice. At 8 weeks of age, Pask
−/− male (Fig. 2a, c, e) and female (Fig. 2b, d) mice displayed 25 ± 5% higher plasma glucose levels after 16 h of fasting than wild-type littermate control mice, but normal glucose tolerance after intraperitoneal injection of the sugar (Fig. 2a–d). Measured after fasting, plasma glucagon was significantly higher in Pask
−/− male mice than in littermate controls (91 ± 5.4 vs 58 ± 3 pg/ml; Fig. 2e).
Islets of Langerhans from Pask−/− mice display impaired glucose-regulated glucagon secretion
We next examined whether the elevated plasma glucagon concentration was due to dysregulation of alpha cell function. Supporting this view, the inhibition of glucagon secretion in response to elevated glucose was impaired in islets from Pask
−/− mice (Fig. 3a). Thus, Pask
−/− islets, in which Pask gene expression was undetectable (Fig. 3e), secreted 2.04 ± 0.2-fold more glucagon at inhibitory (10 mmol/l) glucose concentrations than did islets from wild-type littermates (Fig. 3a), whereas release at stimulatory (0.5 mmol/l) glucose was unaltered. Interestingly, a slight inhibitory effect on glucagon secretion in Pask
−/− islets was still observed at 10 mmol/l glucose (Fig. 3a). Although glucose-stimulated insulin release when normalised to total islet insulin content was not affected by Pask deletion (Fig. 3b), the total amount of insulin per islet was lower (0.38 ± 0.3-fold) in Pask
−/− than in wild-type islets (Fig. 3d), i.e. the amount of insulin release was compromised in Pask
−/− islets, in agreement with published data .
The expression of mouse preproinsulin 2 (Ins2) and Pdx1 genes was strongly impaired in Pask
−/− mouse islets (Fig. 3e), as previously reported [18, 20]. Moreover, the insulin content of whole Pask
−/− mouse pancreases was 1.98 ± 0.3-fold lower than that of wild-type pancreases (67 ± 15 vs 133 ± 12 ng/mg protein for Pask
−/− and wild-type mice, respectively; n = 3 mice for each genotype). While glucagon protein content in Pask
−/− islets (Fig. 3c) was unaffected, preproglucagon (Gcg) gene expression was slightly but significantly increased, as was Prkaa2 mRNA expression (Fig. 3e).
Glucagon release is activated in the absence of Pask in alpha-TC1-9 cells
Since the above experiments using intact islets did not allow ready discrimination between an action of PASK cell-autonomously in the alpha cell and an effect mediated by changes in the release of beta cell-derived factors, we next explored the role of the enzyme in the clonal alpha-TC1-9 cell line . Culture of alpha-TC1-9 cells in the presence of an siRNA against Pask led to near complete ablation of Pask gene expression and protein content (Fig. 4a). Glucagon release from Pask-deficient alpha-TC1-9 cells at inhibitory (10 mmol/l) glucose concentrations was comparable to that observed in control cells at stimulatory (0.5 mmol/l) glucose concentrations (Fig. 4b). By contrast, addition of extracellular insulin (20 nmol/l) led to inhibition of glucagon secretion in control and Pask-silenced alpha-TC1-9 cells, indicating (1) the presence of distinct glucose and insulin signalling pathways in the alpha cell and (2) that the insulin signalling pathway in Pask-silenced alpha-TC1-9 cells was intact (Fig. 4b). Total glucagon protein content, as assessed by radioimmunoassay, was not different between alpha-TC1-9 cells treated with control or anti-Pask siRNA (data not shown).
Glucagon release is inhibited by PASK overexpression in alpha-TC1-9 cells and human islets of Langerhans
The above findings indicated that PASK may be an inhibitor of glucagon release from alpha cells. To test this hypothesis, we next explored the impact of forced activation of the enzyme in these cells. Adenovirus-mediated overexpression of PASK in alpha-TC1-9 cells (Fig. 5a) led to inhibition of glucagon secretion at normally stimulatory (1 mmol/l) glucose concentrations (0.36 ± 0.1-fold vs control).
Similarly, overexpression of PASK in human islets of Langerhans (Fig. 5b) inhibited glucagon secretion at all glucose concentrations tested, while the stimulatory effects of KCl were largely maintained. Interestingly, there was still an apparent effect of glucose on glucagon secretion in human islets overexpressing PASK (Fig. 5b), consistent with a maintained effect on glucagon release mediated by beta cell-derived factors.
Silencing of Pask gene expression in alpha-TC1-9 cells and E13.5 rat pancreatic epithelial explants causes increased AMPK-alpha2 and glucagon gene expression
In an effort to identify the mechanism(s) through which PASK may regulate glucagon secretion, we measured the expression of a number of potential target genes. Pask gene expression was inhibited in alpha-TC1-9 cells treated with a siRNA against Pask (Fig. 6). In line with the above findings in Pask
−/− mouse islets (Fig. 3e), preproglucagon (Gcg) gene expression was increased substantially by Pask ablation in alpha-TC1-9 cells (7.4 ± 1.3-fold vs control; Fig. 6a), although total glucagon protein content was unaltered (Fig. 2c), consistent with the relatively slow turnover of mature glucagon. Prkaa2 (Fig. 6a), but not Prkaa1 (data not shown) gene expression was also increased (4.3 ± 1.5-fold vs control) by Pask silencing.
To determine whether the dysregulation of glucagon gene expression and release in Pask
−/− mice was due to a role of the enzyme in the development of pancreatic alpha and beta cells, we next examined the impact of Pask silencing in developing pancreatic epithelia. E13.5 rat pancreatic epithelial explants , in which Pask gene expression was silenced with an anti-Pask siRNA, developed endocrine buds to an extent indistinguishable from that observed in explants treated with a control (scrambled) siRNA (ESM Fig. 3). However, explants in which Pask was silenced displayed similar increases in Gcg and Prkaa2 gene expression (ESM Fig. 3) to those observed in alpha-TC1-9 cells (Fig. 6). Explants overexpressing Pask did not survive the 10 day culture period.