Histological characterization of retinal degeneration in mucopolysaccharidosis type IIIC.

Heparan-α-glucosaminide N-acetyltransferase (HGSNAT) participates in lysosomal degradation of heparan sulfate. Mutations in the gene encoding this enzyme cause mucopolysaccharidosis IIIC (MPS IIIC) or Sanfilippo syndrome type C. MPS IIIC patients exhibit progressive neurodegeneration, leading to dementia and death in early adulthood. Currently there is no approved treatment for MPS IIIC. Incidences of non-syndromic retinitis pigmentosa and early signs of night blindness are reported in some MPS IIIC patients, however the majority of ocular phenotypes are not well characterized. The goal of this study was to investigate retinal degeneration phenotype in the Hgsnat knockout mouse model of MPS IIIC and a cadaveric human MPS IIIC eye. Cone and rod photoreceptors in the eyes of homozygous 6-month-old Hgsnat knockout mice and their wild-type counterparts were analyzed using cone arrestin, S-opsin, M-opsin and rhodopsin antibodies. Histological observation was performed on the eye from a 35-year-old MPS IIIC donor. We observed a nearly 50% reduction in the rod photoreceptors density in the Hgsnat knockout mice compared to the littermate wild-type controls. Cone photoreceptor density was unaltered at this age. Severe retinal degeneration was also observed in the MPS IIIC donor eye. To our knowledge, this is the first report characterizing ocular phenotypes arising from deleterious variants in the Hgsnat gene associated with MPS IIIC clinical phenotype. Our findings indicate retinal manifestations may be present even before behavioral manifestations. Thus, we speculate that ophthalmological evaluations could be used as diagnostic indicators of early disease, progression, and end-point evaluation for future MPS IIIC therapies.

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.

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 ß-hexosaminidase. Our results demonstrate that rhGNS delivered into CSF is a potential therapeutic option for MPS IIID that is worthy of further development.

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.

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.

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 underwent 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, plasma insulin, and cytokines were measured after 24 h. 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 (blood glucose < 200) and 27% developed severe hyperglycemia (blood glucose ≥ 600). Hyperglycemia was associated with increased inflammation and reduced insulin secretion and sensitivity compared with control mice or CLP mice maintaining euglycemia. Insulin prevented the progression to severe hyperglycemia but was ineffective in reestablishing glycemic control once hyperglycemia had developed. In conclusion, early initiation of clinically relevant low-level dextrose (∼ 20% daily caloric requirements) precipitated hyperglycemia akin to an acute diabetic phenotype in septic mice characterized by decreased insulin sensitivity, decreased insulin secretion, and an increased inflammatory response.

Inorganic nitrite improves components of the metabolic syndrome independent of weight change in a murine model of obesity and insulin resistance.

Nitrite acts as an endocrine source of bioactive nitric oxide, impacting vascular reactivity, angiogenesis and cytoprotection. Nitrite has recently been shown to have a metabolic role although its effects and mechanisms of action in the obese insulin-resistant state are unknown. We examined glucose tolerance and insulin secretion using the frequently sampled intravenous glucose tolerance test and insulin sensitivity using the hyperinsulinaemic euglycaemic clamp in obese male ob(lep) mice administered nitrite (100 mg kg(-1) day(-1) ) or saline (control) for 7 days and compared responses to the known insulin-sensitizing effects of rosiglitazone (6 mg kg(-1) day(-1) ). Under weight-matched conditions, nitrite lowered blood pressure relative to saline and rosiglitazone, whereas only rosiglitazone was effective at reducing hepatic glucose output and basal blood glucose. Both nitrite and rosiglitazone produced improvements, relative to saline, in glucose tolerance (12,524 ± 602, 12,811 ± 692 vs.14,428 ± 335 mg (dl min)(-1) , respectively; P < 0.05) and insulin sensitivity (8.6 ± 0.7, 7.9 ± 0.3 vs. 6.6 ± 0.5 mg kg(-1) min(-1) , respectively; P < 0.001), but there was no effect on insulin secretion. Nitrite exhibited an uncoupling of mitochondrial respiration and a decrease in ATP generation in muscle that was independent of mitochondrial biogenesis or activation of uncoupling proteins. There was no insulin-stimulated phosphorylation of Akt, but nitrite increased the phosphorylation of AMP-activated protein kinase. We conclude that nitrite improves two key components of the metabolic syndrome, blood pressure and insulin sensitivity, independent of weight and with effectiveness comparable to rosiglitazone.

ß-catenin links hepatic metabolic zonation with lipid metabolism and diet-induced obesity in mice.

ß-catenin regulates the establishment of hepatic metabolic zonation. To elucidate the functional significance of liver metabolic zonation in the chronically overfed state in vivo, we fed a high-fat diet (HFD) to hepatocyte-specific ß-catenin transgenic (TG) and knockout (KO) mice. Chow-fed TG and KO mice had normal liver histologic findings and body weight. However, HFD-fed TG mice developed prominent perivenous steatosis with periportal sparing. In contrast, HFD-fed KO mice had increased lobular inflammation and hepatocyte apoptosis. HFD-fed TG mice rapidly developed diet-induced obesity and systemic insulin resistance, but KO mice were resistant to diet-induced obesity. However, ß-catenin did not directly affect hepatic insulin signaling, suggesting that the metabolic effects of ß-catenin occurred via a parallel pathway. Hepatic expression of key glycolytic and lipogenic genes was higher in HFD-fed TG and lower in KO mice compared with wild-type mice. KO mice also exhibited defective hepatic fatty acid oxidation and fasting ketogenesis. Hepatic levels of hypoxia inducible factor-1α, an oxygen-sensitive transcriptional regulator of glycolysis and a known ß-catenin binding partner, were higher in HFD-fed TG and lower in KO mice. KO mice had attenuated perivenous hypoxia, suggesting disruption of the normal sinusoidal oxygen gradient, a major determinant of liver carbohydrate and liver metabolism. Canonical Wnt signaling in hepatocytes is essential for the development of diet-induced fatty liver and obesity.

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.

Time-dependent changes in glucose and insulin regulation during intermittent hypoxia and continuous hypoxia.

Hypoxia manifests in many forms including the short repetitive intermittent hypoxia (IH) of sleep apnoea and the continuous hypoxia (CH) of altitude, both of which may impact metabolic function. Based on our own previous studies and the available literature, we hypothesized that whereas acute exposure to IH and CH would lead to comparable metabolic dysfunction, with longer-term exposure, metabolism would normalize to a greater extent with CH than IH. Studies were conducted in lean C57BL/6J mice exposed to either IH or CH for 1 day or 4 weeks and compared to either intermittent air (IA) or unhandled (UN) controls, respectively. We utilized the frequently sampled intravenous glucose tolerance test and minimal model analyses to determine insulin-dependent (insulin sensitivity; S (I)) and insulin-independent (glucose effectiveness; S (g)) glucose disposal, as well as the insulin response to glucose (acute insulin response to glucose; AIR(g)). Our data show that 1-day exposure impaired the glucose tolerance and caused reductions in S (g) and AIR(g) in both the IH and CH groups, but only IH caused a significant decrease in S (I) (7.5 ± 2.7 vs. 17.0 ± 5.3 µU ml(-1) min(-1); p < 0.05). After 4-week exposure, there was evidence of metabolic adaptation in both hypoxic groups, however, in the CH group, there was a supranormal increase in S (I) relative to both UN and IH groups. We conclude that in lean mice, the marked metabolic dysfunction that occurs with acute exposure to hypoxia is reversed to a greater extent with chronic CH exposure than chronic IH exposure.

Simultaneous measurement of insulin sensitivity, insulin secretion, and the disposition index in conscious unhandled mice.

Of the parameters that determine glucose disposal and progression to diabetes in humans: first-phase insulin secretion, glucose effectiveness (Sg), insulin sensitivity (Si), and the disposition index (DI), only Si can be reliably measured in conscious mice. To determine the importance of the other parameters in murine glucose homeostasis in lean and obese states, we developed the frequently sampled intravenous glucose tolerance test (FSIVGTT) for use in unhandled mice. We validated the conscious FSIVGTT against the euglycemic clamp for measuring Si in lean and obese mice. Insulin-resistant mice had increased first-phase insulin secretion, decreased Sg, and a reduced DI, qualitatively similar to humans. Intriguingly, although insulin secretion explained most of the variation in glucose disposal in lean mice, Sg and the DI more strongly predicted glucose disposal in obese mice. DI curves identified individual diet-induced obese (DIO) mice as having compensated or decompensated insulin secretion. Conscious FSIVGTT opens the door to apply mouse genetics to the determinants of in vivo insulin secretion, Sg, and DI, and further validates the mouse as a model of metabolic disease.

Novel regulation of yolk utilization by thyroid hormone in embryos of the direct developing frog Eleutherodactylus coqui.

Thyroid hormone (TH) is required for metamorphosis of the long, coiled tadpole gut into the short frog gut. Eleutherodactylus coqui, a direct developing frog, lacks a tadpole. Its embryonic gut is a miniature adult form with a mass of yolky cells, called nutritional endoderm, attached to the small intestine. We tested the TH requirement for gut development in E. coqui. Inhibition of TH synthesis with methimazole arrested gut development in its embryonic form. Embryos treated with methimazole failed to utilize the yolk in their nutritional endoderm, and survived for weeks without further development. Conversely, methimazole and 3,3',5-tri-iodo-l-thyronine, the active form of TH, stimulated gut development and utilization and disappearance of the nutritional endoderm. In Xenopus laevis, the receptor for TH, TRß, is upregulated in response to TH. Similarly, EcTRß, the E. coqui ortholog, was upregulated by TH in the gut. EcTRß expression was high in the nutritional endoderm, suggesting a direct role for TH in yolk utilization by these cells. An initial step in the breakdown of yolk in X. laevis is acidification of the yolk platelet. E. coqui embryos in methimazole failed to acidify their yolk platelets, but acidification was stimulated by TH indicating its role in an early step of yolk utilization. In addition to a conserved TH role in gut development, a novel regulatory role for TH in yolk utilization has evolved in these direct developers.

Corticotropin-releasing factor regulates the development in the direct developing frog, Eleutherodactylus coqui.

Direct developing frogs lack a free-living larval phase, such that miniature adults hatch directly from the eggs. Even under such extreme reorganization of the ancestral biphasic developmental pattern, direct developers still undergo thyroid hormone (TH)-dependent post-embryonic development. Hypothalamic regulation of TH synthesis and release plays a central role in controlling the timing of metamorphosis in biphasic developers. In particular, the neuropeptide corticotropin-releasing factor (CRF) regulates TH in tadpoles, but in adults, both thyrotropin-releasing hormone (TRH) and CRF regulate TH. Because direct developers lack a tadpole stage, it was not clear whether hypothalamic regulation of TH would be tadpole-like or adult-like prior to hatching. To test this, we injected pre-hatching Eleutherodactylus coqui daily with CRF, TRH or astressin (a CRF receptor blocker). CRF but not TRH significantly accelerated the developmental rate compared to controls. Astressin-treated animals showed a near complete developmental arrest, which confirmed that development requires CRF. To support the idea that CRF acts to regulate development in E. coqui via thyroid physiology, we showed the TH-direct response gene TRß is up-regulated 24 and 48 h after CRF injection. In addition, treatment with 50 nM T3 (triiodothyronine, the active form of TH) increased the developmental rate similar to CRF injections. Our results extend the evidence for a cryptic metamorphosis in direct developers by showing that neuroendocrine signaling is conserved between biphasic and direct developers. Furthermore, the conserved neuroendocrine regulation implies that changes at the peripheral level of hormone action underlie the evolution of the radically divergent development in direct developers.

Nutritional endoderm in a direct developing frog: a potential parallel to the evolution of the amniote egg.

The egg of the direct-developing frog, Eleutherodactylus coqui, has 20 x the volume as that of the model amphibian, Xenopus laevis. Increased egg size led to the origin of nutritional endoderm, a novel cell type that provides nutrition but does not differentiate into digestive tract tissues. As the E. coqui endoderm develops, a distinct boundary exists between differentiating intestinal cells and large yolky cells, which persists even when yolk platelets are depleted. The yolky cells do not become tissues of the digestive tract and are lost, as shown by histology and lineage tracing. EcSox17, an endodermal transcriptional factor, did not distinguish these two cell types, however. When cleavage of the yolky cells was inhibited, embryogenesis continued, indicating that some degree of incomplete cleavage can be tolerated. The presence of cellularized nutritional endoderm in E. coqui may parallel changes that occurred in the evolution of the amniote egg 360 million years ago.