P < 0.05. ##P < 0.01.

**2. Food restriction induces hypercholesterolemia**

liver, lipolysis in adipose tissue, or increased cholesterol synthesis [15, 17, 19].

LDL cholesterol (mg/dL) 20 1026 ± 147 589 ± 98 702 ± 13

Triglycerides (mg/dL) 20 92 ± 29 53 ± 13 56 ± 9

Bodyweight (kg) 20 4.0 ± 0.2 4.3 ± 0.1 4.0 ± 0.1

dietary lipid lowering for 4 weeks (normal diet) or a restricted diet for 4 weeks (restricted diet).

LDL cholesterol levels are negatively correlated with SQSTM1/p62 protein levels in the liver (**Figure 1**), suggesting stimulation of autophagy as an alternative mechanism for the increase

**Figure 1.** Induction of autophagy in liver of rabbits that were fed 0.3% cholesterol for 20 weeks (baseline), followed by cholesterol withdrawal for 4 weeks either via a normal diet or a restricted diet (20% of normal diet). Liver samples of ten rabbits per group were analyzed by Western blotting for the expression of autophagy marker proteins LC3-II (A) and p62 (B). \*\*P < 0.01, \*\*\*P < 0.001 (One-way ANOVA with post-hoc LSD, n = 10 in each group). (C) Serum LDL-levels show an inverse correlation with liver p62 protein levels (Pearson Correlation Coefficient −0.44, P<0.05).

in serum lipids. SQSTM1/p62 is a scaffold protein that binds directly to the autophagosomal marker Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates via autophagy. Nutrient deprivation is a powerful autophagy-inducing condition [20]. Rabbits that undergo cholesterol withdrawal via a normal diet show high LC3-II levels but unaltered amounts of SQSTM1/p62 (**Figure 1A**), indicating moderate induction of autophagy. In contrast, rabbits undergoing severe food restriction show low levels of LC3-II and a clear reduction of SQSTM1/p62 (**Figure 1B**), which points to strong autophagy stimulation. It has been described that autophagy is strongly involved in managing intracellular lipids [21, 22]. Lipid droplets are taken up by lysosomes, where lysosomal acid lipases hydrolyze cholesteryl esters to generate free cholesterol for ATP-binding cassette transporter 1 (ABCA1)-mediated cholesterol efflux [21]. Impairment of autophagy in macrophages reduces reverse cholesterol transport [21], a condition that refers to net cholesterol flux from the peripheral tissues to the liver (for excretion via the bile). Conversely, pharmacological activation of the autophagy pathway attenuates lipid accumulation [23] and in some conditions (e.g., after treatment with mTOR inhibitors) triggers hypercholesterolemia [24].
