An apolipoprotein B100 mimotope prevents obesity in mice.
An apolipoprotein B100 mimotope prevents obesity in mice.
- 1Department of Molecular & Life Science, College of Science & Technology, Hanyang University (ERICA), Ansan, 426-791, Republic of Korea SJ Biomed Inc., HBI 604, 55 Hanyangdaehak-ro, Ansan, 426-791, Republic of Korea firstname.lastname@example.org email@example.com.
- 2Department of Molecular & Life Science, College of Science & Technology, Hanyang University (ERICA), Ansan, 426-791, Republic of Korea.
- 3Pathology, Korea University Medical School, Ansan Hospital, 123 Jeokgeum-ro, Danwon-gu, Ansan, 425-707, Republic of Korea.
- 4Department of Molecular & Life Science, College of Science & Technology, Hanyang University (ERICA), Ansan, 426-791, Republic of Korea firstname.lastname@example.org email@example.com.
Although apolipoprotein B100 (ApoB100) plays a key role in peripheral fat deposition, it is not considered a suitable therapeutic target in obesity. In the present study we describe a novel ApoB100 mimotope, peptide pB1, and the use of pB1-based vaccine-like formulations (BVFs) against high-fat diet (HFD)-induced obesity. In HFD- compared with chow-fed adolescent mice, BVFs reduced the 3-month body-weight gains attributable to increased dietary fat by 44-65%, and prevented mesenteric fat accumulation and liver steatosis. The body-weight reductions paralleled the titres of pB1-reactive immunoglobulin G (IgG) antibodies, and pB1-reactive antibodies specifically recognized native ApoB100 and a synthetic peptide from the C-terminal half of ApoB100. In cultured 3T3L1 adipocytes, anti-pB1 antibodies increased lipolysis and inhibited low-density lipoprotein (LDL) uptake. In cultured RAW 264.7 macrophages, the same antibodies enhanced LDL uptake (without causing foam cell formation). These findings make ApoB100 a promising target for an immunization strategy against HFD-induced obesity.
KEYWORDS: ApoB100; high-fat-diet-induced obesity; humoral immunity; mimotope
Apolipoprotein B (ApoB) is an abundant blood protein that is essential for forming various types of lipoprotein particles, most notably VLDL (very low density lipoprotein), LDL (low density lipoprotein), and chylomicrones. ApoB occurs in a whole-length version called ApoB100 (dominating in VLDL and LDL) and a C-terminally truncated version called ApoB48 (dominating in chylomicrons); the number meaning that only 48% of the sequence are maintained. The ApoBs have long been a focus of research in the field of atherosclerosis, where one approach has been to create antibodies against subdomains and to test their effect on atherosclerosis.
We have been interested whether an antibody approach towards ApoB epitopes would teach us something about the potential roles of ApoB100 in obesity. At very first glance, it seems obvious that ApoB100 could have a role in obesity. After all, ApoB100 is the defining component of the VLDLs, which are the main transport vehicles that carry neutral fats from liver to peripheral tissues including the white fat. Interfering with that transport by, for example, an ApoB100-neutralizing antibody might be expected to reduce fat deposition in the fat tissue.
However, there is no support for such an idea in the literature. In fact, this idea seems to have rarely been seriously considered. Altered levels of ApoB100 or lipoproteins have often been observed as the consequence of obesity, but never as its cause. At least in mice, a block of VLDL secretion caused a fatty liver, but did not have a significant impact on body weight, even under a high fat diet (1).
While these results looked discouraging, they might merely highlight the complexity and homeostatic potential of body weight regulation: They do not formally rule out the possibility of ApoB100 being able to influence body weight. Notably, the negative results were obtained in a context of genetic changes that primarily impacted ApoB100 sequence or secretion. However, we reasoned that an antibody approach might be able to affect wild type ApoB100 after secretion. By screening a phage display library with a human anti-ApoB100 antibody, we previously discovered a peptide (named pB1) that reacted with anti-ApoB but did not share a sequence homology (i.e., a mimotope). We decided to use this peptide for the vaccination of adolescent mice under a high fat diet. The vaccine was named ApoBMV, for “Apolipoprotein B-100 mimotope peptide vaccine” (Fig. 1). We found that ApoBMV induced ApoB100-reactive antibodies (anti-pB1 Ab) and at the same time significantly inhibited the weight gain caused by the fat diet. Importantly, the antibody titers over time paralleled the anti-obesity effect (2).
We then performed in vitro experiments to get an idea how these results, which appear to contradict the literature consensus, originated (2). The in vitro data suggest that the antibodies acted in two ways.
Firstly, anti-pB1 Ab interfered with LDL uptake by adipocytes (cell line 3T3-L1), a function that is known to require the C-terminal half of ApoB100. In support, antibodies raised against C-terminal and N-terminal portions of ApoB100 did and did not, respectively, reduce LDL uptake. Thus, while this pathway is unlikely to have a major impact on fat mass in vivo, the findings do suggest that the ApoBMV-induced antibody mimics an epitope in the C-terminal half of ApoB100, i.e. the region that is lacking in ApoB48. Moreover, and perhaps more important in vivo, we observed that the ApoBMV-induced antibodies blocked an inhibitory effect of LDL on adipocyte lipolysis, which is performed by hormone-sensitive lipase (HSL) (Fig. 2, ①). The inhibitory effect of LDL on adipocyte lipolysis is also known to be mediated by the interaction of the C-terminal half of ApoB100 with the LDL receptor (3).
Secondly, the same anti-pB1 Ab opsonized (V)LDL for uptake by macrophages (cell line RAW 264.7), an effect that is not observed with native (V)LDLs unless they are oxidized or modified. Thus, in the macrophage experiments, we observed the opposite of what we observed with adipocytes. Importantly, this did not lead to a conversion of the macrophages to foam cells (a phenomenon of macrophage lipid overload), while oxidized LDL (applied even without antibody) caused a foam conversion. To evaluate the possible relevance in vivo, we note that macrophages have the ability to perform fatty acid oxidation, and most importantly, are a highly abundant cell type in the fat tissue of obese animals (4). Thus, it appears that when the lipoproteins are delivered via opsonization to macrophages, the latter do not necessarily become overloaded but can oxidize the lipid, an effect that might well contribute to the prevention of obesity in vivo (Fig. 2, ①). Clearly, direct measurements of macrophage lipid oxidation are warranted. It is also important to mention that macrophages are known to exist in two “polarized” states (M1 and M2), one of which (M1) is proinflammatory and prevails in obesity and is held responsible for negative effects of obesity such as insulin resistance (5). We therefore hypothesize that vaccination facilitates a repolarization towards the M2 phenotype in vivo (Fig. 2, ①), and our very recent unpublished data support this idea.
In summary, our results support the hypothesis that upon vaccination with a mimotope of a peptide from the C-terminal half of ApoB100, the resulting antibodies (i) facilitate adipocyte lipolysis and (ii) re-route the lipoproteins (VLDL and / or LDL) to macrophages, which somehow dispose of them without foam cell formation. Considering that literature shows a remarkable homeostasis of fat in face of altered lipolysis, we consider the second (macrophage-based) mechanism as more likely to be important in vivo.
In addition to these two mechanisms, which already have some support from in vitro experiments, we believe that further, currently speculative, mechanisms may also contribute to the anti-obesity effect of ApoBMV. Firstly, we have to explain the prevention of a fatty liver in ApoBMV-treated, high fat diet-fed mice. It is conceivable that anti-pB1 antibodies interfere with the interaction between ApoB100 and the hepatic LDL receptor (Fig. 2, ②) or hepatic lipase (which is known to interact with the C-terminal half of ApoB100). Secondly, it is conceivable that in the bloodstream of peripheral tissues, the anti-pB1 antibodies interfere with the activation of lipoprotein lipase (LPL), which is known to work in concert with the VLDL receptor / LDL receptor-like receptor (LDLR-LR) on the surface of endothelial cells. As a result, peripheral lipoproteins would be less able to deliver free fatty acids to the peripheral tissues, including fat tissue (Fig. 2, ③).
Our results offer a new tool for dissecting lipoprotein (patho)physiology and suggest a new paradigm for the treatment of obesity, a condition for which effective therapies are still lacking.
(1) Minehira, K., Young, S.G., Villanueva, C.J., Yetukuri, L., Oresic, M., Hellerstein, M.K., Farese, Jr, R.V., Horton, J.D., Preitner, F., Thorens, B. et al. Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice. J. Lipid Res. 49, 2038–2044 (2008).
(2) Kim HJ, Lee HJ, Choi JS, Han J, Kim JY, Na HK, Joung HJ, Kim YS, Binas B. An apolipoprotein B100 mimotope prevents obesity in mice. Clin Sci (Lond), 130(2), 105-16 (2016).
(3) Skogsberg J., et al., ApoB-100-LDL acts as a metabolic signal from liver to peripheral fat causing inhibition of lipolysis in adipocytes PLoS ONE 3(11)e3771 (2008).
(4) Weisberg, S.P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R.L. and Ferrante, A. W. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest. 112, 1796–1808 (2003).
(5) Odegard J., I. and Chawla, A., Pleiotropic actions of insulin resistance and inflammation in metabolic homeostasis. Science 339, 172-177 (2013).