Nitrite Differently Activates cGMP versus cAMP

neonates results in impaired formation of alveoli and lung

Introduction: Multiple metal ions are present in the human

vasculature that is characteristic of bronchopulmonary

diet, with some being required as essential nutrients and all

dysplasia (BPD), which can have long term consequences for

causing toxicity at high intakes. Low level environmental

the development of the respiratory and nervous systems. We

cadmium (Cd) occurs in human diet and accumulates in

have shown that hyperoxia reduces levels of Rev-erbα, a

vivo.

transcriptional

component

repressor

and

circadian

regulator

of

Selenium (Se) is an essential micronutrient, and a of

25

selenoproteins

responsible

for

metabolism, in the mouse lung. We hypothesized that

physiological functions. However, excess Se increases the

reduction of Rev-erbα expression would alter metabolism in

risk of diseases, therefore, determination of adequate Se

a mouse embryonic fibroblast (MEF) model of hyperoxic

amount for human health is critical. Impacts of metals and

exposure. In order to determine changes in metabolic

their interaction on cells and organs need to be addressed.

function and utilization of key substrates resulting from loss

Methods: C57BL6 mice were treated with Se (4 mg

of Rev-erbα, we exposed WT and Rev-erbα KO MEFs to

Na2SeO4/L, 16 weeks), Cd (3.3 mg CdCl2/L, 16 weeks) and

either air or hyperoxia (95% O2/5% CO2) for 24 hours.

Control

cells

Mitochondrial

were

exposed

to

air

24

markers for lung and liver functions were analyzed. Results: Urinary Se was 4-6 folds higher in Se-treated group while no changes were observed in lung and liver. Cd levels

Bioanalyzer. Additionally, a 4 day growth curve was

were elevated in urine, lung and liver in Cd mice. Se-treated

performed

to

determine

and

hours.

utilization were then determined using a Seahorse XF24 air

glycolysis,

for

substrate

in

respiration,

mixture. Metals, redox states, metabolites and phenotypic

cell

mice significant increased body mass than control or Cd

proliferation. Rev-erbα KO MEFs exposed to hyperoxia

differences

in

groups. Cd oxidized Prx3 in lung but not in liver, and co-

exhibited increased oxidative respiration and glycolysis

treatment showed that Se inhibited Prx3 oxidation. Liver

compared to WT cells. Substrate utilization experiments

metabolomics of Se-treated mice showed that altered

showed that immediately following hyperoxic exposure,

metabolome was significantly associated with weight gain,

Rev-erbα KO MEFs were less dependent on glutamine and

and accompanied by decreased levels of bile acids and acyl

fatty acids, but had a higher capacity to metabolize glucose.

carnitines.

Rev-erbα KO MEFs exhibit a higher basal rate of growth in

Discussion: Excess Se disrupts lipids homeostasis. Our study

air compared to WT. In conclusion, disruption of Rev-erbα

showed that mice treated with Se caused higher Cd

in MEFs resulted in increased glycolysis and higher levels of

accumulation in lung and liver, suggesting that excess Se

proliferation. We speculate that these changes may render

could elevate Cd toxicity. The results suggest that presence

the cells more vulnerable to oxidative stress and less viable.

of multiple metal ions in diet could affect human health

We will test this using protein carbonyls as a measurement

potentiating metal toxicity.

of oxidative stress levels and clonogenic assays as a DOI: 10.1016/j.freeradbiomed.2017.10.254

measurement of cell survival. DOI: 10.1016/j.freeradbiomed.2017.10.253

242 241

Nitrite Differently Activates cGMP versus cAMP

Integrative Omics-identified Potential Toxicity of Environmental and Nutritional Metals

1

Emory University, Atlanta, USA

Pride1, Jose Eduardo Tanus-Santos1, and Sruti Shiva1 1

Young-Mi Go1, Xin Hu1, Joshua Chandler1, and Dean P. Jones1

Danielle Guimaraes1, Rafael Portella1, Christelle KamgaUniversity of Pittsburgh, USA

Nitrite is an established signaling molecule that regulates mitochondrial function in hypoxia by inhibiting respiration and mitochondrial oxidant production, but its effects on mitochondrial function in normoxia remain unclear. We

SfRBM 2017

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hypothesize that nitrite mediates differential signaling in

A plethora of metabolic profiling studies have identified

hypoxia

several metabolic signatures in the patient’s biofluids and

and

normoxia

leading

to

modulation

of

mitochondrial function. Here, we demonstrate that nitrite

tissues to be correlated with type 2 diabetes (T2D) and its

switches from stimulation of soluble guanylate cyclase-

comorbidities. However, the association between specific

dependent cGMP generation in hypoxia to cAMP production

metabolite-based markers and occurrence of mitochondrial

in normoxic conditions.

dysfunction among T2D subjects has not been adequately

Nitrite (unlike nitric oxide)

increases cAMP levels in H9C2 cardiomyocytes and in

addressed. Therefore, the current study sought to determine

isolated mitochondria in normoxia leading to protein kinase

whether circulating metabolite signatures in cultured

A (PKA) activation outside and inside the mitochondria.

myotubes and fasting plasma of T2D subjects are associated

This increase in cAMP levels is due to nitrite-dependent

with mitochondrial dysfunction. A cellular disease model

inhibition of phosphodiesterases (PDE2A and PDE4A),

was first established using human myotubes treated with

which degrades cAMP. In addition to increasing PKA

antimycin A, an oxidative phosphorylation inhibitor. The

activation, we observed that nitrite increases the expression

cultured sample of these intracellular-defined metabolites on

of A-kinase anchoring protein (AKAP1), which tethers PKA

treated myotubes was analyzed using mass spectrometry

to the mitochondrial membrane. Consistent with the

based approaches. Further, a targeted metabolic profiling of

mitochondrial targeting of PKA, we show that nitrite

fasting blood plasma from normal (n=83) and T2D (n=92)

induces the phosphorylation of Ser58 on mitochondrial

subjects

complex IV-1 (a known PKA target) and increases its activity

Multivariable-adjusted

by 86%, leading to augmented basal and maximal

analysis was computed to estimate odds ratios (ORs) and

respiration (48% and 29%, respectively compared to

95% confidence intervals for verifying differentiating

control). Pharmacological inhibition of PKA attenuates

metabolites correlated with T2D. Several metabolites were

in

a

cross

sectional

study

conditional

was

logical

validated. regression

IV

considerably altered in cultured myotubes with reduced

activity and phosphorylation. These data demonstrate that

mitochondrial function. Targeted metabolic fingerprinting of

nitrite is a unique signaling molecule able to increase cAMP

fasting blood plasma adjusted for age, sex and body mass

levels in normoxia and show a novel mechanism by which

index (BMI) revealed 20 significant robust metabolites (P

nitrite selectively modulates mitochondrial PKA-dependent

<0.05) with a discriminative power of 87% in the training

signaling. Further, these data show that nitrite is a versatile

set and 82.3% in an independent testing set to be associated

signaling molecule that not only modulates nitrogen

with T2D subjects. These panel metabolites markers

dependent

comprised primarily of branched chain amino acids

nitrite-dependent

increase

in

post-translational

respiration,

modification,

complex

but

also

(leucine, isoleucine and valine), medium-chain acylcarnitine

modulates phosphorylation.

(C6, C8, C10:2, C10:1 and C12:1), free fatty acids (C16:0, C18:0, C18:2, C20:5) and sphingomyelin (d18:2/16:0). Our

DOI: 10.1016/j.freeradbiomed.2017.10.255

finding yields a valuable insight on the identification of circulating selective metabolite signals associated with mitochondrial dysfunction in skeletal muscles that are distinctly found in the blood plasma of T2D subjects. These

243

measures are complementary to the clinical diagnosis of diabetes mellitus and provided an efficient way of depicting

Alterations of Cultured Myotubes and Fasting Plasma Metabolite Profiles Related to Mitochondrial Dysfunction in Type 2

metabolic perturbations and dysregulated mitochondrial metabolism in T2D subjects. DOI: 10.1016/j.freeradbiomed.2017.10.256

Diabetes Subjects Mohamad Hafizi Abu Bakar1, Mohamad Roji Sarmidi2, Hasniza Zaman Huri3, and Nany Hairunisa3 1

Universiti Sains Malaysia, Malaysia

2

Universiti Teknologi Malaysia, Malaysia

3

University Malaya Medical Centre, Malaysia

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