Results from the first controlled trial to assess calorie restriction in healthy humans have confirmed the health benefits of moderately limiting calorie intake over a period of two years. Headed by scientists at Yale School of Medicine and Pennington Biomedical Research Center, the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial found that calorie restriction improved metabolic and immune responses, and also identified a macrophage-produced protein, platelet activating factor acetylhydrolase (PLA2G7), which could feasibly be harnessed to extend health span in humans. The study results showed that expression of the Pla2g7 gene was inhibited in humans undergoing caloric restriction (CR), and deleting the gene in mice improved the animals’ metabolic health.

“Two years of modest calorie restriction reprogrammed the pathways in fat cells that help regulate the way mitochondria generate energy, the body’s anti-inflammatory responses, and potentially longevity,” stated Eric Ravussin, PhD, associate executive director for clinical science at Pennington Biomedical Research Center, reporting on the human study results. “In other words, calorie restriction rewires many of the metabolic and immune responses that boost lifespan and health span.”

“There’s so much debate about what type of diet is better—low carbohydrates or fat, increased protein, intermittent fasting, etc.—and I think time will tell which of these are important,” added Vishwa Deep Dixit, PhD, the Waldemar Von Zedtwitz professor of pathology, immunobiology, and comparative medicine at Yale School of Medicine and senior author of the team’s published study in Science. “CALERIE is a very well-controlled study that shows a simple reduction in calories, and no specific diet, has a remarkable effect in terms of biology and shifting the immuno-metabolic state in a direction that’s protective of human health. So from a public health standpoint, I think it gives hope.”

Dixit, Ravussin, and colleagues reported on findings from the CALERIE study in a paper titled “Caloric restriction in humans reveals immunometaboilc regulators of health span,” in which they concluded, “Collectively, our findings demonstrate that sustained CR in humans activates a core transcriptional program that promotes immune function, reduces inflammation, and reveals PLA2G7 as one of the potential mechanisms to mimic the beneficial effects of CR.”

Decades of research in model organisms including flies, worms, and mice have shown that limiting calorie intake can enhance lifespan, albeit under laboratory conditions. But whether such calorie restriction can do the same for humans hasn’t been clear. And as the authors pointed out, “The extension of life span driven by 40% CR in rodents causes trade-offs in growth, reproduction, and immune defense that make it difficult to identify therapeutically relevant CR-mimetic targets … Furthermore, forced extreme CR in nonconsenting animals may elicit stress responses evidenced by increased production of glucocorticoids, which can further compromise the immune system.”

To address the relevance of CR on human physiology, the CALERIE trial was designed to test the long-term effects of two years of moderate CR on physiology, aging biomarkers, and predictors of healthspan and longevity in healthy volunteers.” The researchers first established baseline calorie intake among more than 200 study participants. Some of the participants then reduced their calorie intake by about 14%, while the rest continued to eat as usual, and the long-term health effects of calorie restriction were measured over the next two years.

Eric Ravussin, PhD, associate executive director for clinical science at Pennington Biomedical Research Center [Pennington Biomedical Research Center]
The overall aim of the trial was to see if calorie restriction is as beneficial for humans as it is for lab animals, said Dixit, And if it is, he noted, researchers wanted to better understand what calorie restriction does to the body specifically that leads to improved health. The level of caloric restriction studied in the trial was also much lower than the forced 25–40% restriction of calories in laboratory animals, the investigators noted, “… and may engage unique mechanisms to maintain homeostasis.”

Given that previous research had shown that CR in mice can increase infections, Dixit, who is also director of the Yale Center for Research on Aging, wanted to determine how calorie restriction might be linked to inflammation and the immune response. “Because we know that chronic low-grade inflammation in humans is a major trigger of many chronic diseases and, therefore, has a negative effect on life span,” he said. “Here we’re asking: What is calorie restriction doing to the immune and metabolic systems and if it is indeed beneficial, how can we harness the endogenous pathways that mimic its effects in humans?”

The investigators started by analyzing the thymus, a gland that sits above the heart and produces T cells, a type of white blood cell and an essential part of the immune system. The thymus ages at a faster rate than other organs. By the time healthy adults reach the age of 40, said Dixit, 70% of the thymus is already fatty and nonfunctional. And as it ages, the thymus produces fewer T cells. “As we get older, we begin to feel the absence of new T cells because the ones we have left aren’t great at fighting new pathogens,” said Dixit. “That’s one of the reasons why elderly people are at greater risk for illness.”

For the study, the research team used magnetic resonance imaging (MRI) to determine if there were functional differences between the thymus glands of those who were restricting calories and those who were not. They found that the thymus glands in participants with limited calorie intake had less fat and greater functional volume after two years of calorie restriction, meaning they were producing more T cells than they were at the start of the study. But participants who weren’t restricting their calories had no change in functional volume. Ravussin noted, “Calorie restriction helps prevent the thymus from shrinking so the person generates more T cells.” The authors further explained, “Compared with baseline, sustained CR for two years significantly (P < 0.05) increased thymic mass as well as total thymic volume in study participants.” Dixit added, “The control participants showed no significant change in thymic volume from baseline to year two. The fact that this organ can be rejuvenated is, in my view, stunning because there is very little evidence of that happening in humans. That this is even possible is very exciting.”

With such a dramatic effect on the thymus, Dixit and colleagues expected to also find effects on the immune cells that the thymus was producing, changes that might underlie the overall benefits of calorie restriction. But when they sequenced the genes in those cells, they found there were no changes in gene expression after two years of calorie restriction. “These data may indicate that 14% CR in healthy humans activates a tissue-protective immunometabolic program that can enhance thymic function without altering the transcriptome of CD4 T cells,” the investigators noted.

When the scientists then looked more closely, they made a surprising discovery. “It turns out that the action was really in the tissue microenvironment not the blood T cells,” Dixit said. The team had studied adipose tissue, or body fat, of participants undergoing calorie restriction at three time points: at the beginning of the study, after one year, and after two years. Body fat is very important, Dixit pointed out, because it hosts a robust immune system. There are several types of immune cells in fat, and when they are aberrantly activated, they become a source of inflammation, he explained.

“We found remarkable changes in the gene expression of adipose tissue after one year that were sustained through year two,” said Dixit. “This revealed some genes that were implicated in extending life in animals but also unique calorie restriction-mimicking targets that may improve metabolic and anti-inflammatory response in humans.”

Recognizing this, the researchers then set out to see if any of the genes they identified in their analysis might be driving some of the beneficial effects of calorie restriction. They honed in on the gene for PLA2G7—or group VII A platelet activating factor acetyl hydrolase—which was one of the genes significantly inhibited following calorie restriction. PLA2G7 is produced by macrophages. “Several proinflammatory-like genes and many transcripts of unknown function in myeloid cells were specifically inhibited after CR. Among the top six genes inhibited at both 1 and 2 years of CR. The phospholipase belonging to group VII A platelet activating factor acetylhydrolase (PLA2G7).”

The change in PLA2G7 gene expression observed in participants who were limiting their calorie intake suggested that the protein might be linked to the effects of calorie restriction. To better understand if PLA2G7 might be causing some of the effects observed with calorie restriction, the researchers also tracked what happened when they used CRISPR technology to delete the Pla2g7 gene in laboratory mice.

“PLA2G7-deficient animals were partially protected from high-fat-diet (HFD)–induced weight gain and increased fat mass,” they reported. “In addition, compared with their littermate controls, the Pla2g7 KO [knockout] mice were partially protected from hepatic steatosis, with reduced expression of interleukin (IL)-1β and caspase-1, which are implicated in controlling obesity-induced lipid metabolism and liver dysfunction.” Interestingly, the reduced weight gain in Pla2g7 KO mice corresponded with increased energy expenditure, and consistent with improved adipose tissue metabolism, the PLA2G7-deficient macrophages also demonstrated lower expression of certain proinflammatory cytokines.

“We found that reducing PLA2G7 in mice yielded benefits that were similar to what we saw with calorie restriction in humans,” said Olga Spadaro, PhD, a former research scientist at the Yale School of Medicine and lead author of the study. Specifically, the thymus glands of these mice were functional for a longer time, the mice were protected from diet-induced weight gain, and they were protected from age-related inflammation.

These effects occurred because PLA2G7 targets a specific mechanism of inflammation called the NLRP3 inflammasome, the researchers said. “Chronic systemic inflammation in aging and Nlrp3 inflammasome activation is associated with age-related functional decline,” they explained. The mouse study results thus indicated that lowering PLA2G7 protected aged animals from inflammation. “We suspect that in a tissue microenvironment such as adipose tissue, where macrophages have to process a complex mixture of lipids, PLA2G7 might be an important regulator of the inflammasome.”

Dixit added, “These findings demonstrate that PLA2G7 is one of the drivers of the effects of calorie restriction. Identifying these drivers helps us understand how the metabolic system and the immune system talk to each other, which can point us to potential targets that can improve immune function, reduce inflammation, and potentially even enhance healthy lifespan.”

For instance, it might be possible to manipulate PLA2G7 and get the benefits of calorie restriction without having to actually restrict calories, which can be harmful for some people, he suggested. “Collectively, our findings demonstrate that sustained CR in humans activates a core transcriptional program that promotes immune function, reduces inflammation, and reveals PLA2G7 as one of the potential mechanisms to mimic the beneficial effects of CR,” the scientists conclude. “The data from this human study are also relevant regarding controversies emerging from animal studies that questioned CR’s effects on health and inflammation.”


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