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. 2004 Jun 1;101(22):8319-24.
doi: 10.1073/pnas.0307737101. Epub 2004 May 17.

Involvement of Per-Arnt-Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose

Gabriela da Silva Xavier  1 , Jared Rutter, Guy A Rutter
Affiliations

Involvement of Per-Arnt-Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose

Gabriela da Silva Xavier et al. Proc Natl Acad Sci U S A. .

Abstract

Per-Arnt-Sim (PAS) domain-containing kinases are common in prokaryotes, but a mammalian counterpart has only recently been described. Although the PAS domain of the mammalian PAS kinase (PASK) is closely related to the bacterial oxygen sensor FixL, it is unclear whether PASK activity is changed in mammalian cells in response to nutrients and might therefore contribute to signal transduction by these or other stimuli. Here, we show that elevated glucose concentrations rapidly increase PASK activity in pancreatic islet beta cells, an event followed by the accumulation of both PASK mRNA and protein. Demonstrating a physiological role for PASK activation, comicroinjection into clonal beta cells of cDNA encoding wild-type PASK, or PASK protein itself, mimics the induction of preproinsulin promoter activity by high glucose concentrations. Conversely, anti-PASK antibodies block promoter activation by the sugar, and the silencing of PASK expression by RNA interference suppresses the up-regulation by glucose of preproinsulin and pancreatic duodenum homeobox 1 gene expression, without affecting glucose-induced changes in the levels of mRNAs encoding glucokinase or uncoupling protein 2. We conclude that PASK is an important metabolic sensor in nutrient-sensitive mammalian cells and plays an unexpected role in the regulation of key genes involved in maintaining the differentiated phenotype of pancreatic beta cells.

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Figures

Fig. 1.
Fig. 1.
Glucose regulates PASK activity and gene expression but not its intracellular localization. (A) MIN6 cells were transfected with pcDNA3 or plasmids pPASK.WT or pPASK.KD, cultured for 16 h in 3 mM glucose (Materials and Methods), then incubated in modified Krebs–Ringer medium for 1 h at the glucose and insulin concentrations indicated. The activities of immunoprecipitated PASK or AMPK were assessed by SAMS peptide assay and are given as means ± SEM of three separate experiments. (B) MIN6 cells were transfected with pPASK.WT, then incubated for 1 h as in A, before immunocytochemical analysis (see Materials and Methods). Scale bar, 10 μm. (C) Untransfected MIN6 cells were cultured for 16 h at 3 mM glucose and then for 6 h at 3 or 30 mM glucose as indicated, before RNA extraction and RT-PCR analysis (30 cycles; see Materials and Methods). The fold increase in PASK mRNA was 14.8 ± 0.4 (mean of three separate experiments involving 30 PCR cycles and 200 ng of template RNA). (D) Immunoblot analysis of endogenous PASK protein. Cells or islets were cultured for 24 h at the indicated glucose concentrations before protein extraction and analysis. PASK migrates as a 142-kDa band. NS, nonspecific. Results in B–D are representative of four independent experiments.
Fig. 2.
Fig. 2.
Regulation of preproinsulin promoter activity by glucose and PASK. MIN6 (A, B, and D) or cultured islet cells (C) were microinjected with plasmid pINS.LucFF and pCMV.RL (see Materials and Methods) plus (A) pcDNA3 (Con) pcDNA3PASK.WT or pcDNA3.PASK.KD, (B) control IgG or anti-PASK antibody (1.0 mg/ml-1), or (C and D) BSA or wild-type PASK protein (1.0 mg/ml-1) before incubation for 6 h followed by imaging of firefly and R. reniformis luciferases (see Materials and Methods). Data are from three experiments on a total of 30–120 single cells per condition. (Bars = 20 μm.)
Fig. 3.
Fig. 3.
Regulation of wild-type and mutated preproinsulin and PDX-1 promoters by PASK. MIN6 cells were injected with unmodified (A) preproinsulin or (B) PDX-1 promoter constructs, with purified wild-type PASK protein or BSA (-), as indicated. Cells were incubated in the presence or absence of diazoxide (DAO, 10 μM), as given. C is as A, but with wild-type or mutant preproinsulin promoters (34): mA3, -220CTCTCCTGACC; mA2, -138CGGACCTTGCAC; mE1, -114GCCCGCTG. D as in C but with wild-type PDX-1 promoter or mutants: Foxa2 binding (area I, region II, a1, -2630GTTTTTGGGGTATTA or a2, -2630GTTTTTGTTGGGGTATCC) (47), PDX-1-binding (area 1, region 1, -2648TATCCTTGG) (48) (mutated residues underlined). See Fig. 2 for other details.
Fig. 4.
Fig. 4.
Involvement of PASK activation in the regulation of endogenous MIN6 β cell genes by glucose. (A and B) Cells were transfected for 72 h with the indicated concentrations of scrambled or PASK siRNA duplexes before (A) measurement of PASK mRNA by RT-PCR or (B) PASK protein after SDS gel electrophoresis and immunoblotting. Quantification by RT-PCR of preproinsulin (C), PDX-1 (D), glucokinase (E), and UCP2 mRNA (F) in MIN6 cells transfected with control (scrambled) siRNA or anti-PASK siRNA (10 pg/ml-1) as indicated. In C–F, cells were cultured at 3 mM glucose for 16 h before culture for 6 h at 3 or 30 mM glucose as indicated. Results are representative of (A and B) or are the means of (C–F) data from three experiments.
Fig. 5.
Fig. 5.
Changes in PASK activity have no effect on the acute stimulation of peptide secretion by glucose. (A) MIN6 cells were transfected with plasmids encoding hGH (1.0 μg/ml-1) and empty vector, pPASK.WT, or pPASK.KD. Transfected cells were cultured for 16 h at 3 mM glucose, before incubation for 20 min in modified modified Krebs–Ringer medium and measurement of released and total cellular hGH by ELISA (see Materials and Methods). (B) MIN6 cells were microinjected with plasmids encoding mitochondrially targeted luciferase and control (black trace) or PASK.WT (light gray) or PASK.KD (dark gray). Injected cells were cultured for 16 h in normal medium, then for 16 h at 3 mM glucose before transfer to modified Krebs–Ringer medium initially containing 3 mM glucose, and imaging; 30 mM glucose was added as indicated. Data are combined from three separate experiments. (C) Preproinsulin promoter activity was assessed in 30–120 single cells (three separate experiments) after microinjection of reporter constructs (Fig. 2) plus either empty vector (50 μg/ml-1) or pPASK.KD, before incubation for 6 h at the indicated concentrations of glucose and in the absence or presence of 20 nM insulin.
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