• 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • br Material and methods br Results


    Material and methods
    Discussion Increasing amount of evidence suggests that in addition to its role in atheroprotection [1,3] HDL is also involved in the regulation of β-cell secretory function and peripheral tissue MF498 sensitivity [27,32,43,44]. To better understand how changes in HDL metabolism may correlate with diet-induced obesity and T2DM we investigated the impact of Apoa1 and Lcat deficiency, two key proteins of peripheral HDL metabolic pathway, on these pathological conditions in mice. Deficiency in Apoa1 results in spherical HDL particles containing mainly esterified cholesterol and Apoe and Apoc2 and some Apoa2 [16] while deficiency in Lcat results in discoidal HDL particles containing primarily free cholesterol, Apoe and barely detectable levels of Apoa1 [45]. The present data show that apoa1 and lcat mice became more sensitive to diet-induced obesity than control C57BL/6 mice, with lcat animals becoming more obese than apoa1 animals (Fig. 1A). HDL from all three mouse strains presented distinct apolipoprotein composition (Fig. 2). HDL from C57BL/6 mice contained predominantly Apoa1, Apoa2 and Apoc2, and lower levels of Apoe. Even though feeding lcat mice western-type diet resulted in an apparent lack of measurable plasma Apoa1, a finding consistent with previous observations [45], the apolipoprotein composition of the HDL in these mice is markedly different from the HDL of apoa1 mice. Specifically, lcat mice had less Apoc2 and higher Apoa2 levels than apoa1 mice (Fig. 2). These structural differences among HDL from the three mouse strains correlated with differences in peripheral postprandial plasma triglyceride and cholesterol tissue deposition (Fig. 3). Nevertheless, they could not account for the observed phenotypic differences in diet-induced obesity. Indeed, measurement of radioactive tracers following gavage administration of olive oil containing [3H]-triolein and [14C]-cholesterol showed a significantly lower dietary triglyceride tissue deposition in WAT of apoa1 and lcat mice, although they accumulated significantly more visceral WAT than control C57BL/6 mice who were the same diet. Interestingly we also observed reduced [3H]-tracer deposition in pancreatic β-islets isolated from apoa1 and lcat mice (Fig. 3A). No difference in WAT [14C]-tracer accumulation was apparent among groups indicating similar post-prandial cholesterol deposition. Nevertheless, apoa1 mice showed higher deposition of [14C]-tracer in BAT and soleus muscle, suggesting increased dietary cholesterol uptake and storage in these tissues (Fig. 3B). The deposition of increased amounts of dietary cholesterol to soleus muscle may explain the development of insulin resistance of this tissue in response to feeding high fat diet, as previously reported [46]. Guided by these results and our previous experience with apolipoprotein E [34], we next hypothesized that Apoa1- or Lcat-deficiency may exert an effect on adipose tissue mitochondrial metabolic activation [34]. When Cytc was analyzed as a marker of oxidative phosphorylation, WAT mitochondrial extracts of apoa1 and C57BL/6 animals displayed similar levels, indicating comparable oxidative phosphorylation activity in this tissue. However, lcat mice expressed lower Cytc levels, suggesting that lack of functional Lcat had a selective negative impact on visceral WAT mitochondrial substrate oxidation towards energy production (heat and ATP). When Ucp1 was analyzed as a marker of non-shivering thermogenesis, WAT mitochondrial extracts, from C57BL/6 mice displayed considerable levels (Fig. 4A, C). However, similar analysis in apoa1 and lcat mice displayed reduced Ucp1 levels, indicative of substantially reduced non-shivering thermogenesis in visceral WAT of these mice compared to the C57BL/6 group (Fig. 4A, C). Of note, Ucp1 levels were much lower in lcat than apoa1 mice suggesting an even lower WAT oxidative phosphorylation capacity in the absence of Lcat. Similar analysis of BAT mitochondrial extracts revealed a rather small increase of Ucp1 levels in apoa1 and lcat mice compared to C57BL/6 mice (Fig. 4B, D). No differences in BAT mitochondrial Cytc levels were found among the three mouse groups. These finding indicate that the increased sensitivity of apoa1 and lcat mice towards diet-induced obesity is associated with reduced WAT mitochondrial non-shivering thermogenesis and that further suppression of WAT oxidative phosphorylation in lcat mice renders them even more sensitive than apoa1 mice to weight-gain. Apparently, the small increase in BAT metabolic activity of apoa1 and lcat mice may not suffice to counteract the massive decline in WAT mitochondrial metabolic activity of these mice when fed high-fat diet (Fig. 6A, C).