g BNip3 localizes to the outer mitochondrial membrane, where it functions in mitophagy and mitochondrial dynamics. While the BNip3 protein is constitutively expressed in adult liver from fed mice, we have shown that its expression is superinduced by fasting of mice, consistent with a role in responses to nutrient deprivation. Loss of BNip3 resulted in increased lipid synthesis in the liver that was associated with elevated ATP levels, reduced AMP-regulated kinase (AMPK) activity, and increased expression of lipogenic enzymes. Conversely, there was reduced ␤-oxidation of fatty acids in BNip3 null liver and also defective glucose output under fasting conditions. These metabolic defects in BNip3 null liver were linked to increased mitochondrial mass and increased hepatocellular respiration in the presence of glucose. However, despite elevated mitochondrial mass, an increased proportion of mitochondria exhibited loss of mitochondrial membrane potential, abnormal structure, and reduced oxygen consumption. Elevated reactive oxygen species, inflammation, and features of steatohepatitis were also observed in the livers of BNip3 null mice. These results identify a role for BNip3 in limiting mitochondrial mass and maintaining mitochondrial integrity in the liver that has consequences for lipid metabolism and disease. Modulation of mitochondrial mass is emerging as a major adaptive response to changes in energy balance arising from deficiencies in oxygen or glucose availability, among other nutrient stresses. For example, nutrient-sensitive changes in PGC-1␣ activity alter expression of genes required for mitochondrial biogenesis, in addition to genes required for fatty acid metabolism (17, 38). While mitochondrial biogenesis increases mitochondrial mass, this is countered by the role of mitophagy in targeting dysfunctional mitochondria for degradation at the autophagosome, resulting in reduced mitochondrial mass (28,29,70). Defects in autophagy have been linked to liver cancer (25,44,65) and have also been shown to promote hepatic insulin resistance (19, 67). However, this cannot be attributed to defective mitochondrial function, since autophagy-deficient liver also exhibits increased endoplasmic reticulum (ER) stress (67), protein aggregation (31), and defective lipidophagy (59). To date, a specific role for mitophagy in preventing hepatic steatosis or other liver pathologies has not been identified.Hypoxia modulates mitochondrial mass through both decreasing mitochondrial biogenesis (74) and increasing mitophagy (3,64,73). These effects are mediated by hypoxia-inducible factor (HIF) transcription factors, acting on the one hand to inhibit Myc-induced expression of PGC-1␤ (74) and on the other to induce expression of the mitochondrial proteins BNIP3 and NIX (3,4,64,73). Initial functional characterization of BNIP3 and NIX indicated that these proteins were loosely conserved members of the BH3-only subgroup of the Bcl-2 family of cell death regulators (7,8,52,68), and indeed, evidence from ischemia-reperfusion injury expe...

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 Å resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene-binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA ∼8,000-times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as substrate analog indicate that the oxidized flavin and substrate analog binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker ∼2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analog for the reduced-FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apo enzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.Flavins are key cofactors in the reductive activation and transfer of oxygen atoms to organic substrates in the biosynthesis of alcohols, aldehydes, and acids (1,2). Through structural studies, dynamic motions of the flavin isalloxazine ring system have been tracked and linked to discrete catalytic steps associated with substrate binding, oxygenation, and product release (3-8). Flavin-dependent epoxidations, which are less commonly encountered, have important roles in the biosynthesis of cholesterol and plant pigments and in the styrene catabolic and detoxification pathway of Pseudomonas bacteria (9-11). The soluble flavoenzyme styrene † This work was supported by NIH grants GM070473 (A. C. R.) and GM081140 (G. T. G). U. E. U. was supported in part by NIH Training Grant GM8061. ‡ The atomic coordinates (code 3IHM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) *Address correspondence to: George T. Gassner, Department of Chemistry and Biochemistry, San Francisco State University, San Francisco CA 94132, Tel: 415-637-1387, Fax: 415-338-2384 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript monooxygenase (SMO), which catalyzes the transformation of styrene to S-styrene oxide, is re...

Nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis, inflammation and fibrosis is an unmet medical need. MEDI0382, a balanced GLP-1/glucagon dual receptor agonist, is under development for the treatment of T2DM. Here we examined the effects of MEDI0382 on NASH compared to liraglutide, a GLP-1 analog. Leptin-deficient ob/ob mice were maintained on high trans-fat, fructose and cholesterol diet for 8 weeks to induce NASH then randomized to four treatment groups: vehicle, MEDI0382 (30 nmol/kg), liraglutide (30 nmol/kg) or vehicle-treated and switched to low-fat diet (LFD). Treatment with MEDI0382 and liraglutide reduced body weight and improved glucose tolerance. Hepatic lipid was reduced by 40% with MEDI0382 treatment (p<0.0001), which was more effective than liraglutide or switch to LFD. Hepatic collagen, quantified by type 1 collagen immunohistochemistry, was increased more than 2-fold with NASH and was reduced by 40% in MEDI0382-treated mice (p=0.005). Consistently, type 1 collagen gene expression increased 2-fold with NASH and was reduced by 85% in MEDI0382-treated livers (p<0.0001). No reductions in collagen were observed with liraglutide or switch to LFD. The NASH score, integrating pathology scores for steatosis, lobular inflammation, fibrosis, hepatocyte ballooning, biliary hyperplasia, and CD68 expression was significantly reduced by MEDI0382 (p<0.0001), greater than liraglutide or switch to LFD. Consistent with histopathology improvements, MEDI0382 treatment also reduced inflammatory gene expression (Tnf, Tgfb1, Il1b, and Ccl2) to a greater extent than liraglutide or LFD switch. In conclusion, MEDI0382 exerted similar metabolic control relative to liraglutide, but exhibited superior effects on primary NASH endpoints. Disclosure M. Beaton: Employee; Self; MedImmune. Employee; Spouse/Partner; MedImmune. S. Guionaud: None. J.P. Conway: None. J. Grimsby: Employee; Self; AstraZeneca. C.J. Rhodes: Stock/Shareholder; Self; AstraZeneca. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.

One proposed mechanism for the effects of bariatric surgery is the enhanced postprandial release of enteroendocrine L-cell hormones, such as PYY and GLP-1. Four week co-administration of Fc-conjugated GLP-1R and Y2R-selective agonists induced weight loss, reduced %HbA1c, and improved glucose tolerance in DIO and KS db/db mice compared to vehicle or monotherapy control groups. To assess the impact of this combination on glucose control, a hyperinsulinemic/euglycemic clamp was performed in KS db/db mice following two week treatment with vehicle or co-administration of GLP-1/Y2R agonists. A vehicle-treated group weight-matched to combination-treated mice was also assessed. Compared to KS db/db vehicle animals, combination-treated mice exhibited a 16-fold greater glucose infusion rate compared to only 4-fold in weight-matched controls. Glucose uptake was increased 4-fold in the skeletal muscle of combination-treated animals, but was unaltered in adipose or brain. Weight-matched controls did not exhibit increased glucose uptake in any tissue. To better characerize the mechanism of Fc-GLP-1/Fc-Y2R action on peripheral glucose metabolism, lean mice were acutely administered Fc-GLP-1 and/or Fc-Y2R, and then sacrificed to assess brain c-Fos activation. Compared to monotherapies, combination administration led to additive c-Fos activation in hindbrain regions. Strikingly, whereas monotherapy had no effect on the PVH, combination administration led to a synergistic 2-fold increase in c-Fos immunoreactivity. These findings implicate a neuronal population in the PVH as a putative driver of the antidiabetic efficacy observed following co-administration of GLP-1R and Y2R selective peptides, and may represent a novel mechanism to improve insulin sensitivity and skeletal muscle glucose uptake. Disclosure B. Boland: Employee; Self; MedImmune. V.G. Howard: Employee; Self; AstraZeneca. Stock/Shareholder; Self; Regeneron Pharmaceuticals, Inc., AstraZeneca. M. Beaton: Employee; Self; MedImmune. Employee; Spouse/Partner; MedImmune. S. Will: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. S. Oldham: Stock/Shareholder; Self; AstraZeneca. Employee; Self; MedImmune. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Stock/Shareholder; Self; AstraZeneca. J. Grimsby: Employee; Self; AstraZeneca.

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