A homozygous variant in RRM2B is associated with severe metabolic acidosis and early neonatal death.

RRM2B encodes the crucial p53-inducible ribonucleotide reductase small subunit 2 homolog (p53R2), which is required for DNA synthesis throughout the cell cycle. Mutations in this gene have been associated with a lethal mitochondrial depletion syndrome. Here we present the case of an infant with a novel homozygous p.Asn221Ser mutation in RRM2B who developed hypotonia, failure to thrive, sensorineural hearing loss, and severe metabolic lactic acidosis, ultimately progressing to death at 3 months of age. Through molecular modeling using the X-ray crystal structure of p53R2, we demonstrate that this mutation likely causes disruption of a highly conserved helix region of the protein by altering intramolecular interactions. This report expands our knowledge of potential pathogenic RRM2B mutations as well as our understanding of the molecular function of p53R2 and its role in the pathogenesis of mitochondrial DNA depletion.

Exercise training prevents skeletal muscle plasma membrane cholesterol accumulation, cortical actin filament loss, and insulin resistance in C57BL/6J mice fed a western-style high-fat diet.

Insulin action and glucose disposal are enhanced by exercise, yet the mechanisms involved remain imperfectly understood. While the causes of skeletal muscle insulin resistance also remain poorly understood, new evidence suggest excess plasma membrane (PM) cholesterol may contribute by damaging the cortical filamentous actin (F-actin) structure essential for GLUT4 glucose transporter redistribution to the PM upon insulin stimulation. Here, we investigated whether PM cholesterol toxicity was mitigated by exercise. Male C57BL/6J mice were placed on low-fat (LF, 10% kCal) or high-fat (HF, 45% kCal) diets for a total of 8 weeks. During the last 3 weeks of this LF/HF diet intervention, all mice were familiarized with a treadmill for 1 week and then either sham-exercised (0 m/min, 10% grade, 50 min) or exercised (13.5 m/min, 10% grade, 50 min) daily for 2 weeks. HF-feeding induced a significant gain in body mass by 3 weeks. Sham or chronic exercise did not affect food consumption, water intake, or body mass gain. Prior to sham and chronic exercise, "pre-intervention" glucose tolerance tests were performed on all animals and demonstrated that HF-fed mice were glucose intolerant. While sham exercise did not affect glucose tolerance in the LF or HF mice, exercised mice showed an improvement in glucose tolerance. Muscle from sham-exercised HF-fed mice showed a significant increase in PM cholesterol, loss of cortical F-actin, and decrease in insulin-stimulated glucose transport compared to sham-exercised LF-fed mice. These HF-fed skeletal muscle membrane/cytoskeletal abnormalities and insulin resistance were improved in exercised mice. These data reveal a new therapeutic aspect of exercise being regulation of skeletal muscle PM cholesterol homeostasis. Further studies on this mechanism of insulin resistance and the benefits of exercise on its prevention are needed.

Effect of Corncob bedding on feed conversion efficiency in a high-fat diet-induced prediabetic model in C57Bl/6J mice.

Laboratory facilities use many varieties of contact bedding, including wood chips, paper products, and corncob, each with its own advantages and disadvantages. Corncob bedding, for example, is often used because of its high absorbency, ability to minimize detectable ammonia, and low cost. However, observations that mice eat the corncob lead to concerns that its use can interfere with dietary studies. We evaluated the effect of corncob bedding on feed conversion (change in body weight relative to the apparent number of kcal consumed over 7 d) in mice. Four groups of mice (6 to 12 per group) were housed in an individually ventilated caging system: (1) low-fat diet housed on recycled paper bedding, (2) low-fat diet housed on corncob bedding, (3) high-fat diet housed on recycled paper bedding, and (4) high-fat diet housed on corncob bedding. After 4 wk of the high-fat diet, feed conversion and percentage body weight change both were lower in corncob-bedded mice compared with paper-bedded mice. Low-fat-fed mice on corncob bedding versus paper bedding did not show statistically significant differences in feed conversion or change in percentage body weight. Average apparent daily feed consumption did not differ among the 4 groups. In conclusion, these data suggest that corncob bedding reduces the efficiency of feed conversion in mice fed a high-fat diet and that other bedding choices should be favored in these models.

Chromium enhances insulin responsiveness via AMPK.

Trivalent chromium (Cr(3+)) is known to improve glucose homeostasis. Cr(3+) has been shown to improve plasma membrane-based aspects of glucose transporter GLUT4 regulation and increase activity of the cellular energy sensor 5' AMP-activated protein kinase (AMPK). However, the mechanism(s) by which Cr(3+) improves insulin responsiveness and whether AMPK mediates this action is not known. In this study we tested if Cr(3+) protected against physiological hyperinsulinemia-induced plasma membrane cholesterol accumulation, cortical filamentous actin (F-actin) loss and insulin resistance in L6 skeletal muscle myotubes. In addition, we performed mechanistic studies to test our hypothesis that AMPK mediates the effects of Cr(3+) on GLUT4 and glucose transport regulation. Hyperinsulinemia-induced insulin-resistant L6 myotubes displayed excess membrane cholesterol and diminished cortical F-actin essential for effective glucose transport regulation. These membrane and cytoskeletal abnormalities were associated with defects in insulin-stimulated GLUT4 translocation and glucose transport. Supplementing the culture medium with pharmacologically relevant doses of Cr(3+) in the picolinate form (CrPic) protected against membrane cholesterol accumulation, F-actin loss, GLUT4 dysregulation and glucose transport dysfunction. Insulin signaling was neither impaired by hyperinsulinemic conditions nor enhanced by CrPic, whereas CrPic increased AMPK signaling. Mechanistically, siRNA-mediated depletion of AMPK abolished the protective effects of CrPic against GLUT4 and glucose transport dysregulation. Together these findings suggest that the micronutrient Cr(3+), via increasing AMPK activity, positively impacts skeletal muscle cell insulin sensitivity and glucose transport regulation.

Hexosamine biosynthesis impairs insulin action via a cholesterolgenic response.

Plasma membrane cholesterol accumulation has been implicated in cellular insulin resistance. Given the role of the hexosamine biosynthesis pathway (HBP) as a sensor of nutrient excess, coupled to its involvement in the development of insulin resistance, we delineated whether excess glucose flux through this pathway provokes a cholesterolgenic response induced by hyperinsulinemia. Exposing 3T3-L1 adipocytes to physiologically relevant doses of hyperinsulinemia (250pM-5000pM) induced a dose-dependent gain in the mRNA/protein levels of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR). These elevations were associated with elevated plasma membrane cholesterol. Mechanistically, hyperinsulinemia increased glucose flux through the HBP and O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), known to activate cholesterolgenic gene products such as the sterol response element-binding protein (SREBP1) and HMGR. Chromatin immunoprecipitation demonstrated that increased O-GlcNAc modification of Sp1 resulted in a higher binding affinity of Sp1 to the promoter regions of SREBP1 and HMGR. Luciferase assays confirmed that HMGR promoter activity was elevated under these conditions and that inhibition of the HBP with 6-diazo-5-oxo-l-norleucine (DON) prevented hyperinsulinemia-induced activation of the HMGR promoter. In addition, both DON and the Sp1 DNA-binding inhibitor mithramycin prevented the hyperinsulinemia-induced increases in HMGR mRNA/protein and plasma membrane cholesterol. In these mithramycin-treated cells, both cortical filamentous actin structure and insulin-stimulated glucose transport were restored. Together, these data suggest a novel mechanism whereby increased HBP activity increases Sp1 transcriptional activation of a cholesterolgenic program, thereby elevating plasma membrane cholesterol and compromising cytoskeletal structure essential for insulin action.

Evidence that chromium modulates cellular cholesterol homeostasis and ABCA1 functionality impaired by hyperinsulinemia--brief report.

OBJECTIVE: Trivalent chromium (Cr3+) is an essential micronutrient. Findings since the 1950s suggest that Cr3+ might benefit cholesterol homeostasis. Here we present mechanistic evidence in support of this role of Cr3+. METHODS AND RESULTS: High-density lipoprotein cholesterol generation in 3T3-L1 adipocytes, which are rendered ineffective by the hyperinsulinemia that is known to accompany disorders of lipid metabolism, was corrected by Cr3+. Mechanistically, Cr3+ reversed hyperinsulinemia-induced cellular cholesterol accrual and associated defects in cholesterol transporter ATP-binding cassette transporter-A1 trafficking and apolipoprotein A1-mediated cholesterol efflux. Moreover, direct activation of AMP-activated protein kinase, which is known to be activated by Cr3+, or inhibition of hexosamine biosynthesis pathway activity, which is known to be elevated by hyperinsulinemia, mimics Cr3+ action. CONCLUSIONS: These findings suggest a mechanism of Cr3+ action that fits with long-standing claims of its role in cholesterol homeostasis. Furthermore, these data imply a mechanistic basis for the coexistence of dyslipidemia with hyperinsulinemia.