A Low-Protein, High-Carbohydrate Diet Increases Fatty Acid Uptake and Reduces Norepinephrine-Induced Lipolysis in Rat Retroperitoneal White Adipose Tissue

A low-protein, high-carbohydrate (LPHC) diet for 15 days increased the lipid content in the carcass and adipose tissues of rats. The aim of this work was to investigate the mechanisms of this lipid increase in the retroperitoneal white adipose tissue (RWAT) of these animals. The LPHC diet induced an approximately two- and tenfold increase in serum corticosterone and TNF-alpha, respectively. The rate of de novo fatty acid (FA) synthesis in vivo was reduced (50%) in LPHC rats, and the lipoprotein lipase activity increased (100%). In addition, glycerokinase activity increased (60%), and the phosphoenolpyruvate carboxykinase content decreased (27%). Basal [U-C-14]-glucose incorporation into glycerol-triacylglycerol did not differ between the groups; however, in the presence of insulin, [U-C-14]-glucose incorporation increased by 124% in adipocytes from only control rats. The reductions in IRS1 and AKT content as well as AKT phosphorylation in the RWAT from LPHC rats and the absence of an insulin response suggest that these adipocytes have reduced insulin sensitivity. The increase in NE turnover by 45% and the lack of a lipolytic response to NE in adipocytes from LPHC rats imply catecholamine resistance. The data reveal that the increase in fat storage in the RWAT of LPHC rats results from an increase in FA uptake from circulating lipoproteins and glycerol phosphorylation, which is accompanied by an impaired lipolysis that is activated by NE.Fundacao de Amparo a Pesquisa do Estado de Mato Grosso (FAP-EMAT)Fundacao de Amparo a Pesquisa do Estado de Mato Grosso (FAPEMAT)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Systolic, diastolic and mean blood pressure.

<p>Graphs show data from the systolic, diastolic and mean blood pressure from sham-operated and operated at 30, 60 and 90 dps (HH and HD groups), n = 6-8/group.</p

Western blotting for calpain-1.

<p>Graphs represent data from Western blotting quantification, n = 5-6/group.</p

Early dystrophin loss is coincident with the transition of compensated cardiac hypertrophy to heart failure - Fig 1

<p>Representative images showing the B-mode long-axis ultrasound (A-E) and pulsed-wave Doppler (F-J) from sham-operated, 30, 60 and 90 dps (HH and HD groups). The B-mode images represent hearts in diastole. Pulsed-wave Doppler shows E peaks in different velocities. The graphs represent data from 3–6 cycles per animal (n = 10-15/group) of the ejection fraction, fractional shortening, E wave velocity and E/E’ ratio. IVS = interventricular septum; LVID = left ventricle internal diameter; LVPW = left ventricle posterior wall. White bars = sham-operated, gray bars = operated; gray bar with stripes = HH group; and black bars = HD group. The values represent the means ± SD.</p

Echocardiographic parameters.

<p>Echocardiographic parameters.</p

The MicroRNA miR-696 is regulated by SNARK and reduces mitochondrial activity in mouse skeletal muscle through Pgc1α inhibition

Objective: MicroRNAs (miRNA) are known to regulate the expression of genes involved in several physiological processes including metabolism, mitochondrial biogenesis, proliferation, differentiation, and cell death. Methods: Using “in silico” analyses, we identified 219 unique miRNAs that potentially bind to the 3′UTR region of a critical mitochondrial regulator, the peroxisome proliferator-activated receptor gamma coactivator (PGC) 1 alpha (Pgc1α). Of the 219 candidate miRNAs, miR-696 had one of the highest interactions at the 3′UTR of Pgc1α, suggesting that miR-696 may be involved in the regulation of Pgc1α. Results: Consistent with this hypothesis, we found that miR-696 was highly expressed in the skeletal muscle of STZ-induced diabetic mice and chronic high-fat-fed mice. C2C12 muscle cells exposed to palmitic acid also exhibited a higher expression of miR-696. This increased expression corresponded with a reduced expression of oxidative metabolism genes and reduced mitochondrial respiration. Importantly, reducing miR-696 reversed decreases in mitochondrial activity in response to palmitic acid. Using C2C12 cells treated with the AMP-activated protein kinase (AMPK) activator AICAR and skeletal muscle from AMPKα2 dominant-negative (DN) mice, we found that the signaling mechanism regulating miR-696 did not involve AMPK. In contrast, overexpression of SNF1-AMPK-related kinase (SNARK) in C2C12 cells increased miR-696 transcription while knockdown of SNARK significantly decreased miR-696. Moreover, muscle-specific transgenic mice overexpressing SNARK exhibited a lower expression of Pgc1α, elevated levels of miR-696, and reduced amounts of spontaneous activity. Conclusions: Our findings demonstrate that metabolic stress increases miR-696 expression in skeletal muscle cells, which in turn inhibits Pgc1α, reducing mitochondrial function. SNARK plays a role in this process as a metabolic stress signaling molecule inducing the expression of miR-696

Western blotting analysis for calpastatin, activated calpain-1 and alpha-fodrin.

<p>Figure shows representative image of Western blotting in the sham, HH and HD groups, n = 5-6/group.</p