Aquaporin 7 is a beta-cell protein and regulator of intraislet glycerol content and glycerol kinase activity, beta-cell mass, and insulin production and secretion

Abstract

To investigate if intracellular glycerol content plays a role in the regulation of insulin secretion in pancreatic beta cells, we studied the expression of the glycerol channels, or aquaglyceroporins, encoded by the aquaporin 3 (Aqp3), Aqp7, and Aqp9 genes in mouse islets. We found expression of Aqp7 only, not that of Aqp3 or Aqp9, in the endocrine pancreas at both the mRNA (by reverse transcription-PCR) and protein (by immunohistochemistry) levels. Immunohistochemistry revealed a complete overlap between insulin and Aqp7 immunostaining in the pancreatic islet. Inactivation of Aqp7 by gene targeting produced viable and healthy mice. Aqp7-/- mice harbored an increased intraislet glycerol concentration with a concomitant increase of the glycerol kinase transcript level and enzyme activity. The islet triglyceride content in the Aqp7-/- mice was also increased compared to that in the Aqp7+/+ mice. Interestingly, Aqp7-/- mice displayed reduced beta-cell mass and insulin content but increased insulin-1 and insulin-2 mRNAs. The reduction of beta-cell mass in Aqp7-/- mice can be explained at least in part by a reduction in cell proliferation through protein kinase C and the c-myc cascade, with a reduction in the transcript levels of these two genes. Concomitantly, there was a decreased rate of apoptosis, as reflected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase 3 and Bax expression in Aqp7-/- mice. Compared with Aqp7+/+ islets, islets isolated from Aqp7-/- mice secreted insulin at a higher rate under basal low-glucose conditions and on exposure to a high (450 mg/dl) glucose concentration. Aqp7-/- mice exhibited normal fasting blood glucose levels but elevated blood insulin levels. Their plasma glucose response to an intraperitoneal (i.p.) glucose tolerance test was normal, but their plasma insulin concentrations were higher than those of wild-type mice during the 2-h test. An i.p. insulin tolerance test showed similar plasma glucose lowering in Aqp7-/- and Aqp7+/+ mice, with no evidence of insulin resistance. In conclusion, we found that pancreatic beta cells express AQP7, which appears to be a key regulator of intraislet glycerol content as well as insulin production and secretion.

FIG. 1.

Gene-targeting strategy for the inactivation of Aqp7 in mice. (A) Restriction maps of the Aqp7 gene (top), the targeting vector (middle), and the targeted recombinant allele (bottom) are shown. The locations of PCR primers 1, 2, and 3 used for genotyping are shown. (B) PCR screening of Aqp7^+/+, Aqp7^+/−, and Aqp7^−/− mice. The PCR product for the wild-type allele is 430 bp, and that for the mutant allele is 208 bp.

FIG. 2.

AQP7 expression in mouse pancreatic islets. (A) RT-PCR analysis of three aquaglyceroporin genes, Aqp3, Aqp7, and Aqp9, in isolated islet, kidney, testis, and liver tissues from wild-type mice. Gapdh was used as an internal control. (B) Confocal laser microscopic analysis of the immunolocalization of AQP3, -7, and -9 in the pancreas, kidneys, liver, and testes (positive control of AQP7). (C and D) Confocal laser microscopic analysis of immunostaining images of AQP7 and 4 islet hormones in the pancreatic islets from wild-type (Aqp7^+/+) (C) and Aqp7^−/− (D) mice. AQP7 expression completely overlaps with that of insulin (Ins) but not that of glucagon (Gcg), somatostatin (Sst), or pancreatic polypeptide (PP). Bars = 100 μm, unless marked otherwise.

FIG. 3.

Aqp7 inactivation in Aqp7^−/− tissues. (A) RT-PCR of Aqp3, -7, and -9 transcripts, using RNAs isolated from the whole pancreas and isolated pancreatic islets from Aqp7^+/+ and Aqp7^−/− mice and from WT β cell, a mouse β-cell line (B47). Gapdh was used as an internal control. (B) AQP8 is expressed in β cells of pancreatic islets.

FIG. 4.

Pancreas and pancreatic islet masses in Aqp7^−/− mice and Aqp7^+/+ mice. (A and B) Whole pancreas wet weights for males (A) and females (B). Values are means ± SD (n = 4). (C) Total islet area. (D) Number of islets in whole pancreas (n = 4). (E and F) Density distributions of TUNEL (E)- and PCNA (F)-positive cells in islets. Values are means ± SD (data are for five sections from each of three animals).

FIG. 5.

Regulation of Aqp7 and c-myc mRNA expression by PKC activator TPA and its inhibitor, H7, in β-cell line B47. Quantitative RT-PCR was performed for Aqp7 (A) and c-myc (B) mRNA expression in B47 β cells in response to 6 h of treatment with 0.1 μM of TPA, 100 μM of H7, or both. Values shown are means ± SD (n = 3). **, P < 0.01; *, P < 0.05.

FIG. 6.

Rate of lipolysis in isolated islets (n = 3). Batches of 100 islets were isolated from Aqp7^−/− and Aqp7^+/+ mice and washed in KRBH buffer, and glycerol (A) and free fatty acid (B) release from these islets over a period of 2 h under basal or forskolin (FK; 2.5 μM)-stimulated conditions was determined using enzymatic kits.

FIG. 7.

GYK activity in pancreases from Aqp7^−/− and Aqp7^+/+ mice. GYK enzyme activity (detailed in Materials and Methods) was measured using protein extracts from total pancreas (A; n = 4) or isolated islets (B; n = 6) of Aqp7^−/− and Aqp7^+/+ mice that were fasted for 18 h. The data are percentages of the control value.

FIG. 8.

Metabolism of islets isolated from Aqp7^−/− and Aqp7^+/+ mice. Insulin secretion (A), CO₂ production (B), and glucose uptake (C) were measured from isolated islets incubated in low (100 mg/dl)- or high (450 mg/dl)-glucose medium. Black bars, islets from Aqp7^−/− mice; white bars, islets from Aqp7^+/+ mice. Values are means ± SD (n = 3).

FIG. 9.

Results of i.p. glucose tolerance (GTT) and insulin tolerance (ITT) tests. Aqp7^−/− and Aqp7^+/+ mice (n = 4) were injected (i.p.) with 1.5 g/kg of body weight of glucose, and plasma glucose (A) and insulin (B) were measured at various times. Similarly, insulin was injected (i.p.) into Aqp7^−/− and Aqp7^+/+ mice (n = 4) at 1 U/kg body weight, and plasma glucose (C) was measured at various times. ⧫, Aqp7^−/− mice; ▪, Aqp7^+/+ mice. Values are means ± SD (n = 4). *, P < 0.05.