Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.

Niemann-Pick Disease, type C1 (NPC1) is an autosomal recessive lipid storage disorder in which a pathological cascade, including neuroinflammation occurs. While data demonstrating neuroinflammation is prevalent in mouse models, data from NPC1 patients is lacking. The current study focuses on identifying potential markers of neuroinflammation in NPC1 from both the Npc1 mouse model and NPC1 patients. We identified in the mouse model significant changes in expression of genes associated with inflammation and compared these results to the pattern of expression in human cortex and cerebellar tissue. From gene expression array analysis, complement 3 (C3) was increased in mouse and human post-mortem NPC1 brain tissues. We also characterized protein levels of inflammatory markers in cerebrospinal fluid (CSF) from NPC1 patients and controls. We found increased levels of interleukin 3, chemokine (C-X-C motif) ligand 5, interleukin 16 and chemokine ligand 3 (CCL3), and decreased levels of interleukin 4, 10, 13 and 12p40 in CSF from NPC1 patients. CSF markers were evaluated with respect to phenotypic severity. Miglustat treatment in NPC1 patients slightly decreased IL-3, IL-10 and IL-13 CSF levels; however, further studies are needed to establish a strong effect of miglustat on inflammation markers. The identification of inflammatory markers with altered levels in the cerebrospinal fluid of NPC1 patients may provide a means to follow secondary events in NPC1 disease during therapeutic trials.

Context Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease, affecting approximately 3 in 10 obese children worldwide. Objective We aimed to investigate the potential relationship between gut microbiota and NAFLD in obese youth, while considering the role of PNPLA3 rs738409, a strong genetic contributor to NAFLD. Design In this cross-sectional study, participants completed abdominal MRI to measure hepatic fat fraction (HFF), oral glucose tolerance test, and PNPLA3 rs738409 genotyping. Fecal samples were collected to analyze the V4 region of the 16S rRNA gene for intestinal bacteria characterization. Setting Yale Pediatric Obesity Clinic. Participants Obese youth (BMI > 95th percentile) with NAFLD (HFF ≥ 5.5%; n=44) and without NAFLD (HFF < 5.5%; n=29). Main Outcome Measure Shannon-Wiener diversity index values and proportional bacterial abundance by NAFLD status and PNPLA3 genotype. Results Subjects with NAFLD had decreased bacterial alpha-diversity compared to those without NAFLD (p=0.013). Subjects with NAFLD showed a higher Firmicutes to Bacteroidetes (F/B) ratio (p=0.019) and lower abundance of Bacteroidetes (p=0.010), Prevotella (p=0.019), Gemmiger (p=0.003), and Oscillospira (p=0.036). F/B ratio, Bacteroidetes, Gemmiger, and Oscillospira were associated with HFF when controlling for group variations. We also observed an additive effect on HFF by PNPLA3 rs738409 and Gemmiger, and PNPLA3 rs738409 and Oscillospira. Conclusions Obese youth with NAFLD have a different gut microbiota composition than those without NAFLD. These differences were still statistically significant when controlling for factors associated with NAFLD, including PNPLA3 rs738409.

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VACTERL/VATER association is a group of congenital malformations characterized by at least 3 of the following findings: vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities. To date, no unifying etiology for VACTERL/VATER association has been established, and there is strong evidence for causal heterogeneity. VACTERL/VATER association has many overlapping characteristics with other congenital disorders that involve multiple malformations. In addition to these other conditions, some of which have known molecular causes, certain aspects of VACTERL/VATER association have similarities with the manifestations of disorders caused by mitochondrial dysfunction. Mitochondrial dysfunction can result from a number of distinct causes and can clinically manifest in diverse presentations; accurate diagnosis can be challenging. Case reports of individuals with VACTERL association and confirmed mitochondrial dysfunction allude to the possibility of mitochondrial involvement in the pathogenesis of VACTERL/VATER association. Further, there is biological plausibility involving mitochondrial dysfunction as a possible etiology related to a diverse group of congenital malformations, including those seen in at least a subset of individuals with VACTERL association.

Highlights:• Loss of mitochondrial phosphoenolpyruvate (PEP) impairs insulin release in vivo.• Pyruvate kinase (PK) activators stimulate beta-cells in preclinical diabetes models.• PEP cycling in vivo depends on PK and mitochondrial PEPCK (PCK2) for insulin release. • Acute and 3-week oral PK activator amplifies insulin release during hyperglycemia. eTOC Blurb:Abudukadier et al. show that small molecule pyruvate kinase activation in vivo and in vitro increases insulin secretion in rodent and human models of diabetes. The phosphoenolpyruvate (PEP) cycling mechanism and its amplification are dependent on mitochondrial PEPCK (PCK2). SummaryThe mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle is an anaplerotic-cataplerotic mitochondrial shuttle utilizing mitochondrial PEPCK (PCK2) and pyruvate kinase (PK). PEP cycling stimulates insulin secretion via OxPhos-independent lowering of ADP by PK. We assess in vivo whether islet PCK2 is necessary for glucose sensing and if speeding the PEP cycle via pharmacological PK activators amplifies insulin secretion. Pck2 -/mice had severely impaired insulin secretion during islet perifusion, oral glucose tolerance tests and hyperglycemic clamps. Acute and chronic pharmacologic PK activator therapy improved islet insulin secretion from normal, high-fat diet (HFD) fed, or Zucker diabetic fatty (ZDF) rats, and glucolipotoxic or diabetic humans. A similar improvement in insulin secretion was observed in regular chow and HFD rats in vivo.Insulin secretion and cytosolic Ca 2+ during PK activation were dependent on PCK2.These data provide a preclinical rationale for strategies, such as PK activation, that target the PEP cycle to improve glucose homeostasis.

Background: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium. The lysosomal system regulates key aspects of iron homeostasis, which prompted us to investigate whether there are hematological abnormalities and iron metabolism defects in NPC1. Methods: Iron-related hematological parameters, systemic and tissue metal ion and relevant hormonal and proteins levels, expression of specific pro-inflammatory mediators and erythrophagocytosis were evaluated in an authentic mouse model and in a large cohort of NPC patients. Results: Significant changes in mean corpuscular volume and corpuscular hemoglobin were detected in Npc1-/- mice from an early age. Hematocrit, red cell distribution width and hemoglobin changes were observed in late-stage disease animals. Systemic iron deficiency, increased circulating hepcidin, decreased ferritin and abnormal pro-inflammatory cytokine levels were also found. Furthermore, there is evidence of defective erythrophagocytosis in Npc1-/- mice and in an in vitro NPC1 cellular model. Comparable hematological changes, including low normal serum iron and transferrin saturation and low cerebrospinal fluid ferritin were confirmed in NPC1 patients. Conclusions: These data suggest loss of iron homeostasis and hematological abnormalities in NPC1 may contribute to the pathophysiology of this disease.

Objective: We have shown that ''euglycemic DKA'' in patients with type 1 diabetes receiving a sodiumglucose cotransporter 2-inhibitor (SGLT2i) is due to normal increases in rates of ketogenesis but blunted increases in plasma glucose levels. In this analysis, we assessed whether rescue treatment of early ketoacidosis with insulin is altered by SGLT2i use. Research Design and Methods: Participants received 0.2 U/kg of aspart insulin after two 6-h interruptions of basal insulin that increased beta-hydroxybutyrate (BHB) by 1.2 -0.7 mmol/L before and by 1.5 -0.2 mmol/L during canagliflozin treatment. BHB and free fatty acid (FFA) were monitored every 30 min for 120 min after receiving a 0.2 U/kg subcutaneous injection of aspart insulin. Results: Ten adults (23 -5 years) were studied. During the 120 min after rescue therapy with insulin, the reductions in BHB and FFA were nearly identical between the pre-and during canagliflozin treatment studies, respectively (-1.27 -0.76 and -1.13 -0.69, P = 0.671 for BHB and -0.50 -0.35 vs. -0.41 -0.41, P = 0.603 for FFA). Conclusion: These data indicate that turning ketogenesis off, as well as on, does not appear to be affected by SGLT2i use.