Expresson of the Endodermal Marker, EcSox17, During Embryogenesis of the Direct Developing Frog, Eleutherodactylus coqui

In the model amphibian, Xenopus laevis, the HMG box transcription factor, Sox17, is expressed throughout the vegetal region that forms the endoderm. It is both necessary and sufficient for the endoderm formation. In order to study endoderm formation in the direct developing frog, Eleutherodactylus coqui, its Sox17 orthologue EcSox17 was cloned by Sean Willamson. To look at the expression of EcSox17, I designed primers and did RT-PCR on whole embryos. EcSox17 was expressed from cleavage to hatching of free-living froglet. EcSox17 RNA was present in the ovaries, an expression which has not been reported in any other vertebrate. Whole mount and bisected in situ hybridizations were done to look at the location of the EcSox17 RNA. EcSox17 was expressed at the blastoporal lip at all times and was not expressed in the yolk-rich vegetal core. RT-PCR on dissected pieces of early gastrulae however, indicated that the vegetal core expressed EcSox17, suggesting its contribution to the developing endoderm. I conclude that EcSox17 expression is ovarian as well as zygotic throughout the embryonic development. The transcript location at the blastoporal lip and perhaps throughout the vegetal region in early gastrula suggests its contribution to the definitive endoderm. Further experimentation is required to assess the function and necessity of EcSox17 in endoderm formation

Role of Thyroid Hormone in the Intestinal Development of Eleutherodactylus coqui

Thyroid hormone (TH) is required for the metamorphosis of the long, coiled tadpole gut in Xenopus laevis into a shorter mature adult gut. Eleutherodactylus coqui, a direct developing frog, lacks a tadpole. Its embryonic gut is a miniature adult form with a mass of yolky endodermal cells attached to the small intestine to provide nutrition. The requirement of TH for the gut development in E. coqui was tested in this study. Inhibition of TH synthesis with methimazole arrested gut development in its embryonic form. T3, the active form of TH, induced gut development. Embryos treated with methimazole failed to utilize the yolk in their nutritional endoderm, and survived for weeks without any further development. Acidification of the yolk platelet is an initial step in the breakdown of yolk in X. laevis. E. coqui embryos in methimazole failed to acidify their yolk platelets, but acidification was stimulated by TH indicating its role in yolk utilization. In X. laevis, TRβ is upregulated in response to TH and induces differentiation of the adult gut. Similarly, EcTRβ, the E. coqui orthologue, was upregulated by TH in the gut. EcTRβ expression was high in the nutritional endodermal cells indicating a direct role for TH in yolk utilization. The low expression level of EcSox17, an endodermal transcription factor, in these nutritional endodermal cells was consistent with the fact that these cells did not contribute to the definitive gut. This study indicated a novel role for TH in yolk utilization in addition to its conserved role in gut development and differentiation

Enzyme Replacement Therapy for Mucopolysaccharidosis IIID using Recombinant Human α-N-Acetylglucosamine-6-Sulfatase in Neonatal Mice

There is currently no cure or effective treatment available for mucopolysaccharidosis type IIID (MPS IIID, Sanfilippo syndrome type D), a lysosomal storage disorder (LSD) caused by the deficiency of α-N-acetylglucosamine-6-sulfatase (GNS). The clinical symptoms of MPS IIID, like other subtypes of Sanfilippo syndrome, are largely localized to the central nervous system (CNS), and any treatments aiming to ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease need to be delivered across the blood–brain barrier. Here, we report a proof-of-concept enzyme replacement therapy (ERT) for MPS IIID using recombinant human α-N-acetylglucosamine-6-sulfatase (rhGNS) via intracerebroventricular (ICV) delivery in a neonatal MPS IIID mouse model. We overexpressed and purified rhGNS from CHO cells with a specific activity of 3.9 × 10⁴ units/mg protein and a maximal enzymatic activity at lysosomal pH (pH 5.6), which was stable for over one month at 4 °C in artificial cerebrospinal fluid (CSF). We demonstrated that rhGNS was taken up by MPS IIID patient fibroblasts via the mannose 6-phosphate (M6P) receptor and reduced intracellular glycosaminoglycans to normal levels. The delivery of 5 μg of rhGNS into the lateral cerebral ventricle of neonatal MPS IIID mice resulted in normalization of the enzymatic activity in brain tissues; rhGNS was found to be enriched in lysosomes in MPS IIID-treated mice relative to the control. Furthermore, a single dose of rhGNS was able to reduce the accumulated heparan sulfate and β-hexosaminidase. Our results demonstrate that rhGNS delivered into CSF is a potential therapeutic option for MPS IIID that is worthy of further development

Exogenous Glucose Administration Impairs Glucose Tolerance and Pancreatic Insulin Secretion during Acute Sepsis in Non-Diabetic Mice

Objectives:The development of hyperglycemia and the use of early parenteral feeding are associated with poor outcomes in critically ill patients. We therefore examined the impact of exogenous glucose administration on the integrated metabolic function of endotoxemic mice using our recently developed frequently sampled intravenous glucose tolerance test (FSIVGTT). We next extended our findings using a cecal ligation and puncture (CLP) sepsis model administered early parenteral glucose support.Methods:Male C57BL/6J mice, 8-12 weeks, were instrumented with chronic indwelling arterial and venous catheters. Endotoxemia was initiated with intra-arterial lipopolysaccharide (LPS; 1 mg/kg) in the presence of saline or glucose infusion (100 μL/hr), and an FSIVGTT was performed after five hours. In a second experiment, catheterized mice underwent CLP and the impact of early parenteral glucose administration on glucose homeostasis and mortality was assessed over 24 hrs.Measurements:And MAIN RESULTS: Administration of LPS alone did not impair metabolic function, whereas glucose administration alone induced an insulin sensitive state. In contrast, LPS and glucose combined caused marked glucose intolerance and insulin resistance and significantly impaired pancreatic insulin secretion. Similarly, CLP mice receiving parenteral glucose developed fulminant hyperglycemia within 18 hrs (all > 600 mg/dl) associated with increased systemic cytokine release and 40% mortality, whereas CLP alone (85 ± 2 mg/dL) or sham mice receiving parenteral glucose (113 ± 3 mg/dL) all survived and were not hyperglycemic. Despite profound hyperglycemia, plasma insulin in the CLP glucose-infused mice (3.7 ± 1.2 ng/ml) was not higher than sham glucose infused mice (2.1 ± 0.3 ng/ml).Conclusions:The combination of parenteral glucose support and the systemic inflammatory response in the acute phase of sepsis induces profound insulin resistance and impairs compensatory pancreatic insulin secretion, leading to the development of fulminant hyperglycemia. © 2013 Watanabe et al

Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese hamster ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-α-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with nine different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igκ light chain and human chymotrypsinogen B1 signal peptides, whereas Igκ light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use

Nocturnal Hypoxia Improves Glucose Disposal, Decreases Mitochondrial Efficiency, and Increases Reactive Oxygen Species in the Muscle and Liver of C57BL/6J Mice Independent of Weight Change

Although acute exposure to hypoxia can disrupt metabolism, longer-term exposure may normalize glucose homeostasis or even improve glucose disposal in the presence of obesity. We examined the effects of two-week exposure to room air (Air), continuous 10% oxygen (C10%), and 12 hr nocturnal periods of 10% oxygen (N10%) on glucose disposal, insulin responsiveness, and mitochondrial function in lean and obese C57BL/6J mice. Both C10% and N10% improved glucose disposal relative to Air in lean and obese mice without evidence of an increase in insulin responsiveness; however, only the metabolic improvements with N10% exposure occurred in the absence of confounding effects of weight loss. In lean mice, N10% exposure caused a decreased respiratory control ratio (RCR) and increased reactive oxygen species (ROS) production in the mitochondria of the muscle and liver compared to Air-exposed mice. In the absence of hypoxia, obese mice exhibited a decreased RCR in the muscle and increased ROS production in the liver compared to lean mice; however, any additional effects of hypoxia in the presence of obesity were minimal. Our data suggest that the development of mitochondrial inefficiency may contribute to metabolic adaptions to hypoxia, independent of weight, and metabolic adaptations to adiposity, independent of hypoxia

Early Initiation of Low-Level Parenteral Dextrose Induces an Accelerated Diabetic Phenotype in Septic C57BL/6J Mice

Development of hyperglycemia during sepsis is associated with increased morbidity and mortality. Nutritional support is common practice in the intensive care unit, but the metabolic effects are not well understood. The purpose of this study is to determine the effect of early low level calorie provision on the development of hyperglycemia in a clinically relevant murine model of sepsis. C57BL/6J mice un-derwent femoral arterial and venous catheterization followed by cecal ligation and puncture (CLP) or sham surgery and low-dose intravenous dextrose or saline infusion. Blood glucose (BG), plasma insulin, and cytokines were measured after 24 hours. Additional septic mice underwent hyperinsulinemic euglycemic clamps or received intravenous insulin concurrent with dextrose to determine whole body insulin sensitivity and test the efficacy of insulin to reverse hyperglycemia. Neither dextrose infusion nor CLP alone induced hyperglycemia. Early initiation of low level dextrose in septic mice produced a variable glycemic response; 49% maintained euglycemia (BGThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Stimulation of the endogenous incretin glucose-dependent insulinotropic peptide by enteral dextrose improves glucose homeostasis and inflammation in murine endotoxemia.

Loss of glucose homeostasis during sepsis is associated with increased organ dysfunction and higher mortality. Novel therapeutic strategies to promote euglycemia in sepsis are needed. We have previously shown that early low-level intravenous (IV) dextrose suppresses pancreatic insulin secretion and induces insulin resistance in septic mice, resulting in profound hyperglycemia and worsened systemic inflammation. In this study, we hypothesized that administration of low-level dextrose via the enteral route would stimulate intestinal incretin hormone production, potentiate insulin secretion in a glucose-dependent manner, and thereby improve glycemic control in the acute phase of sepsis. We administered IV or enteral dextrose to 10-week-old male C57BL/6J mice exposed to bacterial endotoxin and measured incretin hormone release, glucose disposal, and proinflammatory cytokine production. Compared with IV administration, enteral dextrose increased circulating levels of the incretin hormone glucose-dependent insulinotropic peptide (GIP) associated with increased insulin release and insulin sensitivity, improved mean arterial pressure, and decreased proinflammatory cytokines in endotoxemic mice. Exogenous GIP rescued glucose metabolism, improved blood pressure, and increased insulin release in endotoxemic mice receiving IV dextrose, whereas pharmacologic inhibition of GIP signaling abrogated the beneficial effects of enteral dextrose. Thus, stimulation of endogenous GIP secretion by early enteral dextrose maintains glucose homeostasis and attenuates the systemic inflammatory response in endotoxemic mice and may provide a therapeutic target for improving glycemic control and clinical outcomes in patients with sepsis