Liver-targeted Angptl4 silencing by antisense oligonucleotide treatment attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice.

BACKGROUND AND AIMS: Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid uptake from triglyceride-rich lipoproteins such as VLDL. While pharmacological inhibition of ANGPTL3 is being evaluated as lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aimed to compare the therapeutic potential of liver-specific Angptl3 and Angptl4 silencing to attenuate hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established humanized model for lipoprotein metabolism. METHODS AND RESULTS: Mice were subcutaneously injected twice-weekly with saline or liver-targeted antisense oligonucleotides against Angptl3, Angptl4, both, or a scrambled oligonucleotide. Plasma lipid levels, VLDL clearance and hepatic VLDL production were determined, and atherosclerosis development was assessed. For toxicological evaluation, cynomolgus monkeys were treated with three dosages of liver-targeted ANGPTL4-silencing oligonucleotides.Liver-targeted Angptl4 silencing reduced plasma triglycerides (-48%) and total cholesterol (-56%), explained by higher VLDL-derived fatty acid uptake by brown adipose tissue and lower VLDL production by the liver. Accordingly, Angptl4 silencing reduced atherosclerotic lesion size (-86%) and improved lesion stability. Hepatic Angptl3 silencing similarly attenuated hyperlipidemia and atherosclerosis development. While Angptl3 and Angptl4 silencing lowered plasma triglycerides in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma triglycerides independent of nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects. CONCLUSIONS: Liver-targeted Angptl4 silencing potently attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, and liver-targeted ANGPTL4 silencing is well-tolerated in non-human primates. These data warrant further clinical development of liver-targeted ANGPTL4 silencing.

Doublecortin-like knockdown in mice attenuates obesity by stimulating energy expenditure in adipose tissue.

Crosstalk between peripheral metabolic organs and the central nervous system is essential for body weight control. At the base of the hypothalamus, ß-tanycytes surround the portal capillaries and function as gatekeepers to facilitate transfer of substances from the circulation into the cerebrospinal fluid and vice versa. Here, we investigated the role of the neuroplasticity gene doublecortin-like (DCL), highly expressed by ß-tanycytes, in body weight control and whole-body energy metabolism. We demonstrated that DCL-knockdown through a doxycycline-inducible shRNA expression system prevents body weight gain by reducing adiposity in mice. DCL-knockdown slightly increased whole-body energy expenditure possibly as a result of elevated circulating thyroid hormones. In white adipose tissue (WAT) triglyceride uptake was increased while the average adipocyte cell size was reduced. At histological level we observed clear signs of browning, and thus increased thermogenesis in WAT. We found no indications for stimulated thermogenesis in brown adipose tissue (BAT). Altogether, we demonstrate an important, though subtle, role of tanycytic DCL in body weight control through regulation of energy expenditure, and specifically WAT browning. Elucidating mechanisms underlying the role of DCL in regulating brain-peripheral crosstalk further might identify new treatment targets for obesity.

Time-restricted feeding attenuates hypercholesterolaemia and atherosclerosis development during circadian disturbance in APOE∗3-Leiden.CETP mice.

BACKGROUND: Circadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has been associated with elevated risk for atherosclerotic cardiovascular disease (asCVD) in humans, but evidence for the effectiveness of prevention strategies is lacking. METHODS: Here, we applied time-restricted feeding (TRF) as a strategy to counteract atherosclerosis development during CD in female APOE∗3-Leiden.CETP mice, a well-established model for humanized lipoprotein metabolism. Control groups were subjected to a fixed 12:12 h light-dark cycle, while CD groups were subjected to 6-h phase advancement every 3 days. Groups had either ad libitum (AL) access to food or were subjected to TRF with restricted food access to the dark phase. FINDINGS: TRF did not prevent the increase in the relative abundance of circulating inflammatory monocytes and elevation of (postprandial) plasma triglycerides during CD. Nonetheless, TRF reduced atherosclerotic lesion size and prevented an elevation in macrophage content of atherosclerotic lesions during CD, while it increased the relative abundance of anti-inflammatory monocytes, prevented activation of T cells, and lowered plasma total cholesterol levels and markers of hepatic cholesterol synthesis. These effects were independent of total food intake. INTERPRETATION: We propose that time restricted eating could be a promising strategy for the primary prevention of asCVD risk in shift workers, which warrants future study in humans. FUNDING: This work was funded by the Novo Nordisk Foundation, the Netherlands Ministry of Social Affairs and Employment, Amsterdam Cardiovascular Sciences, and the Dutch Heart Foundation.

A single day of high-fat diet feeding induces lipid accumulation and insulin resistance in brown adipose tissue in mice.

Brown adipose tissue (BAT) catabolizes glucose and fatty acids to produce heat and thereby contributes to energy expenditure. Long-term high-fat diet (HFD) feeding results in so-called 'whitening' of BAT characterized by increased lipid deposition, mitochondrial dysfunction, and reduced fat oxidation. The aim of the current study was to unravel the rate and related mechanisms by which HFD induces BAT whitening and insulin resistance. Wild-type mice were fed a HFD for 0, 1, 3, or 7 days. Within 1 day of HFD, BAT weight and lipid content were increased. HFD also immediately reduced insulin-stimulated glucose uptake by BAT, indicating rapid induction of insulin resistance. This was accompanied by a tendency toward a reduced uptake of triglyceride-derived fatty acids by BAT. Mitochondrial mass and Ucp1 expression were unaltered, whereas after 3 days of HFD, markers of mitochondrial dynamics suggested induction of a more fused mitochondrial network. Additionally, HFD also increased macrophage markers in BAT after 3 days of HFD. Counterintuitively, the switch to HFD was accompanied by an acute rise in core body temperature. We showed that a single day of HFD feeding is sufficient to induce the first signs of whitening and insulin resistance in BAT, which reduces the uptake of glucose and triglyceride-derived fatty acids. BAT whitening and insulin resistance are likely sustained by reduced mitochondrial oxidation due to changes in mitochondrial dynamics and macrophage infiltration, respectively. Likely, the switch to HFD swiftly induces thermogenesis in other metabolic organs, which allows attenuation of BAT thermogenesis.