a CF: cystic fibrosis.
b UMC: University Medical Center.
c CFTR: cystic fibrosis transmembrane conductance regulator.
Open-ended questionnaire analysis resulted in 28 subthemes. Most questionnaire findings were included in the qualitative part of this study. Findings that were not included either had high agreement rates (eg, no people with CF or parents felt too closely watched by their CF team) or required no further qualitative explanations (eg, the devices that people wanted to be able to access the RMP on). Qualitative analyses of interviews resulted in 380 specific codes divided into 7 themes and 12 subthemes. The themes and subthemes are presented in Multimedia Appendix 6 .
Between the second quarter of 2020 and the end of 2022, a total of 608 people with CF were enrolled on the program, and a total of 9418 lung function tests and 2631 symptom surveys were conducted. Questionnaire respondents reported that they used the program weekly (7/72, 10%), monthly (19/72, 26%), during symptoms (28/72, 39%), or rarely (18/72, 25%). Figure 1 shows the monthly enrollment rate, lung function rate, and symptom survey rate for all users enrolled in the program between January 2020 and December 2022. In interviews, HCPs reported a reduction in use frequency over time and especially after the introduction of a new modulator therapy (elexacaftor/tezacaftor/ivacaftor [ETI]). HCPs generally categorized people with CF into 4 different user groups: those who had never started using the program, those who started but discontinued using the program due to difficulties (technical, psychosocial, or otherwise), those who used the program only on indication, or those who used the program regularly. Consequently, HCPs doubted whether the program was suitable for everyone ( Textbox 1 , quote 1).
Overall, usability was well rated. Most users found the RMP easy to use (HCPs: 16/24, 67%; people with CF: 55/72, 76%; P =.42), and almost all respondents thought that most users would be able to quickly learn to use the RMP (HCPs: 23/24, 96%; people with CF: 60/72, 83%; P =.17). The most reported negative aspect of the RMP’s usability were technical errors related to the portable spirometer. In questionnaires, people with CF reported that they experienced technical errors sometimes (14/72, 19%), often (7/72, 10%), or very frequently (2/72, 3%). Moreover, 42% (10/24) of HCPs and 29% (21/72) of people with CF had doubts about the reliability of the home spirometry measurements, and 25% (6/24) of HCPs compared to 74% (53/72) of people with CF thought that people with CF used a good lung function technique at home.
In interviews, both people with CF and HCPs reported that home measurements were generally lower than hospital measurements or more variable. The mouthpiece of the home spirometer was often found difficult to use, especially by children. Some people with CF reported missing the support of a pulmonary function technician at home ( Textbox 1 , quote 2). Others found the home spirometer easy and quick to use and found that good education and experience helped improve technique and interpretation. HCPs reported that technical and reliability issues had caused some people with CF to lose motivation to use the program ( Textbox 1 , quote 3). For the HCPs themselves, the reproducibility of measurements, the flow-volume loop, and context (eg, increased symptoms, deviation from baseline, or own assessment of technique) were important to interpret results.
People with CF suggested adding percentage predicted values for home spirometry outcomes, and HCPs suggested better integration of the program into existing electronic health records and better education and communication about the program and new developments.
Questionnaire results showed that most people with CF and HCPs found the program a good addition to daily CF care (people with CF: 44/72, 61%; HCPs: 21/24, 88%; P =.02) or their job (HCPs: 21/24, 88%). Less than half of HCPs felt that the program improved work performance (5/24, 21%), productivity (8/24, 33%), or effectiveness (10/24, 42%). On the other hand, HCPs found that the RMP helped recognize early deterioration of people with CF (16/24, 67%) and gave more insights into people with CF’s disease courses (18/24, 75%). In interviews, HCPs reported that most benefits were present at the patient level rather than at a work performance level ( Textbox 1 , quote 4). However, HCPs especially appreciated being able to remotely monitor acute deterioration, which facilitated remote triage and adjustment and follow-up of treatments.
A total of 49% (35/72) of people with CF (people with CF: 22/54, 41%; parents of people with CF: 13/18, 72%; P =.03) believed that they were able to recognize deterioration sooner than without the RMP, 49% (35/72) believed that they needed less in-person outpatient visits when they were doing well, 36% (26/72) felt more in control, and 35% (25/72) felt more motivated for their treatment. From qualitative analyses, the most important benefits for people with CF included improved understanding of their condition, improved control over their condition, better symptom perception, and reductions in unscheduled and routine outpatient visits ( Textbox 1 , quote 5). Some parents of people with CF mentioned that the program improved communication with their children about their condition. Benefits varied across people with CF.
According to questionnaire results, 17% (12/72) of people with CF experienced stress due to the RMP. Qualitative analyses revealed positive psychosocial effects such as reassurance about health status and less anxiety before outpatient visits ( Textbox 1 , quote 6). However, for others, the program caused negative psychosocial effects such as increased stress and worries, confrontations between parents and children who disliked home measurements, frustrations with technical errors or bad outcomes, fears of deterioration, increased disease burden, and confrontation with health identities. HCPs were mostly aware of these effects, which sometimes required intervention ( Textbox 1 , quote 7). Most interviewed people with CF believed that the benefits outweighed the negative effects, whereas others had not experienced any psychosocial effects.
In both the questionnaire and interviews, the presence of symptoms was found to be an important incentive to use the RMP regularly. People with CF reported that a future reduction in outpatient visits would be a strong incentive for self-monitoring also during periods of well-being ( Textbox 1 , quote 8). Other incentives included agreements with and encouragement of HCPs regarding measurement frequency, creating a habit or setting reminders, living far away from the hospital, intrinsic motivation and a perceived necessity for self-monitoring, the fact that measurements are easy and quick to perform, and individually experienced benefits. Ultimately, people with CF wanted an adequate balance between monitoring burden and the benefits of use.
Stable periods were accompanied by little motivation for frequent self-monitoring. For many, these stable periods were more prevalent after initiation of ETI. Other disincentives for regular self-monitoring included having a good symptom perception, unclear implementation and goals of remote monitoring, the existence of other communication media (eg, direct phone lines or email), little intrinsic motivation, other health priorities, technical errors or doubts about reliability of outcomes, remote monitoring being mostly supplemental to regular care without reductions in therapy burden elsewhere, and the aforementioned negative psychosocial effects ( Textbox 1 , quote 9).
A total of 79% (19/24) of HCPs and 75% (54/72) of people with CF wanted more care remotely in the future rather than physically when people with CF were feeling well (ie, hybrid care). Although some people with CF wanted to keep using the RMP as supplemental to routine care, many people with CF and HCPs also supported hybrid care in interviews, especially in adult CF care. The most important driver was improvement in people with CF’s condition due to new modulator drugs. Hybrid care was thought to help people with CF reclaim agency over their lives and reduce the burden of hospital visits. However, there was a consensus among interviewed participants that in-person care could not be replaced fully by remote care and that there was a strong need for individualized approaches ( Textbox 1 , quote 10). In pediatrics, it was mentioned that evaluating the physical and psychosocial development of children was also important during outpatient follow-up and that this might not be fully replaceable by digital care.
Table 2 shows additional remote monitoring functions identified as useful for CF care in questionnaires. Additional remote microbiology assessments was the only repeated suggestion for future additions in interviews. One pulmonologist mentioned that, when additional measurements are required to assess patients remotely, it would be better to see the patient in person. Interviewees showed little support for pulmonary exacerbation prediction models requiring high self-monitoring frequencies since the introduction of ETI, but some acknowledged that they would have used these models before they started ETI ( Textbox 1 , quote 11).
HCPs (n=24), n (%) | People with CF (n=72), n (%) | value | CF group, n (%) | value | ||
People with CF (n=54) | Parents (n=18) | |||||
Monitoring of lung function | 22 (92) | 66 (92) | >.99 | 51 (94) | 15 (83) | .16 |
Monitoring of pulmonary symptoms | 17 (71) | 26 (36) | .004 | 19 (35) | 7 (39) | .78 |
A function that alerts people with CF when they are not doing well | 15 (63) | 20 (28) | .003 | 14 (26) | 6 (33) | .56 |
A function that alerts HCPs when people with CF are not doing well | 13 (54) | 32 (44) | .48 | 21 (39) | 11 (61) | .11 |
Monitoring gastrointestinal symptoms | 6 (25) | 15 (21) | .78 | 10 (19) | 5 (28) | .50 |
Tracking dietary requirements from the dietician or pancreatic enzyme use | 6 (25) | 7 (10) | .08 | 6 (11) | 1 (6) | .67 |
Tracking physiotherapy exercises or activity (eg, number of steps) | 5 (21) | 9 (13) | .33 | 9 (17) | 0 (0) | .10 |
Tracking medication use or side effects | 13 (54) | 24 (33) | .09 | 17 (31) | 7 (39) | .58 |
Tracking how people with CF feel with regard to their CF (eg, fear or confidence) | 11 (46) | 13 (18) | .01 | 10 (19) | 3 (17) | >.99 |
As little functions as possible | 2 (8) | 4 (6) | .64 | 3 (6) | 1 (6) | >.99 |
a Proportions of respondents who selected the functions were compared to each other using the Fisher exact test (health care professionals vs people with CF and people with CF vs parents of people with CF).
b HCP: health care professional.
c Significant difference.
Only 54% (13/24) of HCPs reported that it was easy to predict beforehand which people with CF would benefit from these programs. HCPs identified several requirements for remote monitoring in interviews: regular home measurements to maintain baseline values, a reliable lung function technique, motivation of people with CF, adequate technology savviness or (digital) health literacy, and adequate psychosocial coping strategies. For hybrid care, requirements for patients were found to be stricter and included a stable condition, adequate symptom recognition, alertness and responsibility during deterioration, and adequate therapy adherence. Therefore, an important prerequisite to be able to assess eligibility was being well acquainted with patients and maintaining this relationship remotely ( Textbox 1 , quote 12).
To optimize remote monitoring, requirements for HCPs included a more coaching role, adopting shared decision-making in deciding remote monitoring goals and methods, a change in mindset, including remote monitoring in regular follow-up conversations, clear department policies with a division of tasks and adequate safety nets, better education of (new) employees on remote monitoring, and improved communication between HCPs and developers. In most centers, the availability of motivated specialist nurses was essential as they performed most remote monitoring tasks ( Textbox 1 , quote 13).
This study identified experiences, future perspectives, and use behavior of remote monitoring in CF care. Our results show broad support among both HCPs and people with CF for remote monitoring. Benefits ranged from improved symptom perception to reduced routine outpatient care. Lung function home measurements were most appreciated. Pitfalls included negative psychosocial effects and the potential to lose sight of patients. Multiple incentives or disincentives to use the program were identified, such as the severity of physical symptoms. Future perspectives were centered on hybrid care models, personalized remote monitoring strategies, and balancing experienced benefits and monitoring burden.
A recent study identified similar patient benefits of remote monitoring, such as improved control and symptom recognition [ 24 ]. In previous work, we also found these benefits in pediatric asthma [ 12 ]. Unfortunately, these benefits have received little attention in the literature, potentially because they have not been systematically identified, lack good objective end points, or are hard to express in financial savings. Nevertheless, these benefits can be supportive to individual patients or parents. Future research should distinguish the varieties of individual patient benefits as well as identify objective end points. One possible example of end points could be personalized electronic patient-reported outcome measures [ 25 ].
The literature suggests that between a third and a half of people with CF are eager to replace regular care with remote care [ 11 , 26 ]. In our study, >75% of HCPs and people with CF wanted more remote care and less in-person care during periods of well-being. Presently, a minimum of 4 outpatient visits annually is a quality criterion for CF outpatient care in the Netherlands and according to the European CF Society best practice guideline [ 27 ]. Most interviewees considered a minimum of 2 annual outpatient visits feasible and desirable when supplemented with remote monitoring. These findings are in line with those of Hendra et al [ 28 ]. Our findings may be explained by the improved physical condition of our respondents as 85% used ETI and by the overrepresentation of adults in our study. Parents and HCPs in pediatrics also encouraged hybrid care models but were more reluctant to reduce follow-up visits because of safety and the specialty’s broader developmental focus. Nevertheless, a reduction of 50% in regular outpatient visits with remote monitoring has already been proven safe and efficient for pediatric asthma [ 13 , 14 ].
An important benefit not previously identified was the ability of people with CF to objectify symptoms at home, which helped guide HCPs and people with CF through periods of deterioration and recovery. Although the reliability of lung function outcomes was frequently questioned, especially by HCPs, adequate training, education, and regular practice could mostly overcome this issue. Consequently, remote monitoring has potential to reduce unscheduled health care consumption (eg, outpatient visits and emergency department visits), but this needs to be studied further.
Up to 20% of questionnaire respondents experienced increased stress from using the program. For some, the psychological burden of home monitoring required intervention. These results are more discouraging than previously reported acceptance rates [ 5 , 11 , 24 , 29 ]. This might be due to selection bias or a lack of good psychosocial screening methods. These findings emphasize that HCPs need to make time for education, regular evaluation, and support. This also requires motivation and new digital health skills for HCPs and especially for CF nurses, who played a crucial role in the remote monitoring of people with CF. Moreover, it illustrates that not everyone might benefit from remote monitoring. Patients should be responsible and motivated and have sufficient (digital) health literacy and adequate coping strategies. Importantly, these interventions should not be forced on patients but should be evaluated on a patient-by-patient basis through open communication and shared decision-making. This requires an adequate relationship between patients and HCPs not just at the initiation of remote monitoring but also throughout the complete patient journey, where a long-term relationship is required to evaluate the success of remote monitoring and make adaptions to the individual strategy accordingly. Maintaining this relationship will pose a new challenge in the era of digital health as the nexus between patients and HCPs will be increasingly distant in both time and space.
Up to 40% of respondents only used the program during periods of increased symptoms. People with CF likely prioritize reducing their disease burden as they improve physically with new modulator drugs [ 8 , 30 , 31 ]. Therefore, people with CF might be disincentivized to regularly self-monitor when it is mostly supplemental to regular care. In addition, similarly to the findings by Simpson et al [ 32 ], people with CF identified less monitoring functions as useful than HCPs. These findings were reinforced by the objective observation that home measurement rates declined after COVID-19 lockdowns and after the introduction of ETI. These disincentives offer an explanation for the low monitoring adherence in many studies [ 5 , 11 , 24 , 29 , 33 ]. It also corresponds to the negative change in attitude of people with CF toward frequent monitoring to predict deterioration after starting ETI. Nonetheless, many people with CF have no access to modulator therapies or have disappointing responses, and they might still benefit from future prediction models. Generally, a balance must be struck between individual benefits and the burden of remote monitoring.
Our mixed methods design enabled the integration of multiple sources of data from a large group of people with CF. To prevent both interpretation and confirmation bias, 2 experienced qualitative researchers (YE and MH) with different backgrounds (psychology [YE] and nursing sciences [MH]) were involved in the collection and analysis of qualitative data. These researchers were not involved in the RMP. To our knowledge this is the most comprehensive appraisal of remote monitoring in CF within long-term, multicenter care. Recently, there have been increasing calls for studies such as this one [ 5 ]. Moreover, the overlap in findings with those of other studies in pediatric and adult asthma suggests that our results translate well to other pulmonary diseases [ 12 , 14 ].
The COVID-19 pandemic was the direct cause for the initiation of the RMP. Hence, the time of implementation was turbulent and not representative of regular CF care before the pandemic. This should be considered in interpreting the results. For example, few HCPs found that the RMP improved their work performance and productivity, but this might also be explained by a preoccupation with COVID-19–related care and managing the overburdened health system. Consequently, the full potential of the RMP might not have been reached as there was little time to create clear remote monitoring strategies and policies even though they were found to be important prerequisites for successful implementation. We initially aimed to include more medical doctors in interviews because we aimed for equal representation of different HCPs involved in the remote monitoring of people with CF. However, data saturation was reached after 7 interviews. Possibly, CF nurses were more likely to respond to interview invitations as their role was crucial in the implementation of remote monitoring in routine care. Finally, because we recruited participants through informed e-consent to our program, we were unable to reach people with CF who stopped using the program. HCPs were added to provide some insights into this population.
The results of this study can be used to synthesize 4 future directions of remote monitoring in CF care, which are summarized in Textbox 2 (sensible strategies for remote monitoring). These strategies can be used simultaneously and alternately depending on active needs, capacities, and available resources of people with CF, HCPs, and the overarching health system. In more practical terms, this means that the different sensible strategies should be implemented based on what resources are available to support remote monitoring within the local context and what local priorities for digital support of the health system are in place. At a patient level, the sensible strategies should translate to an iterative process of shared decision-making on remote monitoring goals, methods, and evaluation and adaption that evolves alongside the changing and dynamic needs of patients. This requires clear remote monitoring policies and tasks and should foster the continuing relationship between patients and HCPs.
The sensible strategies provide a first goal-based frame to conceptualize remote monitoring in clinical practice while recognizing the need for flexibility in applying these principles to local contexts and individual patients. Consequently, the sensible strategies can also be applied in resource-scarce settings where conventional communication media (eg, telephone or SMS text messages) are more accessible than digital programs. As at least patient-driven and care-driven approaches have been identified in previous work in pediatric asthma, it is probable that the sensible strategies can also be used for other CRDs [ 12 , 14 ].
We recognize that the sensible strategies might not be complete, but it is the first goal-based classification of remote monitoring strategies that originated within routine, long-term CRD care. These strategies have the potential to be a first step in resolving the disconnect between current research efforts and clinical applicability as the strategies allow researchers to align their efforts with a clinically relevant strategy that is accompanied by clinical considerations. Moreover, the strategies will empower patients, clinicians, and policy makers to make informed decisions about the use of remote monitoring within their own contexts. Nevertheless, future efforts are needed to make the sensible strategies more actionable by expanding them, validating them, and defining potential end points while acknowledging the inequities in available resources for digital health worldwide.
Remote monitoring can offer a range of benefits for HCPs and people with CF at both the individual and collective levels. It is essential to integrate remote monitoring strategies into care models according to local capacities and needs to maximize benefits while ensuring feasibility. Many previous studies have focused on predicting deterioration. These interventions are complex and expensive and require significant long-term effort from patients. Therefore, they will not be beneficial for everyone or always feasible in daily practice. The sensible strategies for remote monitoring in chronic respiratory diseases in Textbox 2 can help facilitate the integration of remote monitoring into the care models of CF and other CRDs as it aims to help clinicians, researchers, and policy makers align remote monitoring with local demands and capacities. Future research should pay more attention to the other sensible strategies as they better correspond to value-based health care and could provide immediate support for patients, HCPs, and the overarching health systems.
The authors would like to thank the Dutch Cystic Fibrosis Foundation for donating the portable spirometers and for providing valuable insights and guidance in the development of the questionnaires. They would like to thank the Dutch Cystic Fibrosis Foundation and the Steering Group for supplying the Dutch Cystic Fibrosis Registry data used in this study. They would also like to thank all people with cystic fibrosis and health care professionals who participated in the interviews. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors MCO, LSvdW, JR, and PJFMM are involved in the development of the software used in this study. The software is property of the Radboud University Medical Center and, hence, does not generate any profits for the authors. No generative artificial intelligence was used at any stage of this study.
Anonymized data from this study will be made available upon reasonable request.
All authors were involved in the conceptualization of this study and recruitment of participants. MCO, YE, MH, JR, and PJFMM were responsible for the study design. MCO managed the project administration, visualization of results, writing of the manuscript, and quantitative analyses supervised by PJFMM and JR. MCO and YE conducted the formal analysis of the qualitative results supervised by MH, JR, and PJFMM. All authors were involved in reviewing the manuscript.
None declared.
STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) checklist.
Details of the remote monitoring program.
Questionnaires including answers (translated).
Interview guides (translated into English).
COREQ (Consolidated Criteria for Reporting Qualitative Research) checklist.
Themes and subthemes.
cystic fibrosis |
cystic fibrosis transmembrane conductance regulator |
Consolidated Criteria for Reporting Qualitative Research |
chronic respiratory disease |
elexacaftor/tezacaftor/ivacaftor |
health care professional |
remote monitoring program |
technology acceptance model |
Edited by S Ma, T Leung; submitted 28.11.23; peer-reviewed by T Sirari, H Oh; comments to author 18.03.24; revised version received 09.04.24; accepted 27.05.24; published 06.08.24.
©Martinus C Oppelaar, Yvette Emond, Michiel A G E Bannier, Monique H E Reijers, Hester van der Vaart, Renske van der Meer, Josje Altenburg, Lennart Conemans, Bart L Rottier, Marianne Nuijsink, Lara S van den Wijngaart, Peter J F M Merkus, Maud Heinen, Jolt Roukema. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 06.08.2024.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research (ISSN 1438-8871), is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included.
Journal of Animal Science and Biotechnology volume 15 , Article number: 105 ( 2024 ) Cite this article
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In the realm of swine production, optimizing body composition and reducing excessive fat accumulation is critical for enhancing both economic efficiency and meat quality. Despite the acknowledged impact of dietary calcium (Ca) and phosphorus (P) on lipid metabolism, the precise mechanisms behind their synergistic effects on fat metabolism remain elusive.
Research observations have shown a decreasing trend in the percentage of crude fat in carcasses with increased calcium and phosphorus content in feed. Concurrently, serum glucose concentrations significantly decreased, though differences in other lipid metabolism-related indicators were not significant across groups. Under conditions of low calcium and phosphorus, there is a significant suppression in the expression of FABPs, CD36 and PPARγ in the jejunum and ileum, leading to inhibited intestinal lipid absorption. Concurrently, this results in a marked increase in lipid accumulation in the liver. Conversely, higher levels of dietary calcium and phosphorus promoted intestinal lipid absorption and reduced liver lipid accumulation, with these changes being facilitated through the activation of the CAMKK2/AMPK signaling pathway by high-calcium-phosphorus diets. Additionally, the levels of calcium and phosphorus in the diet significantly altered the composition of liver lipids and the gut microbiota, increasing α-diversity and affecting the abundance of specific bacterial families related to lipid metabolism.
The evidence we provide indicates that the levels of calcium and phosphorus in the diet alter body fat content and lipid metabolism by modulating the response of the gut-liver axis to lipids. These effects are closely associated with the activation of the CAMKK2/AMPK signaling pathway.
Calcium and phosphorus, fundamental to the mineral nutrient spectrum, play an indispensable role in bone development and act as critical secondary messengers in cellular signaling. The significance of these minerals transcends the mere provision of skeletal integrity, extending to a broad spectrum of physiological processes, including muscle contraction, the facilitation of neurotransmitter dissemination, hormone secretion, and the regulation of body weight equilibrium. These roles underscore the indispensable contribution of calcium and phosphorus to both developmental and homeostatic mechanisms within the biological system [ 1 , 2 ]. In the field of lipid metabolism, calcium and phosphorus are essential for the maintenance of a healthy state. Calcium augments energy utilization and weight regulation through the facilitation of lipolysis, amplification of insulin sensitivity, meticulous regulation of fatty acid oxidation, and nuanced modulation of cholesterol levels. On the other hand, phosphorus occupies a central position in energy metabolism. The role of phosphorus in ATP production is well-established, significantly impacting lipid synthesis and catabolism, lipoprotein metabolism, and, through the regulation of hormones like insulin, subsequently influencing the overarching lipid equilibrium. Calcium supplementation has been shown to mitigate organ fat accumulation induced by a high-fat diet and to enhance intramuscular fat storage in livestock, indicating its potential in improving meat quality and animal health [ 3 ]. Conversely, whilst phosphorus supplementation was able to reduce overall lipid content, it also reduced intramuscular fat content, demonstrating its subtle effects on lipid distribution within muscle tissue [ 1 , 4 ]. Previous studies have focused on the dual regulation of calcium and phosphorus on muscle performance, leaving a gap in understanding the effects of dietary calcium and phosphorus changes on overall lipid metabolism and the mechanisms involved. This knowledge gap underscores the need for a more comprehensive exploration of how these minerals interact in complex networks of lipid metabolism that may provide new nutritional strategies for animal health and production performance.
In the animal body, lipids play a multifaceted and critical role, not only as a major energy reservoir, but also influencing livestock production and meat quality. However, excessive accumulation of fat not only reduces lean meat percentage and meat quality, but also affects feed conversion efficiency and may even lead to serious health problems such as fatty liver [ 5 ]. Therefore, understanding and controlling fat accumulation and its metabolic processes are important for improving meat quality, enhancing animal health, and improving feed efficiency. As a central regulator of intracellular energy metabolism, 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is essential for maintaining the stable availability of glucose, glycogen and fatty acids. In addition, AMPK plays a key role in signaling pathways that sense intracellular lysosomal and nuclear DNA damage [ 6 ]. Studies have shown that AMPK has an important role in the regulation of lipid and glucose metabolism in the heart, hypothalamus, adipose tissue, muscle, and liver. Calcium has a marked inhibitory effect on endogenous lipid production in the liver through activation of the AMPK pathway, but the deeper molecular mechanisms have not been elucidated [ 7 ]. As an upstream signal of AMPK, calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) can enhance its regulatory effect on lipid metabolism by activating AMPK, and conversely AMPK activation can influence Ca 2+ signaling and modulate CAMKK2 activity. The function of CAMKK2, especially in regulating lipid metabolism has been demonstrated in several studies [ 8 , 9 , 10 ]. Both genetic deletion and pharmacological inhibition of CAMKK2 significantly reduces de novo fatty acid synthesis and may bring about amelioration of high-fat diet-induced fatty liver, reduced insulin sensitivity, and prostate cancer cell proliferation [ 11 , 12 ].
Although calcium and phosphorus, as key mineral nutrients, play important roles in maintaining the body’s lipid metabolism homeostasis, the understanding of how different dietary calcium and phosphorus intakes specifically affect lipid metabolism and their molecular mechanisms is incomplete. In view of this, the aim of this study was to investigate the effects of different calcium and phosphorus levels (normal, low and high) on lipid metabolism and the mechanisms behind them using a weaned piglet model. In particular, this study focused on the interaction between intestine and liver during lipid absorption and processing, revealing how increased calcium and phosphorus levels ameliorate disordered lipid metabolism by activating the AMPK/CAMKK2 pathway in the intestine-liver axis. Further, this study also examined the correlation between gut microbiota and liver lipid composition, exploring how the intestine-liver axis serves as a key mechanism by which dietary calcium and phosphorus regulate lipid metabolism. This comprehensive study not only deepens our understanding of the role of calcium and phosphorus in the regulation of lipid metabolism, but also provides new insights into how to optimize lipid metabolism by modulating calcium and phosphorus intake in the diet.
Ethics statement.
All animal experimental procedures were approved by the Laboratory Animal Welfare Ethics Committee of the Institute of Animal Science, Guangdong Provincial Academy of Agricultural Sciences, in accordance with the current animal protection law (Ethical approval code: GDIAS20221103).
Seventy-two weaned piglets (Duroc × Landrace × Large White), with an initial body weight of 7.23 ± 0.92 kg and aged 25 d, were randomly assigned to 3 treatment groups. Each treatment included 6 replicate pens, with each pen housing 4 piglets (2 castrated males and 2 females). No significant differences in body weight were observed between pens ( P > 0.05). The experiment was divided into two phases: the 7–11 kg phase and the 12–25 kg phase. The study demonstrated that weaned piglets exhibited higher average daily gain and feed conversion ratio when the dietary ratio of standardized total tract digestible (STTD) calcium to STTD phosphorus was 1.2 [ 13 ]. Therefore, the NRC (2012) [ 14 ] recommended STTD P levels (0.34% to 0.42%) were used as the control group, with the difference in the recommended STTD P levels between the two phases serving as the concentration gradient. Accordingly, this study adjusted the dietary calcium and phosphorus levels based on the NRC (2012) [ 14 ] nutritional guidelines to maintain a constant STTD Ca to STTD P ratio of 1.2. The STTD Ca:STTD P ratios for the 3 groups were 0.504%:0.42% (CON), 0.216%:0.18% (LCAP), and 0.696%:0.58% (HCAP). The specific dietary composition and nutritional levels are detailed in Table 1 . Additionally, to eliminate the influence of phytase on the experiment, no phytase was added to the diets. The feed was prepared as pelleted feed, with pelleting temperatures ranging from 80 to 83 °C and a die aperture diameter of 3.5 mm. For the first phase, 200 kg of feed was produced per batch, while 135 kg was produced per batch for the second phase. Animals were housed in individual pens of equal size. The trial period lasted for 42 d, during which the piglets had ad libitum access to feed and were provided with ample clean water. All pigs were vaccinated according to standard immunization protocols.
After a 12-h fast starting at 20:00 on d 42 of the trial, each piglet was weighed individually at 8:00 on d 43. From each group, 3 female piglets and 3 castrated male piglets closest to the group’s average body weight were selected, totaling 6 piglets per group and 18 piglets in all. Prior to slaughter, 10 mL of blood was collected from the anterior vena cava of each of the 18 selected piglets into anticoagulant tubes, and an additional 10 mL was collected into non-anticoagulant tubes and allowed to stand for 1 h. The samples were then centrifuged at 3,500 r/min and 4 °C for 12 min to separate serum and plasma. These were aliquoted into sterile 1.5 mL EP tubes, with each tube containing 500 μL, and stored at −20 °C for subsequent biochemical analysis of serum indicators. The piglets were anesthetized using electrical stunning after blood collection, followed by euthanasia. Upon opening the abdominal cavity, 1 cm ring-shaped sections from the mid-jejunum and distal ileum, as well as 3 cm 3 of liver tissue, were collected. These samples were gently rinsed with phosphate-buffered saline (PBS) using a syringe, then quickly frozen in liquid nitrogen and stored at −80 °C for subsequent research. Additionally, chyme from the colon was collected, rapidly frozen in liquid nitrogen, and stored at −80 °C for further experiments. Portions of the liver and intestinal tissues were fixed in 4% paraformaldehyde for subsequent histological examination.
In summary, on d 43 of the experiment, after collecting the required samples from each slaughtered piglet, all remaining blood, intestinal and liver tissues, and other abdominal organs, along with the entire body, were processed into a bone and meat mixture using grinders of various sizes. The chopped samples were thoroughly homogenized in a blender. Wet samples were taken using the quartering method, spread evenly on petri dishes, dried in an oven at 65 °C for 72 h, vacuum-packed, and stored at −20 °C until further analysis. The total fat content of carcass was determined by Soxhlet extraction method. Briefly, three replicates of each sample were taken and about 30 g of carcass samples were ground into minced meat, dried in a vacuum freeze dryer and then ground into powder. 1 g of dried meat sample (accurate to 0.0001 g) was wrapped in filter paper into a cylindrical filter cup and extracted with n-hexane at 140 °C for 50 min in a Soxhlet extraction unit (SE-A6, ALVA, Jinan, China). After air-drying for 10 min and baking at 102 °C for 30 min, the extracted oil-containing aluminium cups were accurately weighed (to the nearest 0.0001 g) when the aluminium cups were cooled to room temperature. Total carcass fat (%) = (weight of oil-containing aluminium cups after extraction − weight of empty aluminium cups)/weight of air-dried meat samples × 100%.
Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose (GLU) concentrations were determined using an automated biochemical analyser (iMagic-M7, ICUBIO, Shenzhen, China). Intestinal wall samples, liver samples and carcass samples were homogenised in saline solution (1:9) and sediment was removed by centrifugation (3,000 r/min, 10 min) to obtain 10% tissue homogenate. The TC and TG levels in the intestines, liver and carcass were all measured using colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
The liver tissues of piglets fixed in 4% paraformaldehyde were frozen and embedded. The cryosections were cut into 10 μm thickness and stained with Oil red O (G1015, Servicebio, Wuhan, China). The sections were washed with 85% propylene glycol and then with distilled water before being stained with hematoxylin. The staining process was repeated after each wash. The presence of lipid droplets was indicated by a red stain. Sections were examined under a microscope (B80i, Nikon, Tokyo, Japan), photographed and recorded using Image-Pro Plus 6.0 software for subsequent comparison.
Total RNA from intestine and liver tissues was extracted with TRIzol reagent (Takara Bio, Shiga, Japan). 1 μg of RNA was then reverse transcribed to cDNA using the PrimeScript RT kit with cDNA Eraser (Takara Bio) according to the kit instructions. Quantitative PCR (qPCR) was conducted on a CFX Connect Detection System (Bio-Rad, Hercules, CA, USA) using a SYBR Green mixing kit (Takara Bio, Shiga, Japan) in a reaction volume of 20 μL. The 20 μL of reaction system showed as below, SYBR Premix Ex Taq (10 μL), upstream primer (0.4 μL), upstream primer (0.4 μL), downstream primer (0.4 μL), ROX II dye (0.4 μL), cDNA samples (2 μL), and ultrapure water (6.8 μL).
The real-time quantitative PCR reaction conditions were: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 5 s, annealing at 60 °C for 34 s, extension at 95 °C for 15 s, and the number of cycles of amplification was 40. The results were calculated by the 2 −ΔΔCt method, and all the primer sequences required for qPCR in this study were shown in Table S1 .
The samples were separated by SDS-PAGE and the protein was transferred to PVDF membrane (MilliporeSigma, Burlington, MA, USA). The following antibodies were used in western blotting, such as β-actin (Affinity, T0022, 1:3,000), PPARγ (Abcam, 209350, 1:1,000), SIRT1 (Cell Signaling Technology, 9475S, 1:1,000), AMPKα (Cell Signaling Technology, #5831, 1:1,000), phospho-AMPKα (Thr172; Cell Signaling Technology, #2535, 1:1,000), CAMKK2 (Proteintech, 111549-1-AP, 1:1,000), phospho-CAMKK2 (Ser511) (Cell Signaling Technology, #12818, 1:1,000), DGAT1 (Abcam, ab54037, 1:1,000), ACC1 (Cell Signaling Technology, #4190, 1:1,000), SREPB1C (Abcam, ab28481, 1:1,000), and FASN (Cell Signaling Technology, 3180S, 1:1,000).
Fixed intestinal tissues were embedded in paraffin and cut into 4 μm sections, then dewaxed, rehydrated, and treated in microwave oven with EDTA-containing antigen retrieval buffer (pH 8.0, Beyotime Biotechnology Co., Ltd., Shanghai, China). Afterwards, sections were blocked with 5% fetal bovine serum (Bioss, Beijing, China) for 1 h at room temperature and then incubated overnight (1:500 dilution) at 4 °C with rabbit anti-AMPKα, CD36, FABP4 (Service Bio, Wuhan, China). After rinsing with PBS, they were incubated with Alexa Fluor 555 (BBI, Shanghai, China)-conjugated goat anti-rabbit secondary antibody for 30 min at room temperature in the dark. In addition, images were obtained under a microscope with magnification of 200 (Nikon, Tokyo, Japan) and positive results were quantified using ImageJ 1.54 software.
The total lipids were extracted from the livers of three groups of piglets. After thawing, liver tissue (20 mg) was homogenized in 1 mL mixture (methanol, MTBE, and internal standard mixture). After that, ultrasound was performed at 4 °C for 20 min and then left at room temperature for 30 min. The solution was centrifuged at 10 °C at 14,000 × g for 15 min to obtain an upper organic solvent layer and dried under nitrogen. Samples were analyzed using a high-performance liquid chromatography system (Nexera UHPLC LC-30A, Shimadzu, Kyoto, Japan) and LC separation was performed on a column (Acquity Premiercsh C18, 1.7 μm × 2.1 mm × 100 mm, Waters, Milford, USA). The lipid extract was redissolved in 200 mL of 90% isopropanol/acetonitrile, centrifuged at 14,000 × g for 15 min, and finally injected with 3 mL of the sample. Solvent A was acetonitrile–water (6:4, v/v) containing 0.1% formic acid and 0.1 mmol/L ammonium formate, and solvent B was acetonitrile-isopropanol (1:9, v/v) containing 0.1% formic acid and 0.1 mmol/L ammonium formate. The initial flow rate was 300 μL/min with 40% solvent B. Hold for 3.5 min, then linearly increase to 75% of solvent B within 9.5 min, then linearly increase to 99% of solvent B within 6 min, then equilibrate in 40% of solvent B for 5 min. The mass spectra were obtained by Q-exactive Plus in both positive and negative modes. All measured ESI parameters were optimized and preset as follows: source temperature, 300 °C; capillary temperature, 350 °C, ion spray voltage set to 3,000 V, and S-Lens RF level set to 50%, respectively, the scanning range of the instrument is set to m/z 200–1,800. Lipid search was used for peak recognition, peak extraction, and lipid identification (secondary identification). Fatty acid composition expressed as a percentage of total fatty acids.
Total genomic DNA of the gut microbiota was extracted from samples of colon contents using a DNA isolation kit (Omega Bio-Tek, Norcross, GA, USA). DNA concentration was determined using a Nanodrop instrument (Thermo Fisher Scientific, Waltham, MA, USA). DNA integrity was assessed using 2% agar gel electrophoresis. Universal forward primer (5'-CCTAYGGGGRBGCASCAG-3') and reverse primer (5'-GGACTACNNGGGGTATCTAAT-3') were used to amplify the V3-V4 region of the bacterial 16S rRNA gene. Sequencing was performed on the Illumina MiSeq/NovaSeq platform. The DADA2 module in QIIME2 software (Version QIIME2-202202) was used for noise reduction. Microbial composition diversity was analysed using QIIME and R software. PICRUSt2 software was used for KEGG pathway analysis.
Short-chain fatty acids in colon contents were determined using gas chromatography. To prepare the sample, 50 mg of colon contents were mixed with 250 µL of ultrapure water for 5 min. The suspension was then centrifuged at 5,000 r/min for 30 min. Next, 1 mL of supernatant was transferred to a 2-mL PE tube and mixed with 200 μL of 42 mmol/L crotonic acid and 200 μL of 10% metaphosphoric acid solution. The PE tubes were refrigerated overnight at 4 °C and then centrifuged at 10,000 r/min for 10 min at 4 °C. The resulting supernatant was mixed with an equal amount of ether and extracted for 5 min. The ether layer was aspirated using a disposable syringe, filtered through a 0.22-μm organic membrane, and then injected into a brown vial for injection. Short-chain fatty acids (acetic, propionic, butyric, valeric, isobutyric and isovaleric acids, with crotonic acid as an internal standard) were measured using a gas chromatograph and a mass spectrometry detector (7890A and 5975C Inert XL EI/CI Mass Detectors, Agilent Technologies, Santa Clara, CA, USA). Detection of short-chain fatty acids was performed according to a previous GC procedure [ 15 ].
The results were statistically analyzed using SPSS 21.0 statistical analysis software, and the data were expressed as mean ± standard deviation. Statistical treatment was performed using one-way analysis of variance (ANOVA) followed by LSD post-test. P < 0.05 was considered statistically significant.
In a six-week dietary intervention study (Fig. 1 A), we systematically evaluated the effects of dietary calcium and phosphorus on lipid homeostasis in piglets. We first measured the percentage of carcass crude fat and total triglyceride levels to assess the direct impact of dietary calcium and phosphorus on lipid accumulation. The results showed that, compared to the LCAP group, the HCAP group had significantly lower percentages of carcass crude fat (Fig. 1 B) and total carcass triglycerides (Fig. 1 C) ( P < 0.05). Further analysis of piglet serum biochemical parameters (Fig. 1 D) revealed that piglets fed the high calcium and phosphorus diet had significantly lower serum glucose concentrations than the LCAP group ( P < 0.05), while their serum total cholesterol concentrations were significantly higher. Other lipid metabolism-related indicators showed no significant differences among the 3 groups.
Dietary calcium and phosphorus levels affect carcass and blood lipids. A Experimental procedure. Created with BioRender.com . B Percentage of total fat in carcass. C Total triglyceride content in carcass. D Serum glucose, total triglyceride, total cholesterol, HDL-C, and LDL-C levels, and the ratio of HDL-C to LDL-C. Data are means and standard errors for 6 pigs per treatment. Different letters indicate significant differences ( P < 0.05). CON, control group; LCAP, low-calcium-phosphorus group; HCAP, high-calcium-phosphorus group
By measuring the lipid content of the jejunum and ileum (Fig. 2 A and B), we found that the levels of triglyceride and cholesterol within the intestinal tissues were proportional to the Ca and P content in the feed, particularly in the HCAP group, the levels of triglyceride in jejunum and ileum were significantly increased ( P < 0.05). Further exploring the regulation of intestinal lipid metabolic pathways, we analyzed the expression of key lipid metabolism genes. The results showed that LCAP significantly inhibited the expression of lipid transport and absorption related genes such as FABP2 , CD36 , APOB , APOA1 , as well as FABP3 and FABP4 in jejunum and ileum (Fig. 2 C and D; P < 0.05). Notably, both high and low calcium-phosphorus diets appeared to suppress FATP4 expression in the jejunum. Meanwhile, low calcium-phosphorus levels also significantly suppressed the expression of fat synthesis-related genes, such as PPARγ and ACC ( P < 0.05), whereas DGAT2 expression tended to decrease but did not reach a significant level. By immunofluorescence technique, we further validated the expression profiles of CD36 and FABP4 proteins in jejunum and ileum (Fig. 2 E), consistent with the results of gene expression analysis. These results together revealed that dietary calcium and phosphorus content had significant effects on intestinal lipid absorption and metabolism.
Effect of dietary calcium and phosphorus levels on intestinal lipid absorption. A and B Total triglyceride and total cholesterol levels in the jejunum ileum. C and D Lipid transport ( CD36 , FABP1 , FABP2 , FABP3 , FABP4 , APOA1 , APOB ) and lipid synthesis ( DGAT2 , ACC , PPARγ ) gene expression abundance in the jejunum and ileum. Data are means and standard errors for 6 pigs per treatment. Different letters indicate significant differences ( P < 0.05). CON, control group; LCAP, low calcium and phosphorus group; HCAP, high calcium and phosphorus group; CD36, cluster of differentiation 36; FABP1, fatty acid binding protein 1; FABP2, fatty acid binding protein 2; FABP3, fatty acid binding protein 3; FABP4, fatty acid binding protein 4; APOA1, apolipoprotein A1; APOB, apolipoprotein B; DGAT2, diacylglycerol O-acyltransferase 2; ACC, acetyl CoA carboxylase; PPARγ, peroxisome proliferator-activated receptor gamma
In order to study the mechanism of calcium and phosphorus regulating lipid absorption, several key parameters of AMPK/CAMKK2 pathway were measured. The expression of AMPKα and its upstream and downstream regulatory genes in the jejunum was significantly suppressed in the low-calcium-phosphate diet group (Fig. 3 A; P < 0.05). The western blot analysis indicated that the low-calcium-phosphorus diet significantly suppressed the protein levels of phosphorylated AMPKα (P-AMPKα) and sirtuin 1 (SIRT1) in the jejunum (Fig. 3 B; P < 0.05). However, there was no significant difference in the level of phosphorylation of CAMKK2 (P-CAMKK2). The results of immunofluorescence were consistent with the immunoblot analysis, further validating this finding. The results of ileal analysis also supported these findings, showing that the expression of AMPK and its upstream and downstream genes was significantly suppressed under low calcium-phosphorus conditions (Fig. 3 D; P < 0.05). The low calcium-phosphorus diet resulted in a significant decrease in P-AMPKα protein levels (Fig. 3 E and F; P < 0.05), whereas the expression of P-CAMKK2 and SIRT1 proteins showed a decreasing trend but did not reach statistical significance.
Dietary calcium and phosphorus levels regulate intestinal lipid metabolism through the CAMKK2/AMPK pathway. A Expression abundance of AMPKα and its upstream and downstream genes in the jejunum. B Quantification of AMPKα signaling-associated protein bands and protein expression abundance in the jejunum and monitoring with β-actin. C Immunofluorescence validation of jejunal AMPKα protein. D Expression abundance of AMPKα and its upstream and downstream genes in the ileum. E Quantification of AMPKα signaling-associated protein bands and protein expression abundance in ileum and monitoring with β-actin. F Immunofluorescence validation of ileal AMPKα protein. Data are means and standard errors for 6 pigs per treatment. Different letters indicate significant differences ( P < 0.05). CON, control group; LCAP, low calcium-phosphorus group; HCAP, high calcium-phosphorus group; AMPKα, AMP-activated protein kinase alpha; CAMKK2, calcium/calmodulin-dependent protein kinase kinase 2; SIRT1, sirtuin 1
We first determined the effect on liver TG deposition and showed that low calcium and phosphorus significantly increased TG accumulation in the liver (Fig. 4 A; P < 0.05), but had no significant effect on TC levels (Fig. 4 B). Oil red O staining further confirmed a significant increase in fat deposition in the liver of piglets in the LCAP group (Fig. 4 C), although liver index did not show a statistical difference (Fig. 4 D). To deeply explore the effects of calcium and phosphorus levels on the regulatory mechanisms of liver lipid metabolism, we analyzed the gene expression of key enzymes and transcriptional regulators of lipid metabolism in the liver. Low calcium and phosphorus levels significantly up-regulated the expression of lipid synthesis-related genes SCD and FASN (Fig. 4 E; P < 0.05), while DGAT1 and SREBP1C showed no significant changes. Meanwhile, the expression of lipolysis-related genes was inhibited and the expression of lipid transport gene CD36 was significantly decreased ( P < 0.05). Immunoblotting results were consistent with gene expression analysis showing that low calcium and phosphorus levels promoted the expression of FASN protein (Fig. 4 F and H), whereas ACC1 and SREBP1-C tended to be elevated (Fig. 4 G and J). There was no significant change in DGAT1 protein expression (Fig. 4 I).
Dietary calcium and phosphorus content regulates liver lipid metabolism through the CAMKK2/AMPK pathway. A Liver total cholesterol content. B Liver total triglyceride content. C Oil red O staining showing liver lipid accumulation. D Liver index. E Liver lipid synthesis ( SCD , FASN , DGAT1 , SREPB1C ), lipid hydrolysis and oxidation ( ATGC , PPARα , CPT1A , PRDM16 ), and lipid transport ( CD36 , FABP4 ) gene expression abundance. F Liver lipid synthesis-related protein bands. G – J Protein expression associated with liver lipid synthesis. K Liver CAMKK2/AMPK pathway gene expression abundance. L – N Protein levels of p-AMPK and p-CAMKK2 were examined by protein blot analysis and monitored with AMPK and CAMKK2, respectively. Data are means and standard errors of 6 pigs per treatment; different letters indicate significant differences ( P < 0.05); CON, control; LCAP, low calcium-phosphorus group; HCAP, high calcium-phosphorus group; SCD, stearoyl-CoA desaturase; FASN, fatty acid synthase; DGAT1, diacylglycerol O-acyltransferase 1; SREBP1C, sterol regulatory element-binding protein 1C; ATGC, Acyl-CoA thioesterase 4; PPARα, peroxisome proliferator-activated receptor alpha; CPT1A, carnitine palmitoyltransferase 1A; PRDM16, PR domain containing 16; CD36, cluster of differentiation 36; FABP4, fatty acid-binding protein 4
In this study, we report for the first time the effect of dietary calcium and phosphorus levels on liver lipidomic characteristics. Using UPLC-MS non-targeted lipidomic analysis, we detected more than 45 different lipid classes and 3,429 different lipid molecules in 18 liver samples from three groups (6 samples per group). By principal component analysis (PCA), we observed significant differences in liver lipid metabolites among the three groups (Fig. 5 A).
Liver untargeted lipidomic analysis. A PCA analysis. ( B and C ) OPLS-DA analysis of HCAP vs. CON group and LCAP vs. CON group. D Content of glycerolipids, glycerophospholipids, sphingolipids, and fatty acyl groups in CON, LCAP, and HCAP groups; n = 6; error lines represent SEM. E Volcano plots of two-by-two comparisons between the three groups. F Metabolic pathway analysis of differential lipids using MetaboAnalyst. G Heat map of relative abundance of glycerol ester differential lipids in the three groups. H Heat map of relative abundance of glycerophospholipid differential lipids in the three groups. Data are means and standard errors for 6 pigs per treatment; different letters indicate significant differences ( P < 0.05)
Further orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that the changes of calcium and phosphorus levels had significantly different effects on liver lipid profiles, this is evident from the predicted separation of principal component 1 (Fig. 5 B and C). At the lipid class level, different calcium and phosphorus levels in feeds resulted in changes in the content of specific lipid classes (Fig. 5 D). We found a trend of increased monoradylglycerols (MG) and TG content in the glyceride category in the LCAP group compared with the CON group ( P = 0.09). In the glycerophospholipid category, the levels of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and sphingosine (SPH) were significantly increased, while lysophosphatidylglycerol (LPG) was significantly decreased ( P < 0.05). In the category of nerve sphingolipids, monosyalilated ganglioside M3(GM3) was significantly increased ( P < 0.05), while the content of SM also tended to increase ( P = 0.076). We further analyzed differences in lipid metabolites among the three groups using OPLS-DA VIP > 1 and P -value < 0.05 as screening criteria for significant differences. The results showed that there were 31 differential lipid metabolites in the LCAP group compared with the control group, 22 differential lipid metabolites in the HCAP group compared with the control group, and 8 differential lipid metabolites in the LCAP group compared with the HCAP Group (Fig. 5 E). By analyzing the KEGG pathway for these differential lipid metabolites, we found that calcium and phosphorus levels significantly affected both the glycerophospholipid pathway and the glyceride pathway (Fig. 5 F). The analysis of lipids showing significant differences between the glyceride pathway (Fig. 5 G) and the glycerophospholipid pathway (Fig. 5 H) as shown in the heatmap further confirmed the elevation of the glyceride and glycerophospholipid categories in the LCAP group; This suggests that low levels of calcium and phosphorus disrupt liver lipid metabolism.
In order to explore the effects of different dietary calcium and phosphorus levels on the intestinal microbial composition of weaned piglets, we collected colonic content samples from CON, LCAP and HCAP groups, and analyzed them against the bacterial 16S rDNA V3–V4 region using high-throughput sequencing to assess the effects of calcium and phosphorus levels on the structure of intestinal microbial communities. The results of α diversity analysis showed that the CHAO1 index and observed features index in both HCAP and LCAP groups increased, while the goods coverage index decreased, it was suggested that changes in calcium and phosphorus levels in feed might have increased the diversity of gut microbes (Fig. 6 A).
Dietary calcium and phosphorus levels influence the structure of the gut microbiota. A α-diversity of the gut flora. B β-diversity: principal coordinate analysis (PCoA) based on OTU abundance per weaned piglet. C Venn diagram based on OTU levels. D and E Relative abundance of gut microbiota at the phylum level and family level, respectively. F Heat map based on genus level. G Changes in flora associated with lipid metabolism: level of Firmicutes/Bacteroidetes, Alloprevotella abundance, Prevotella_9 abundance, Lactobacillus abundance. H Linear discriminant analysis of effect size (LEfSe) from gate level to genus level (LDA > 3.0). Data are expressed as mean ± SEM and compared with one-way analysis of variance (ANOVA) by Tukey’s multiple comparisons post-test
The PCoA analysis further confirmed the significant isolation of the flora composition among the three groups (Fig. 6 B). Venn diagram revealed an increase in the total number of OTUs due to changes in calcium and phosphorus levels (Fig. 6 C). Phyla-level microbial relative abundance analysis showed that although the relative abundance of Firmicutes did not change and the ratio of Firmicutes/Bacteroidetes did not differ significantly, the relative abundance of Bacteroidota decreased with increasing levels of calcium and phosphorus (Fig. 6 D). Family-level relative abundance (Fig. 6 E) showed that Prevotellaceae, Streptococcaceae, and Veillonellaceae were negatively correlated with calcium and phosphorus concentrations. Compositional analyses at the genus level revealed significantly lower relative abundance of Prevotella_9 in the HCAP group compared to the CON group, and significantly lower relative abundance of Alloprevotella compared to the LCAP group ( P < 0.05) (Fig. 6 G). LEfSe analyses identified microbial communities that significantly differed across dietary calcium and phosphorus levels. Micrococcales and g_Lactococcus were dominant in the CON group, whereas g_Alloprevotella , g_Streptococcus , and f_Streptococcaceae were dominant in the LCAP group. Tissierellales, Peptostreptococcaceae, Terrisporobacter , and Prevotellaceae_NK3B31_group , on the other hand, were the characteristic flora of the HCAP group (Fig. 6 H). KEGG pathway analyses showed that, compared with the CON and LCAP groups, the HCAP group had a higher predominance in amino acid metabolism, carbohydrate metabolism, lipid metabolism, membrane transport and metabolism pathways were up-regulated in abundance, suggesting that increased calcium and phosphorus levels in the feed contributed to improved lipid metabolism (Fig. 7 A). The results of short-chain fatty acid (SCFA) content showed that low calcium-phosphorus levels significantly reduced the concentrations of isobutyric acid and isovaleric acids, while high calcium-phosphorus levels reduced the concentration of acetic acid (Fig. 7 B), further confirming that calcium-phosphorus levels in feeds have a significant regulatory effect on intestinal metabolites.
Link between microbiome and lipidomics at different dietary calcium and phosphorus levels. A Heat map showing the gut microbiota predicting changes in KEGG pathways at different calcium and phosphorus levels. B SCFA ( n = 6 per group) including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid pentanoic acid, hexanoic acid, and total short-chain fatty acids were determined in the colonic contents of weaned piglets by gas chromatography; different letters indicate significant differences ( P < 0.05). C Heatmap showing Spearman correlation analysis between gut microbiota and serum lipid metabolism indices. Red represents positive correlations and blue represents negative correlations. Significant correlations are marked with * ( P < 0.05), ** ( P < 0.01) and *** ( P < 0.001). D Relationship between discriminatory colonic contents microbial OTUs and significantly different lipid molecules at different calcium and phosphorus levels. The size of the dot for each genus shows the average relative abundance. Dots represent faecal microbiomes and square dots represent lipid molecules. Transparency of the line indicates the negative logarithm of the correlation P -value (Spearman’s) (bottomed out at 10), the green line indicates a negative correlation, the orange line indicates a positive correlation, and the width of the line indicates a large correlation (Spearman’s)
By Spearman’s correlation coefficient analysis, we explored the relationship between gut microbial communities and serum glycolipid metabolic indices, which were presented as a heat map (Fig. 7 C). At the genus level, HDL-C showed significant negative correlations with Lachnospiraceae_NK4A136_group , Selenomonas , and Subdoligranulum ( P < 0.0001 to P < 0.001), whereas it showed a positive correlation with Streptococcus ( P < 0.05). In addition, serum TG showed negative correlation with Alloprevotella , Anaerovibrio , Megasphaera ( P < 0.05) and positive correlation with Lachnospiraceae_AC2044_group , Terrisporobacter ( P < 0.05). Through secreting metabolites into the blood, the gut microbiota is involved in the occurrence and progression of diseases [ 16 ]. Spearman correlation analysis revealed a correlation between 21 faecal OTUs and 19 significantly different lipid molecules (Fig. 7 D). Significantly different lipid molecules TG (18:2_13:0_18:2), Cer (d36:0), and PE (18:1_22:1) correlated with a wide range of differentiated bacterial genera. Among them Lachnospiraceae_NK4A136_group , which was previously found to be negatively correlated with serum HDL-C concentration, was significantly positively correlated with the lipid molecule Cer (d46:7). Alloprevotella , which correlated with serum total cholesterol and HDL-C/LDL-C ratio, correlated with TG (18:1_18:1_18:2).
In this study, we systematically investigated for the first time the effects of different calcium and phosphorus levels in the same proportion of feed on lipid metabolism in weaned piglets. It reveals the disorder of lipid metabolism that may result from low calcium-phosphorus levels and elucidates the mechanism of how high-calcium-phosphorus feeds improve lipid metabolism by activating specific signaling pathways. Our results showed that low calcium and phosphorus diets caused significant lipid accumulation in the body and liver of piglets, which was mainly attributed to increased intestinal lipid absorption and abnormal liver lipid accumulation. This finding is consistent with the view of the intestine and liver as the main sites of lipid metabolism and emphasis the importance of regulating the lipid processing capacity of these two organs to maintain lipid homeostasis. Furthermore, our study also showed that by providing a high-calcium phosphate diet, the CAMKK2-AMPKα pathway can be activated, promoting intestinal lipid absorption and expression of transporters, increasing oxidative hydrolysis of renal lipids; At the same time reduce the synthesis of liver lipids, thus effectively improve the lipid metabolic disorder. Combined with the correlation analysis between gut microbial composition and serum lipid metabolism indexes, this study further reveals the important role of gut microbes in the regulation of lipid metabolism. The association of specific microbiota with lipid metabolism-related indicators reinforces the idea that the gut microbiota may be involved in the regulation of lipid metabolism by influencing the secretion patterns of host metabolites. In conclusion, the present study not only highlights the effects of calcium and phosphorus intake on lipid metabolism, but also reveals the potential molecular mechanisms of lipid metabolism regulation, which provides a scientific basis for the development of future nutritional intervention strategies for lipid metabolism disorders. With the further understanding of the mechanism of lipid metabolism, the regulation of calcium and phosphorus intake may become an effective way to improve the disorder of lipid metabolism and prevent related metabolic diseases.
Calcium and phosphorus are not only the most abundant minerals in mammals, but are also involved in a variety of key physiological roles, including lipid metabolism and the synthesis and maintenance of bone structure. As a major energy source and active endocrine organ in the body, lipids play a crucial role in the integrity of cell membranes, protection of vital organs, hormone synthesis, and absorption and transport of vitamins, as well as directly affecting livestock and poultry performance and meat quality [ 17 ]. In this study, we found that low calcium and phosphorus diets increased carcass TG and fat percentage, although not significantly, compared with normal calcium and phosphorus levels. This finding echoes previous studies in which calcium supplementation was found to promote muscle fat accumulation [ 18 ], whereas phosphorus supplementation showed the opposite effect [ 4 ]. When calcium and phosphorus were supplemented simultaneously, however, a reduction in muscle lipid accumulation was observed [ 19 ], which is consistent with our experimental results. Notably, adjustment of calcium and phosphorus levels in the feed significantly affected blood glucose concentrations, where high calcium and phosphorus levels significantly reduced blood glucose levels, suggesting that calcium and phosphorus supplementation may play a role in regulating glucose metabolism. Furthermore, we also observed an effect of calcium and phosphorus supplementation on the HDL-C to LDL-C ratio, consistent with Zhang et al. [ 20 ]. Although no significant changes were found in other serum lipid metabolic indexes, the decrease of HDL-C/LDL-C ratio may reflect the changes of lipid metabolism. LDL-C plays a key role in cholesterol transport and the synthesis of cell membranes and certain hormones, while HDL-C is responsible for transporting cholesterol in tissues and maintaining the stability of the cardiovascular system. Therefore, changes in the HDL-C/LDL-C ratio may affect cardiovascular health, suggesting a possible role for calcium and phosphorus in maintaining cardiovascular stability [ 21 ].
As the main site of dietary fat absorption, the intestine plays a central role in the overall lipid metabolism. Recent studies have revealed an association between dysregulation of intestinal lipid metabolism and systemic lipid metabolic diseases [ 22 ]. Nevertheless, the specific effects of dietary calcium-phosphorus ratios on intestinal lipid metabolism and their underlying mechanisms are not fully understood. The intestinal lipid accumulation, lipid absorption (CD36, FABP2, FABP3, and FABP4), lipid synthesis (DGAT1, DGAT2, ACC, and PPAR γ) gene and protein expression were detected. Here we reveal a correlation between changes in feed calcium and phosphorus levels and intestinal lipid absorption, pointing to a link between enhanced fatty acid uptake capacity and elevated calcium and phosphorus levels. Furthermore, we observed that low calcium and phosphorus levels promote abnormal accumulation of liver lipids, which results from increased lipid synthesis and inhibition of lipid transport and oxidative hydrolysis of key enzyme activities. In contrast, increased calcium and phosphorus levels mitigated this phenomenon, consistent with previous findings that altering calcium or phosphorus levels alone affects lipid accumulation [ 23 ]. These findings emphasis the important influence of the dietary calcium-phosphorus ratio on the lipid metabolism capacity of the intestine-liver axis. Previous studies on rodents [ 24 ], poultry [ 4 ], fish [ 25 ], and pigs [ 23 ] have all shown that, calcium or phosphorus supplementation alone reduced fat accumulation in the liver and intestines, and we also altered calcium and phosphorus levels in the liver to produce changes consistent with them. The occurrence of this phenomenon may be related to the fact that calcium and phosphorus levels regulate beta oxidation of fatty acids, which is the main pathway of fatty acid degradation. However, the lipid accumulation in the intestine was contrary to previous reports, and the mechanisms need to be further explored because of the paucity of studies on the effects of dietary Ca and P levels on lipid metabolism.
AMPK plays a crucial role in the maintenance of cellular energy homeostasis, not only regulating glycolipid metabolism, but also being involved in the modulation of appetite and anorexia signaling [ 6 ]. Although the inhibitory effect of calcium on endogenous lipid production in the liver by activating the AMPK pathway has been reported, its underlying molecular mechanisms are still at the forefront of exploration [ 7 ]. CAMKK2, an upstream kinase of AMPK, plays a critical role in regulating lipid metabolism by phosphorylating AMPK at the Thr172 site. Furthermore, the importance of CAMKK2’s function in regulating lipid metabolism was demonstrated by the ability of its deletion or pharmacological inhibition to reduce ab initio lipogenesis, which shows potential therapeutic value in ameliorating high-fat diet-induced fatty liver, insulin sensitivity problems [ 11 , 12 ]. As previously reported, elevated levels of dietary calcium and phosphorus increase intracytoplasmic calcium ion concentrations [ 25 , 26 ] and serum lipocalin concentrations [ 27 , 28 ], and lipocalin induces activation of the lipocalin receptor 1 (AdipoR1), which drives CAMKK2 to increase AMPK activity by activating extracellular Ca 2+ efflux [ 29 ]. We further hypothesized that dietary calcium and phosphorus levels might regulate intestinal-liver axis lipid metabolism through the CAMKK2/AMPK signaling pathway. We observed that low calcium-phosphorus levels decreased mRNA and protein levels of intestinal CAMKK2 and AMPKα, accompanied by a decrease in lipid deposition, while high calcium-phosphorus levels reversed this trend. This phenomenon may be related to the upregulation of AMPK activity, which enhances the uptake capacity of long-chain fatty acids (LCFA) in the gut by promoting the expression and membrane translocation of CD36 and FATP4 proteins; This leads to lipid accumulation [ 30 ]. Furthermore, we found that dietary calcium and phosphorus supplementation activated the liver CAMKK2/AMPK signaling pathway to mitigate abnormal accumulation of liver lipids by reducing lipid synthesis and enhancing lipid hydrolytic oxidation processes; This process involves the upregulation of SIRT1, a signaling molecule downstream of AMPK. Our results suggest that dietary calcium and phosphorus levels regulate intestinal-liver axis lipid metabolism through the CAMKK2/AMPK signaling pathway.
Given that Ca and P supplementation is known to influence lipid metabolism in skeletal muscle and adipose tissue [ 1 , 18 ], the present study examined the effects of changes in calcium and phosphorus dietary levels on liver lipid composition, which is important for understanding the role of calcium and phosphorus in overall metabolic regulation. Our findings reveal that low calcium phosphorus intake is associated with increased levels of triglycerides (MG and TG) and certain phospholipids (PIP 2 and SPH) and sphingolipids (GM3 and SM) in the liver; The concentrations of LPG and Ceramide (Cer) were also reduced. This finding implies that dietary calcium and phosphorus levels are negatively correlated with specific lipid classes in the liver. Through the KEGG pathway analysis of differential lipids, we further verified that these lipids are mainly involved in glyceride and glycerophospholipid metabolic pathways. The increase of glycerides, especially MG and TG, may reflect the accumulation of intermediate products during lipolysis and the enhancement of activity of TG biosynthesis pathway, this is consistent with existing studies of the effects of calcium and phosphorus supplementation alone on lipid metabolism. In addition, the up-regulation of PIP 2 and SPH, as well as the increase in GM3 and SM, may involve changes in cell signaling, cell proliferation and apoptosis, as well as cell membrane composition and function. In particular, changes in SPH and SM implicate a potential role for sphingolipid metabolism in regulating energy homeostasis and AMPK expression. The changes of SPH and SM may affect the energy metabolism of liver by affecting the physical properties of cell membrane and cell signaling pathway [ 31 ]. In particular, SM (d18:1/16:0) was found to enhance ATP production and reduce AMPK expression by activating glycolytic pathways [ 32 ]. This provides new insights into the interaction between lipid metabolism and energy sensing.
To establish a potential link between dietary Ca, P, and the microbiome and specific lipid species, we performed a microbiome analysis of colonic contents in the LCAP, HCAP, and CON groups. We found that both the LCAP and HCAP groups increased the alpha diversity of the colonic microbiota compared to the CON group. This may reflect changes in microbial community structure leading to an increase in the diversity of harmful bacteria [ 33 ]. Indeed, a closer examination of the individual bacterial composition of LCAP and HCAP revealed an increased abundance of bacteria belonging to the Enterobacteriaceae, Streptococcaceae, and Clostridiaceae, which are families of bacteria that contain pathogenic bacteria [ 34 ]. The β-diversity analysis further revealed significant differences in microbiome structure among the three groups, consistent with previous findings [ 35 ]. Our examination of diet-related phylum abundance showed that changes in dietary calcium and phosphorus did not alter the abundance of the Firmicutes, but the abundance of the Bacteroidetes declined with increasing Ca and P levels, with the Firmicutes/Bacteroidetes ratio being lowest in the LCAP group. Further analyses of specific ASVs showed that bacteria from the Prevotellaceae and Veillonellaceae families decreased with increasing calcium and phosphorus levels, consistent with their association in obesity pathology [ 36 , 37 ]. Strikingly, the significant reductions in Alloprevotella and Prevotella in the HCAP group compared with the LCAP group were associated with improvements in liver steatosis and other lipid metabolic markers. Alloprevotella and Prevotella_9 belong to the genus Mycobacterium , which is a group of two in the genus Prevotella . Several reports have linked Prevotella to a range of diseases, including advanced liver fibrosis, cirrhosis, insulin resistance, type 2 diabetes, inflammation and obesity [ 38 , 39 ]. Furthermore, high abundance of Prevotella copri in a porcine model was associated with elevated concentrations of obesity-related serum metabolites, which were significantly correlated with fat accumulation in pigs [ 40 ]. These results support our observations in this study. Our results provide new insights into the potential role of the gut microbiota in influencing lipid metabolism through calcium and phosphorus levels.
The endocrine function of the gut plays a key role in the regulation of lipid metabolism, in particular, short-chain fatty acid produced by the fermentation of gut microbes (SCFA) has a significant effect on lipid biosynthesis through the intestine-liver axis [ 41 , 42 ]. We observed a decreasing trend in total SCFA levels in colonic contents with increasing dietary calcium and phosphorus levels. This finding echoes previous studies, which have shown that the majority of liver lipogenesis is dependent on microbial SCFA from the colon, which are key components of fatty acid biosynthesis [ 43 ]. Further analyses of specific SCFA concentrations revealed significant reductions in acetic acid concentrations in the HCAP group as well as isobutyric and isovaleric acid concentrations in the LCAP group. Acetic acid as the main energy source of the liver, isobutyric acid and isovalerate play critical roles in maintaining intestinal health, providing energy, having anti-inflammatory and immunomodulatory effects, and promoting the integrity of gut barrier function [ 44 ]. Their decline in LCAP seems to be explained by an increase in pathogenic bacteria. Collectively, our results suggest that dietary calcium and phosphorus levels may provide a feasible path to improve liver steatosis and optimize lipid metabolic indices by influencing SCFA production.
In summary, our results indicate that when the STTD Ca: STTD P ratio is 0.216%:0.18% (LCAP), it reduces intestinal lipid absorption and leads to abnormal lipid accumulation in the liver and carcass. In contrast, supplementing calcium and phosphorus can improve lipid metabolism by promoting intestinal lipid transport and liver lipid hydrolysis and oxidation through the activation of the CAMKK2/AMPK pathway. Changes in dietary calcium and phosphorus levels can also regulate the gut microbiota. Overall, we report on the response of intestinal-liver axis lipid metabolism to dietary calcium and phosphorus levels and its regulatory mechanisms, and reveal how these changes affect liver lipid composition and the characteristics of the intestinal microbiome (Fig. 8 ). These findings underscore the importance of precisely managing dietary mineral levels to optimize lipid metabolism and enhance the overall health of animals.
Schematic diagram of dietary calcium and phosphorus levels regulating lipid metabolism in weaned piglets. Calcium and phosphorus in the diet regulate intestinal lipid absorption and hepatic lipid metabolism through the CAMKK2/AMPK axis and alter the gut microbiota to influence lipid metabolism. Created with BioRender.com
The data analyzed during the current study are available from the corresponding author on reasonable request.
Acetyl-CoA carboxylase 1
5′-Adenosine monophosphate-activated protein kinase
AMP-activated protein kinase alpha
Calcium/calmodulin-dependent protein kinase kinase 2
Cluster of differentiation 36
Carnitine palmitoyltransferase 1a
Diacylglycerol O-acyltransferase 1
Fatty acid binding protein 2
Fatty acid binding protein 3
Fatty acid synthase
Fatty acid transport protein 4
Monosyalilated ganglioside M3
High-density lipoprotein cholesterol
Low-density lipoprotein cholesterol
Lysophosphatidylglycerol
Monoradylglycerols
Nonalcoholic fatty liver disease
Operational taxonomic unit
Phosphorylated AMPKα
Phosphorylated CAMKK2
Phosphatidylinositol 4,5-bisphosphate
Peroxisome proliferator-activated receptor γ
PR domain-containing protein 16
Stearoyl-CoA desaturase
Short chain fatty acid
Sphingomyelin
Sphingosine
Sterol regulatory element-binding protein 1c
Standardized total tract digestibility
Total cholesterol
Triglyceride
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We would like to thank the researchers in our laboratory for their efforts and all the staff at the Baiyun Teaching Experimental Base of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences for their selfless support.
This study was financially supported by the National Key Research and Development Program of China (2021YFD1300402), Research Fund of Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture (2022ZD003, 2021TDQD002), the earmarked fund for China Agriculture Research System (CARS-35), and Special Project for Rural Revitalization Strategy in Guangdong Province (2023TS-3-1).
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State Key Laboratory of Swine and Poultry Breeding Industry, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture and Rural Affairs, Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, 510642, China
Zhenyan Miao, Yanjie Sun, Zhangjian Feng, Qiwen Wu, Xuefen Yang, Li Wang, Zongyong Jiang & Hongbo Yi
College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China
Zhenyan Miao, Zhangjian Feng & Ying Li
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ZM: Conceptualization, Investigation, Methodology, Data curation, Project administration, Visualization, Writing-original draft. YS: Data curation, Visualization, Writing-original draft. ZF: Investigation, Investigation, Data curation. QW, XY, LW: Data curation, Visualization, Writing-original draft. YL: Investigation, Methodology, Data curation. ZJ, HY: Conceptualization, Investigation, Methodology, Writing-review and editing.
Correspondence to Zongyong Jiang or Hongbo Yi .
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Additional file 1: table s1..
The primer sequence of qPCR.
Uncropped and unprocessed images of the complete gel and blot in Fig. 3B, 3E, 4F and 4L.
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Miao, Z., Sun, Y., Feng, Z. et al. CAMKK2-AMPK axis endows dietary calcium and phosphorus levels with regulatory effects on lipid metabolism in weaned piglets. J Animal Sci Biotechnol 15 , 105 (2024). https://doi.org/10.1186/s40104-024-01061-0
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Although chimeric antigen receptor (CAR) T cell therapies have demonstrated promising clinical outcomes, durable remissions remain limited. To extend the efficacy of CAR T cells, we develop a CAR enhancer (CAR-E), comprising a CAR T cell antigen fused to an immunomodulatory molecule. Here we demonstrate this strategy using B cell maturation antigen (BCMA) CAR T cells for the treatment of multiple myeloma, with a CAR-E consisting of the BCMA fused to a low-affinity interleukin 2 (IL-2). This selectively induces IL-2 signaling in CAR T cells upon antigen–CAR binding, enhancing T cell activation and antitumor activity while reducing IL-2-associated toxicities. We show that the BCMA CAR-E selectively binds CAR T cells and increases CAR T cell proliferation, clearance of tumor cells and development of memory CAR T cells. The memory cells retain the ability to re-expand upon restimulation, effectively controlling tumor growth upon rechallenge. Mechanistic studies reveal the involvement of both CAR and IL-2 receptor endodomains in the CAR-E mechanism of action. The CAR-E approach avoids the need for specific engineering and enables CAR T cell therapy with lower cell doses.
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Data availability.
All relevant data are provided in this paper and Supplementary Information . RNAseq data generated in this study are available from the National Center for Biotechnology Information Sequence Read Archive ( PRJNA1118916 ). The Ensembl H . sapiens database ( https://useast.ensembl.org/Homo_sapiens/Info/Index ) was used to identify RNA transcripts. Any remaining raw data are available from the corresponding author upon reasonable request.
No custom code was developed for the analyses of the results.
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Funding was provided by the Dana-Farber Cancer Institute Innovation Research Fund Award (M.R.), the Parker Institute for Cancer Immunotherapy (M.R., Grant no. C-03160), a Blavatnik Therapeutics Challenge Award (M.R., Grant no. 223813.5124476.0008) and an American Cancer Society postdoctoral fellowship (T.R., Grant no. PF-20-015-01-CCE). We thank the Boston University Micro and Nano Imaging Core Facility and Photonics Center for providing optical imaging support through Natural Science Foundation Major Research Instrumentation Awards 2215990 and DP2HL168562 (H.T.N.). We are grateful to I. Rubin-Bejerano, M. Namchuk, E. Smith and M. Goudarzi for helpful discussions. We thank Louise Clark for her technical assistance. We thank the National Cancer Institute-sponsored Biological Resource Branch Preclinical Biologics Repository for providing the anti-CD3 antibody (OK3) and IL-2, IL-7 and IL-15. We extend our gratitude to the microscopy core and the flow cytometry core within the Department of Cancer Immunology, as well as the dedicated animal facility staff at the Dana-Farber Cancer Institute and the Boston Children’s Hospital, for their invaluable support throughout this project.
These authors contributed equally: Shreya R. Mantri, Heydar Moravej, Benjamin B. V. Louis.
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA
Taha Rakhshandehroo, Shreya R. Mantri, Heydar Moravej, Benjamin B. V. Louis, Ali Salehi Farid, Leila Munaretto, Radia M. M. Khan, Alexandra Wolff, Zoe Farkash, Min Cong, Adrien Kuhnast, Ali Nili, Uk-Jae Lee, Harris H. Allen, Lea Berland, Ester Simkova, Safak C. Uslu, Soheil Tavakolpour, Jennifer E. Rowley, Elisabeth Codet, Haneyeh Shahbazian, Jessika Baral, Jason Pyrdol, Kai W. Wucherpfennig & Mohammad Rashidian
Department of Biomedical Engineering, Boston University, Boston, MA, USA
Kathryn Regan, Jennifer E. Rowley & Hadi T. Nia
Harvard Medical School, Boston, MA, USA
Jessika Baral, Caron A. Jacobson, Omar Nadeem, Kai W. Wucherpfennig & Mohammad Rashidian
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
Caron A. Jacobson & Omar Nadeem
Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA
Kai W. Wucherpfennig & Mohammad Rashidian
Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
Mohammad Rashidian
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T.R., S.R.M., H.M., B.B.V.L., A.S.F., L.M., K.R., R.K., A.W., Z.F., M.C., A.K., A.N., U.-J.L., H.H.A., L.B., E.S., S.C.U., J.B., S.T., J.E.R., E.C., H.S. and J.P. designed and performed experiments and analyzed the results. C.A.J., O.N., H.T.N. and K.W.W. provided specific experimental advice and technical support. M.R. planned the experiments, analyzed the data and wrote the paper with help from the other authors.
Correspondence to Mohammad Rashidian .
Competing interests.
T.R. and M.R. are inventors on a related patent application. M.R. is the scientific founder of Koi Biotherapeutics. C.A.J. serves as a consultant for Kite/Gilead, Novartis, BMS, Sana, Synthekine, Janssen, Miltenyi, Caribou, Galapagos, ADC Therapeutics, AstraZeneca and Abbvie, and receives research funding from Kite/Gilead. O.N. receives research support from Takeda and Janssen, participates on advisory boards for Bristol Myers Squibb, Janssen, Sanofi, Takeda and GPCR Therapeutics, and receives honoraria from Pfizer. The other authors declare no competing interests.
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Rakhshandehroo, T., Mantri, S.R., Moravej, H. et al. A CAR enhancer increases the activity and persistence of CAR T cells. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02339-4
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