Aquaporin-9 (AQP9) is an aquaglyceroporin membrane channel shown biophysically to conduct water, glycerol, and other small solutes. Because the physiological role/s of AQP9 remain undefined and the expression sites of AQP9 remain incomplete and conflicting, we generated AQP9 knockout mice. In the absence of physiological stress, knockout mice did not display any visible behavioral or severe physical abnormalities. Immunohistochemical analyses using multiple antibodies revealed AQP9 specific labeling in hepatocytes, epididymis, vas deferens, and in epidermis of wild type mice, but a complete absence of labeling in AQP9 ؊/؊ mice. In brain, no detectable labeling was observed. Compared with control mice, plasma levels of glycerol and triglycerides were markedly increased in AQP9 ؊/؊ mice, whereas glucose, urea, free fatty acids, alkaline phosphatase, and cholesterol were not significantly different. Oral administration of glycerol to fasted mice resulted in an acute rise in blood glucose levels in both AQP9 ؊/؊ and AQP9 ؉/؊ mice, revealing no defect in utilization of exogenous glycerol as a gluconeogenic substrate and indicating a high gluconeogenic capacity in nonhepatic organs. Obese Lepr db /Lepr db AQP9 ؊/؊ and obese Lepr db /Lepr db AQP9 ؉/؊ mice showed similar body weight, whereas the glycerol levels in obese Lepr db /Lepr db AQP9 ؊/؊ mice were dramatically increased. Consistent with a role of AQP9 in hepatic uptake of glycerol, blood glucose levels were significantly reduced in Lepr db /Lepr db AQP9 ؊/؊ mice compared with Lepr db / Lepr db AQP9 ؉/؊ in response to 3 h of fasting. Thus, AQP9 is important for hepatic glycerol metabolism and may play a role in glycerol and glucose metabolism in diabetes mellitus.aquaglyceroporin ͉ diabetes mellitus ͉ leptin receptor

Enteroendocrine cells such as duodenal cholecystokinin (CCK cells) are generally thought to be confined to certain segments of the gastrointestinal (GI) tract and to store and release peptides derived from only a single peptide precursor. In the current study, however, transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the CCK promoter demonstrated a distribution pattern of CCK-eGFP positive cells that extended throughout the intestine. Quantitative PCR and liquid chromatography-mass spectrometry proteomic analyses of isolated, FACS-purified CCK-eGFP-positive cells demonstrated expression of not only CCK but also glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), peptide YY (PYY), neurotensin, and secretin, but not somatostatin. Immunohistochemistry confirmed this expression pattern. The broad coexpression phenomenon was observed both in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two peptide precursors in addition to CCK. The coexpression pattern was further confirmed through a cell ablation study based on expression of the human diphtheria toxin receptor under the control of the proglucagon promoter, in which activation of the receptor resulted in a marked reduction not only in GLP-1 cells, but also PYY, neurotensin, GIP, CCK, and secretin cells, whereas somatostatin cells were spared. Key elements of the coexpression pattern were confirmed by immunohistochemical double staining in human small intestine. It is concluded that a lineage of mature enteroendocrine cells have the ability to coexpress members of a group of functionally related peptides: CCK, secretin, GIP, GLP-1, PYY, and neurotensin, suggesting a potential therapeutic target for the treatment and prevention of diabetes and obesity.

Pregnancy-associated plasma protein A (PAPPA) is a metzincin superfamily metalloproteinase in the insulin-like growth factor (IGF) system. PAPPA increases IGF bioavailability and mitogenic effectiveness in vitro through regulated cleavage of IGF-binding protein 4 (IGFBP4). To determine its function in vivo, we generated PAPPA-null mice by gene targeting. Mice homozygous for targeted disruption of the PAPPA gene were viable but 60% the size of wild-type littermates at birth. The impact of the mutation was exerted during the early embryonic period prior to organogenesis, resulting in proportional dwarfism. PAPPA, IGF2 and IGFBP4 transcripts co-localized in wild-type embryos, and expression of IGF2 and IGFBP4 mRNA was not altered in PAPPA-deficient embryos. However,IGFBP4 proteolytic activity was completely lacking in fibroblasts derived from PAPPA-deficient embryos, and IGFBP4 effectively inhibited IGF-stimulated mitogenesis in these cells. These results provide the first direct evidence that PAPPA is an essential growth regulatory factor in vivo, and suggest a novel mechanism for regulated IGF bioavailability during early fetal development.

Cerebral water accumulation was studied during induction of brain edema in dystrophin-null transgenic mice (mdx-␤geo) and control mice. Immunofluorescence and immunoelectron microscopic analyses of dystrophin-null brains revealed a dramatic reduction of AQP4 (aquaporin-4) in astroglial end-feet surrounding capillaries (blood-brain barrier) and at the glia limitans (cerebrospinal fluidbrain interface). The AQP4 protein is mislocalized, because immunoblotting showed that the total AQP4 protein abundance was unaltered. Brain edema was induced by i.p. injection of distilled water and 8-deamino-arginine vasopressin. Changes in cerebral water compartments were assessed by diffusion-weighted MRI with determination of the apparent diffusion coefficient (ADC). In dystrophin-null mice and control mice, ADC gradually decreased by 5-6% from baseline levels during the first 35 min, indicating the initial phase of intracellular water accumulation is similar in the two groups. At this point, the control mice sustained an abrupt, rapid decline in ADC to 58% ؎ 2.2% of the baseline at 52.5 min, and all of the animals were dead by 56 min. After a consistent delay, the dystrophin-null mice sustained a similar decline in ADC to 55% ؎ 3.4% at 66.5 min, when all of the mice were dead. These results demonstrate that dystrophin is necessary for polarized distribution of AQP4 protein in brain where facilitated movements of water occur across the blood-brain barrier and cerebrospinal fluid-brain interface. Moreover, these results predict that interference with the subcellular localization of AQP4 may have therapeutic potential for delaying the onset of impending brain edema. Brain edema is associated with many intracranial neuropathological states, such as head trauma, ischemic brain injury, neoplasms, and metabolic diseases including systemic hyponatremia. Although much investigation has addressed the underlying molecular mechanisms and pathophysiology of brain edema, little is known about the regulation of water transport across the blood-brain barrier, the cerebrospinal fluid (CSF)-brain interface, and between extracellular and intracellular compartments in brain parenchyma.It is well recognized that the aquaporin family of water channel proteins is the major pathway by which water rapidly crosses cell membranes (1). Manley et al. recently reported that AQP4 (aquaporin-4; ref. 18) negatively influences the outcome from brain edema (2), and other recent studies also have suggested that AQP4 contributes to the development of brain edema (3, 4). These observations are consistent with the distribution of AQP4 in brain, because the protein is expressed abundantly in a highly polarized distribution in ependymal cells and astroglial membranes facing capillaries and forming the glia limitans (5).Decreased expression of AQP4 protein without changes in the AQP4 mRNA levels was reported recently in brains of Dmd mdx mice (C57BL10 ScSn mdx), a strain carrying a spontaneous mutation that prevents expression of the longest isoform of dystrophin (6)....