Lowering of plasma cholesterol by a vegan diet is not related to changes in circulating PCSK9 Aim: To test the hypothesis that reduced circulating PCSK9 levels explain the reduction of LDL cholesterol following a 12 month vegan diet intervention demonstrated in paper III.
Of 66 patients with rheumatoid arthritis enrolled, 38 were randomized to a vegan diet and 28 to a non–vegan diet for a period of one year. Twenty-two patients in the vegan diet group and 25 in the non-vegan group completed the study, representing a dropout rate of 42% and 11 %, respectively, and were included in our analyses. All patients were sampled at baseline, and after 3 and 12 months on their respective diet.
In the vegan diet group, total cholesterol was reduced after both 3 and 12 months by 13% and 12%, respectively, as compared to baseline. VLDL cholesterol was reduced after 3 and 12 months by 28% and 31%, respectively. LDL cholesterol was reduced after 3 months by 7%, dropping from 2. 0 (±0.81) to 1.9 (±0.70) mmol/L. HDL cholesterol increased after 12 month by 11%. No alterations in bile acid synthesis, cholesterol synthesis, or circulating PCSK9 levels were observed during the regimen. In the non-vegan group there were no significant changes in the cholesterol variables during the study.
Neither circulating PCSK9 levels nor altered cholesterol or bile acid synthesis explain the beneficial effects on serum lipid profiles observed after a vegan diet for one year in patients with rheumatoid arthritis.
5 GENERAL DISCUSSION
Cholesterol and bile acid homeostasis is under strict regulation from multiple mechanisms.
The bile acid activated nuclear receptor FXR is central to maintain bile acid homeostasis via regulation of transporters in their enterohepatic circulation and of key enzymes in bile acid synthesis. In mice, numerous proteins have been proposed to be of regulatory importance following genetic gain- and loss-of-function experiments, such as FXR [85], SHP [86, 87], bile acid transporters including BSEP [116, 117] and ASBT, factors influencing conjugation [118, 119], klotho and FGF15. Additional proteins discussed as of possible relevance to the regulation of bile acid synthesis are hepatic SHP2 [120] and intestinal Diet1 [121].The physiological importance of these pathways in humans remains to be determined.
In paper I we studied the importance of circulating FGF19 as a regulator of bile acid
synthesis during different cholestyramine treatment regimens. All observations in paper I are not fully compatible with the thinking that FGF19 is the major suppressor of bile acid
synthesis in humans. During the normalization period, both after a single day of
cholestyramine treatment and after long-time treatment, there were notable discrepancies between serum levels of FGF19 and the level of bile acid synthesis. Although the results from studying the onset of treatment would be well compatible with the concept that lowering of FGF19 results in a stimulation of bile acid synthesis, these observations indicate that an increased synthesis may remain also when FGF19 levels are normalized. This suggests that cholestyramine treatment may induce bile acid synthesis also via more direct hepatic pathways, independent of the circulating levels of FGF19. An alternative explanation for these discrepancies between the level of bile acid synthesis and FGF19 serum levels could be that the interaction between FGF19 and its receptor FGFR4 is modulated by cofactors which could modify the effect of circulating FGF19.
In response to nutritional and hormonal signals, the liver regulates lipid and carbohydrate metabolic pathways to maintain homeostasis. In paper I, we further studied if changes in the presumed metabolic regulator FGF19 could explain the change in TG levels during
cholestyramine treatment. We found that the elevated levels of TGs observed in healthy individuals developed unrelated to circulating levels of FGF19 or to the level of bile acid synthesis per se. During the night after a single day of treatment, TG strongly increased, peaked and declined to be doubled before breakfast. Further, glucose and insulin transiently increased during the night. In the morning after a single day of treatment FGF19 levels was strongly reduced compared to baseline, while BA synthesis (reflected by C4c levels) and cholesterol synthesis (reflected by lathosterol/c levels) were increased. In the experiment where weekly step wise increases of cholestyramine doses were used, circulating FGF19 was accordingly reduced while the levels of TGs, glucose or insulin were unchanged. In the one day treatment experiment, total serum bile acids were strongly reduced concomitantly with elevated levels of TGs. In addition, bile acid composition was altered with an enlarged percent of DCA, displaying a pattern similar to one described among patients with type 2 diabetes [122, 123]. Moreover, altered diurnal rhythms – non steady-state situations – may
directly or indirectly influence levels of triglycerides and glucose. Furthermore, mechanisms regulating apoB assembly and secretion are not fully defined and PCSK9 may have a
regulatory role on triglyceride rich lipoprotein production both in intestine [124] and liver [125].
By analyzing samples from studies of different experimental situations we have demonstrated that fasting strongly reduces circulating PCSK9 in healthy humans. This occurs
concomitantly with a suppressed cholesterol synthesis, as monitored by lathosterol
concentrations. Despite these pronounced dynamic changes, LDL cholesterol levels were not reduced. SREBP-2 activates genes required to generate PCSK9, HMG-CoA and LDLR. The strong correlation between PCSK9 and lathosterol observed in our studies makes it
reasonable to believe that hepatic PCSK9 and HMG-CoA reductase are regulated by a common mechanism in these situations. In contrast, during long time cholestyramine treatment and in response to a ketogenic diet, PCSK9 and HMG-CoA may not correlate. To separate transcription of genes regulated in a coordinated manner may sometimes be
advantageous. The efficiency of statins would be improved if regulation of PCSK9 and LDLR were separated [126]. During statin treatment a reduction in cholesterol synthesis (enzyme activity) together with an increase in the number of LDLRs and levels of circulating PCSK9 is demonstrated. What appears as a discrepancy between expression of PCSK9, HMG-CoA reductase and LDLR may not be a discrepancy at mRNA level, as statins inhibit the enzyme thereby stimulating its gene expression due to reduced hepatic cholesterol. The relationship between circulating PCSK9 and LDLR may be more complex due to the fact that 20-40 % of plasma PCSK9 may be bound to apoB in LDL [127-129]. Thus, a reduction of hepatic LDLRs would result in a reduced clearance of PCSK9, leading to its accumulation in plasma. Accordingly, subjects with FH – particularly homozygotes - show significantly higher levels of circulating PCSK9 compared to healthy subjects [130]. Moreover, we could demonstrate that humans treated with GH had reduced serum PCSK9 levels (paper II), which could contribute to the LDL-lowering effect of GH in humans [107]. Circulating PCSK9 is also reduced by thyroid hormone, which may likely contribute to the lower plasma LDLC levels in hyperthyroidism [131]. Also increased levels of estrogen reduce both PCSK9 and LDL cholesterol in women [112], and variations in endogenous estrogen during the menstrual cycle may contribute to the intraindividual variation in PCSK9 and LDLC in normal women [57].
Diet composition and eating patterns influence cholesterol metabolism. Knowledge of underlying mechanisms provides the ability to modulate regulation and homeostasis in desired direction. A ketogenic diet (paper II) increased cholesterol synthesis by 24% and total plasma cholesterol by 37%, but did not influence levels of circulating PCSK9. A vegan diet (paper III) reduced total-C after 3 and 12 months. We further investigated the possible mechanism(s) implicated in this dietary effect, but could not find evidence for any
involvement of PCSK9 or altered bile acid or cholesterol synthesis (paper IV). However, a randomized isocaloric trial for 10 weeks (HEPFAT) showed that PUFA, in contrast to SFA, decreased circulating PCSK9 in parallel with LDL cholesterol [132]. A Mediterranean diet
for 5 weeks in men with metabolic syndrome reduced LDL cholesterol and circulating
PCSK9 [133]. Further studies on how diet may influence LDL metabolism through effects on PCSK9 in humans will be of great interest. Moreover, the gut microbiome responds to an altered diet [134]. The microbiome plays a part in in controlling the composition of the acid pool, and hence also modulates bile acid signaling [135-137].
6 CONCLUSIONS
From the studies following conclusions can be drawn
Circulating FGF19 is markedly influenced by the transintestinal flux of bile acids, whereas its proposed role in the suppression of BA synthesis and TG levels may not always apply.
Circulating PCSK9 has a diurnal variation and is strongly reduced during fasting in humans. These changes may relate to diurnal oscillations in hepatic intracellular cholesterol levels.
A vegan diet reduced levels of oxLDL and LDL-cholesterol, and raised anti-PC IgA and IgM levels in patients with RA.
Changes in circulating PCSK9 or alterations in the synthesis of cholesterol or bile acids do not contribute to the improved lipid profile observed during a vegan diet.
7 ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to everyone who in various ways has contributed to this work. For discussions, encouragement, criticism, technical support, practical help, it was all essential. In particular, I would like to thank the following people:
First of all, my supervisor Mats Rudling for introducing me to this intriguing and interesting area of research. You have taught me a lot during these past years about how to conduct and present research – thank you!
I would like to thank my co-supervisor Bo Angelin, for all support and for generously sharing your extensive knowledge and for valuable discussions.
All co-authors: Ingiäld Hafström, Johan Frostegård, Guoqing Cao, Maria Dahlin, Ann-Charlotte Elkan, Mats Eriksson, Cecilia Gälman, Björn Kolsrud, Robert J. Konrad, Suzanne Lind, Lena Beckman (Persson), Bo Ringertz, Sara Straniero, Lars Ståhle, Jason S. Troutt, Håkan Wallén and Per Åmark, for contributions to the papers in this thesis.
Lena Emtestam for all your administrative help and for always having the right answer.
Ingela Arvidsson and Lisbet Benthin at Metabollab for inestimable technical support.
Britt-Marie Leijonhufvud, Katarina Hertel, Yvonne Widlund, Ewa Steninger and Sabine Süllow-Barin at Patient research center for their assistance with collection of blood samples and for taking excellent care of the participants in the studies.
A special thanks to Camilla Pramfalk and Ylva Bonde!
Friends and colleagues: Amani Al-Khaifi, Amit Laskar, Daniela Strodthoff, Johanna Apro, Moumita Ghosh, Sara Straniero, and former members of the group Lena Beckman, Thomas Lundåsen, Cecilia Gälman and Manuela Matasconi, it has been fun working with you, discussing science and many other aspects of life.
Special thanks also to Anna Ehrlund, Lisa-Mari Mörk, Lise-Lotte Vedin (I will very soon buy a new harness and a pair of climbing shoes), Patricia Humire, Per Antonsson and Eckardt Treuter for introducing me to Tomas.
All former and present colleagues and friends at Novum (none mentioned, none forgotten).
To the Ekström and Palosaari families, Birgitta and Arne thank you for a warm welcome.
Loving and supporting family; my parents Catharina and Tom, my brothers Daniel and Edvin. Thanks for your never ending love, support and energy.
Anne, Rebecka, Nick, Cornelia, Wilhelm and Neo.
Thea och Ellen, underbara älskade barn, tack för att ni finns!
Tomas, my life-companion, for endless love and support.
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