Effects of long-term dietary nitrate supplementation in mice.

BACKGROUND: Inorganic nitrate (NO3(-)) is a precursor of nitric oxide (NO) in the body and a large number of short-term studies with dietary nitrate supplementation in animals and humans show beneficial effects on cardiovascular health, exercise efficiency, host defense and ischemia reperfusion injury. In contrast, there is a long withstanding concern regarding the putative adverse effects of chronic nitrate exposure related to cancer and adverse hormonal effects. To address these concerns we performed in mice, a physiological and biochemical multi-analysis on the effects of long-term dietary nitrate supplementation. DESIGN: 7 week-old C57BL/6 mice were put on a low-nitrate chow and at 20 weeks-old were treated with NaNO3 (1 mmol/L) or NaCl (1 mmol/L, control) in the drinking water. The groups were monitored for weight gain, food and water consumption, blood pressure, glucose metabolism, body composition and oxygen consumption until one group was reduced to eight animals due to death or illness. At that point remaining animals were sacrificed and blood and tissues were analyzed with respect to metabolism, cardiovascular function, inflammation, and oxidative stress. RESULTS: Animals were supplemented for 17 months before final sacrifice. Body composition, oxygen consumption, blood pressure, glucose tolerance were measured during the experiment, and vascular reactivity and muscle mitochondrial efficiency measured at the end of the experiment with no differences identified between groups. Nitrate supplementation was associated with improved insulin response, decreased plasma IL-10 and a trend towards improved survival. CONCLUSIONS: Long term dietary nitrate in mice, at levels similar to the upper intake range in the western society, is not detrimental.

Spatial and temporal regulation of GLUT4 translocation by flotillin-1 and caveolin-3 in skeletal muscle cells.

Skeletal muscle tissue is one of the main sites where glucose uptake occurs in response to insulin. The glucose transporter type-4 (GLUT4) is primarily responsible for the insulin-stimulated increase in glucose uptake. Upon insulin stimulation, GLUT4 is recruited from intracellular reserves to the plasma membrane. The molecular mechanisms that regulate the translocation of GLUT4 to the sarcolemma remain to be fully identified. Here, we demonstrate that GLUT4 is localized to perinuclear stores that contain flotillin-1, a marker of lipid rafts, in skeletal muscle cells. Stimulation with insulin for 10 min results in the translocation of flotillin-1/GLUT4-containing domains to the plasma membrane in a PI3K- and PKCzeta-dependent manner. We also demonstrate that caveolin-3, a marker of caveolae, is required for the insulin receptor-mediated activation of the PI3K-dependent pathway, which occurs 2 min after insulin stimulation. In fact, we demonstrate that lack of caveolin-3 significantly reduces insulin-stimulated glucose uptake in caveolin-3 null myotubes by inhibiting both PI3K and Akt, as well as the movement of GLUT4 to the plasma membrane. Interestingly, caveolin-3 moves away from the plasma membrane toward the cytoplasm 5 min after insulin stimulation and temporarily interacts with flotillin-1/GLUT4-containing domains before they reach the sarcolemma, with the consequent movement of the insulin receptor from caveolin-3-containing domains to flotillin-1-containing domains. Such translocation temporally matches the insulin-stimulated movement of Cbl and CrkII in flotillin-1/GLUT4-containing domains, as well as the activation of the GDP-GTP exchange factor C3G. Disruption of flotillin-1-based domains prevents the activation of C3G, movement of GLUT4 to the sarcolemma, and glucose uptake in response to insulin. Thus, the activation of the Cbl/C3G/TC10-dependent pathway, which occurs before flotillin-1/GLUT4-containing domains reach the plasma membrane, is flotillin-1 mediated and follows the activation of the PI3K-mediated signaling. Taken together, these results indicate that flotillin-1 and caveolin-3 may regulate muscle energy metabolism through the spatial and temporal segregation of key components of the insulin signaling.