Caffeine is a stimulant LDLR-mediated cholesterol clearance is improved by SREBP2-induced hepatic PCSK9 expression

Received: 03-Mar-2022, Manuscript No. pulcj-22-4615; Editor assigned: 05-Mar-2022, Pre QC No. pulcj-22-4615(PQ); Accepted Date: Mar 15, 2022; Reviewed: 11-Mar-2022 QC No. pulcj-22-4615(Q); Revised: 13-Mar-2022, Manuscript No. pulcj-22-4615(R); Published: 26-Mar-2022, DOI: 10.37532/pulcj.22.6(2).13-14

Citation: Fasolato E. Caffeine is a stimulant LDLR-mediated cholesterol clearance is improved by SREBP2-induced hepatic PCSK9 expression. Clin Cardiol J. 2022; 6(2):13-14.

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Abstract

Caffeine (CF) appears to lower the risk of Cardiovascular Disease (CVD). The process by which this occurs, however, has yet to be discovered. The influence of CF on the expression of two genuine regulators of circulating Low-density Lipoprotein Cholesterol (LDLc) levels, the Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) and the Low-density Lipoprotein Receptor (LDLR), was studied in this study (LDLR). Following the discovery that CF decreased circulating PCSK9 levels while increasing hepatic LDLR expression, further CF-derived analogues with higher PCSK9 inhibitory potency than CF were created. The effect of CF on decreasing PCSK9 was later validated in a group of healthy individuals. We show that CF increases hepatic Endoplasmic Reticulum (ER) Ca²⁺ levels, which inhibits transcriptional activation of the Sterol Regulatory Element-binding Protein 2 (SREBP2), which regulates PCSK9, resulting in increased LDLR expression and LDLc clearance. Our findings reveal ER Ca²⁺ as a master regulator of cholesterol metabolism and a mechanism through which CF may protect against CVD.

Key Words

Cardiovascular disease; Low-density lipoprotein receptor; Sterol regulatory element-binding protein 2

Introduction

LDLC (Low-Density Lipoprotein Cholesterol) levels in the blood are strongly connected to the development of Cardiovascular Disease (CVD). Despite the approval of various LDLc-lowering medicines, many patients are unable to achieve their LDLc-lowering goals due to intolerance, side effects, or simply the high cost of pharmaceuticals. The Endoplasmic Reticulum (ER)resident transcription factor Sterol Regulatory Element-binding Protein 2 (SREBP2) is an essential regulator of LDLc. Reduced intracellular cholesterol and the decrease of ER Ca²⁺ activate SREBP2;, causing it to translocate to the nucleus and induce cholesterol regulating genes such PCSK9;, LDLR, and HMG-Co; reductase (HMGR). PCSK9; inhibits the ability of metabolically active organs, such as the liver, to remove excess LDLc from the bloodstream [1]. Anti-PCSK9; antibodies, based on these important discoveries, are now accessible to individuals at high risk of CVD, resulting in a 60%-70% reduction in LDLc levels6. Anti-PCSK9 antibodies are effective, but their high cost and/or requirement for subcutaneous delivery limits their access to patients globally. In order to produce more cost-effective medicines, greater research into the molecular mechanisms that affect the production and secretion of PCSK9 from hepatocytes is required. Caffeine (CF), also known as 1,3,7-trimethylxanthine, is a central nervous system stimulant found in a variety of plants and is usually found in coffee and tea. The majority of published literature shows that the average adult caffeine drinker consumes between 400 mg and 600 mg of caffeine per day, and organisations such as Health Canada and the Food and Drug Administration have concluded that such doses are not linked to toxicity, cardiovascular effects, bone status, calcium imbalance, behaviour, cancer incidence, or effects on male fertility. On the contrary, mounting data suggests that moderate to high levels of CF (>600 mg) eaten daily in the form of non-alcoholic beverages are linked to a lower risk of cardiovascular disease [2]. Although biochemical investigations have demonstrated that CF elevates intracellular Ca²⁺ levels and causes vascular endothelium vasodilation via nitric oxide release, a biological activity known to be cardioprotective, molecular mechanisms supporting clinical proof are still absent.

IN HEPATOCYTES, CF INHIBITS PCSK9 EXPRESSION AND SECRETION

To begin our research, we treated cultured immortalised hepatocytes known to express and secrete PCSK913, such as HuH7 and HepG2 cells, as well as primary mouse and human hepatocytes (PMH and PHH, respectively) 
with CF for 24 hours and measured PCSK9 expression using immunoblots and real-time PCR. These preliminary studies suggested that CF decreased PCSK9 protein and mRNA transcript levels. CF also reduced PCSK9 expression induced by thapsigargin, a SERCA pump antagonist and wellknown ER stress-inducing drug. Because sterol depletion is a well-known activator of SREBP2 activation, cells were additionally given CF in the presence and absence of U18666A (U18), a pharmacological drug that depletes intracellular sterols. CF inhibited U18-induced PCSK9 expression in the same way that TG did [3]. Importantly, CF inhibited PCSK9 secretion in HuH7 and HepG2 cultured hepatocytes, as well as PMH and PHH. By assessing levels of recombinant PCSK9 in the presence and absence of CF, control studies showed that CF did not interfere with the ELISA. To confirm that CF was not influencing global protein production, a Coomassie stain of electrophoretically resolved medium taken from these cells was employed. After then, HepG2 cells were given an increasing dose of CF. In the presence of ActD, CF fails to suppress PCSK9 mRNA and secreted protein, implying that CF has a transcriptiondependent effect on PCSK9. CF also failed to suppress PCSK9 secretion in cells transfected with a CMV-driven PCSK9 vector, corroborating these findings. These findings show that CF can significantly reduce PCSK9 expression at the mRNA and protein levels in a variety of cultured hepatocyte cell types at nanomolar concentrations, implying that the suppression occurs at the transcriptional level [4]. 

IN HEPATOCYTES, CF INHIBITS SREBP2 ACTIVITY 

ER stress, specifically depletion of ER Ca²⁺, enhances the activation of SREBP2 and the production of PCSK9, as our research group has previously showed. As a result, we investigated the impact of CF on TG-induced SREBP2 activation. We found that CF inhibited the expression of SREBP2 in PMHs and PHHs, as well as in HepG2 cells, which is consistent with earlier research. CF also blocked the expression of a downstream target of SREBP2 transcriptional activity in PMHs, HMGR, as well as SREBP1, the isoform known to regulate fatty acid synthesis. In the absence of TG, the effect of CF on the expression of hepatocyte nuclear factor 1a, a liver-expressed transcription factor likewise known to influence PCSK9 expression, was investigated, but no significant difference was found. In HuH cells transfected with a plasmid encoding GFP driven by the sterol regulatory element, SREBP2 activity was evaluated at the protein level. In the presence and absence of TG, CF inhibited the nuclear/activated isoform of SREBP2 (nSREBP2; 60 kDa) and the expression of SRE-driven GFP, which was consistent with the real-time PCR results. Immunofluorescent labelling was used to visualise GFP expression, which was then quantified using ImageJ software. SREBP2 immunofluorescence labelling in cells treated with TG in the presence and absence of CF revealed that CF inhibited SREBP2 re-localization from the perinuclear area to the nucleus. Given SREBP2’s well-known function in PCSK9 transcriptional control, our findings suggest that CF suppresses PCSK9 expression and secretion via inhibiting de novo synthesis. Following that, PMHs were isolated from Wildtype (WT) and Ampk1/ mice. PCSK9 expression and secretion were reduced in these hepatocytes after treatment with CF, indicating that AMPK is not directly implicated in CF-mediated PCSK9 suppression. CDN1163 (CDN), a pharmacologic drug known to raise ER Ca²⁺ levels by triggering SERCA pump activation, produced a comparable outcome in hepatocytes. 

PCSK9 EXPRESSION AND SECRETION ARE INFLUENCED BY ER Ca²⁺ 

CF’s potential to enhance intracellular Ca²⁺ levels is well-studied among its various intracellular effects. We hypothesised that (a) CF may raise ER Ca²⁺ levels, and (b) additional drugs known to enhance ER Ca²⁺ levels may also prevent SREBP2 activation and PCSK9 expression, based on our earlier findings that ER Ca²⁺ depletion increases SREBP2 activation. To test this idea, we used the high-affinity fluorescent Ca²⁺ indicator Fura-2-AM to look at cytosolic Ca²⁺ levels in CF-treated cells. In immortalised hepatocytes, CF dramatically raised cytosolic Ca²⁺ levels, which was consistent with prior research. D1ER, a genetically encoded ER-resident fluorescence resonance energy transfer (FRET)-based calreticulin chameleon Ca²⁺ sensor that increases in fluorescence intensity upon Ca²⁺ binding, was then used to monitor ER Ca²⁺ levels in cells transfected with it. Mag-Fluo-4, a lowaffinity Ca²⁺ indicator, was also used to measure ER Ca²⁺ and fluorescence intensity changes in response to Ca²⁺ binding [5]. Using a fluorescent spectrophotometer and a fluorescent microscope, the fluorescence intensity of cells treated with CF and control agents, TG and CDN, was measured and visualised. We discovered that, in addition to elevated cytosolic Ca²⁺ levels, CF also enhanced ER Ca²⁺ levels. The control agent CDN increased ER Ca2+ levels, as expected, but TG decreased ER Ca²⁺ levels. The high-affinity Ca²⁺ dye Fura-2-AM was used to evaluate ER Ca²⁺ concentration indirectly. HuH7 cells were pretreated with CF for 24 hours before being exposed to a high dosage of TG, which causes ER Ca²⁺ to be lost spontaneously. When cells were exposed to TG after being pretreated with CF, they showed enhanced ER Ca²⁺ efflux compared to cells treated with the vehicle control. We also discovered that CF promoted and TG inhibited the protein production of calnexin, an ER-resident protein with a high Ca²⁺ binding capability [6]. ELISAs were used to measure secreted PCSK9 levels in the medium of cells treated with Ca²⁺-modulating drugs. We found that high-dose ryanodine, CDN, and 2APB inhibited PCSK9 secretion, which was consistent with realtime PCR results. PCSK9 secretion was likewise prevented by overexpression of calnexin and loss-of-function ryanodine receptor mutants (RyR2E4872A and RyR2A4860G), which have previously been demonstrated to raise ER Ca²⁺ levels. We found that, in contrast to its influence on PCSK9 mRNA transcript levels, TG inhibited PCSK9 secretion, which is consistent with our earlier findings [7]. Sterol deprivation via treatment with U18, which hasn’t been shown to influence ER Ca²⁺ levels, resulted in enhanced PCSK9 secretion, which was in line with our prior findings. Finally, tests were performed with HepG2 cells cultured in Ca²⁺-deficient media for 48 hours to establish that CF prevented PCSK9 secretion in a Ca²⁺ dependent manner. We previously showed that this therapy causes significant ER stress, which accounts for the observed decrease in secreted PCSK9 levels in the absence of CF. Importantly, these findings show that CF has no effect on PCSK9 production in cells that have been depleted of Ca²⁺. Overall, these findings show that ER Ca²⁺ levels influence not only the production of ER stress indicators, but also the regulation of PCSK9 and SREBP2 [8].

Discussion

Others have looked into the impact of CF on the vascular system and CVD in the past. Because CF is commonly consumed in the form of beverages with variable doses and often mixed with adulterants such as dairy and sugar products, the results of such trials can be difficult to understand and often change. A new meta-analysis gives a comprehensive overview of the current body of knowledge on the effects of CF intake on cardiovascular outcomes, including total CVD. Surprisingly, the majority of research looked at, which included tens of thousands to hundreds of thousands of individuals, found that consuming CF reduced the risk of cardiovascular disease. The inhibition of adenosine receptors, GABA receptors, and phosphodiesterase enzymes, as well as generating intracellular Ca²⁺ transients through increasing RyRmediated Calcium-induced Calcium Release (CICR)11, are all established molecular targets for CF. Although the above interactions do not directly support our finding that CF raised ER Ca²⁺ levels, CF is also known to decrease ER Ca²⁺ release by inhibiting the IP3-receptor. CF has also been reported to bind to hepatic RyR, presumably blocking RyR-mediated Ca²⁺ release. The determination of the molecular processes relating to CF’s protective effect on the vascular system is difficult given the wide range of targets known to interact with it [9]. The ER functions as a vital and dynamic Ca²⁺ reserve, capable of extruding Ca²⁺ for signalling and/or excitatory reasons, as well as eliminating excess cytosolic Ca²⁺ after periods of excitation. The quantity of ER-resident low-affinity/high-capacity Ca²⁺ binding proteins determines the ER’s Ca²⁺ sequestering capability, which greatly exceeds that of the cytoplasm. Chaperone function is also aided by the interaction of Ca²⁺ with these proteins. Chaperones lose their folding capacity when ER Ca²⁺ is depleted, and misfolded polypeptides accumulate in the ER. The UPR is then activated in order to restore ER folding capacity and Ca²⁺ levels by increasing the quantity of ER-resident chaperones. Given the observed reduction in UPR marker expression by drugs that activate SERCA and increase ER Ca²⁺ influx or those capable of preventing leakage from either IP3R or RyR, boosting ER Ca²⁺ levels appears to have a net beneficial effect on ER homeostasis. We also discovered that CF protected cultured hepatocytes against TG-induced ER stress and lowered the expression of a number of ER chaperones in the livers of mice, which is consistent with our findings. PCSK9 promotes the beginning and progression of CVD by enhancing LDLR degradation, which is one of the most difficult and costly health care issues that society faces today. Understanding the regulatory mechanisms that regulate PCSK9 production and secretion from hepatocytes could contribute in the development of anti-PCSK9 medicines that are less expensive than those currently available. Overall, our findings support a concept in which small compounds such as CF, which can raise ER Ca²⁺ levels, can inhibit SREBP2 activation by improving GRP78 chaperone activity and binding ability [10].

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