Glucose restriction increases life expectancy by inducing mitochondrial respiration and increasing oxidative stress

Reducing caloric intake (CR) is a long-standing means of increasing the life expectancy of animals, including mammals. It is currently unknown whether CR can increase a person’s life expectancy. However, a 2006 study on healthy overweight people found that six-month calorie restriction improves longevity biomarkers: reduces fasting insulin and internal body temperature, and reduces DNA damage.

The idea that life expectancy depends on the number of calories consumed is not new. In 1908, Max Rubner proposed that the maximum life span is inversely proportional to metabolized food energy. In 1928, this hypothesis was extended by Raymond Pearl, who suggested that an increased metabolic rate shortens life expectancy. In the process of metabolism, reactive oxygen species (ROS) are formed. They are produced by the mitochondria – the parts of the cell that have energy. The scientists suggested that with increased mitochondrial metabolism, more ROS would be released. ROS accumulation will cause oxidative damage and lead to neurodegenerative diseases, diabetes, cancer, and premature aging.

Cellular stresses such as hypoxia, infections, growth factor stimulation, and starvation increase ROS production.

Historically, reactive oxygen species were thought to cause only cell damage and have no physiological function. However, a growing body of evidence suggests that ROS is critical for healthy cell function. Moreover, increased mitochondrial metabolism, which produces more ROS and other stressors, increases protection against stress and possibly prolongs life.

This fact is confirmed by the fact that physical activity increases the formation of ROS, and at the same time, it increases life expectancy. Furthermore, recent evidence suggests that dietary supplements with antioxidants can shorten life expectancy.

Scientists have studied how life expectancy is affected by limiting the use of glucose. In Western countries, glucose is the basis of nutrition. In particular, sugar accounts for 15.8% of Americans ‘ daily calorie intake. To investigate the role of glucose metabolism in longevity, the researchers exposed the nematode Caenorhabditis elegans to a chemical glycolysis inhibitor, causing a metabolic condition that resembles dietary glucose restriction.

Glucose restriction in C. elegans:

  • Increased mitochondrial respiration, which increased oxidative stress. This stress was eliminated by pretreatment with various antioxidants.
  • Increased the production of ROS, which increased the ability to antioxidant defense.
  • Increased the maximum life expectancy by 25% and the average life expectancy by 17%. Antioxidants such as ascorbic acid (vitamin C) and Trolox (a derivative of vitamin E) reversed the increase in life expectancy, while none of these antioxidants alone had a significant effect on life expectancy.

These latest findings suggest that the widespread use of antioxidants as dietary supplements for humans may cause undesirable effects, i.e., reduced cellular and systemic stress tolerance, reducing life expectancy.

The physiological role of mitochondrial reactive oxygen species

Reactive oxygen species are essential for healthy cell function. Reactive oxygen species are now thought to act as signaling molecules that regulate a wide range of physiological processes, including adaptation to hypoxia, autophagy, immunity, stem cell differentiation, and longevity.

Mitochondria intensively secrete ROS in response to stress. ROS act as a signaling intermediate to facilitate cellular adaptation to this stress. It is believed that in the absence of cellular stress, there is a tonal level of ROS that supports homeostasis, while after pressure, this level of ROS can fluctuate, changing the signaling pathways.

Mitochondrial ROS regulate adaptation to hypoxia

Oxygen is crucial for the survival of aerobic organisms because of its role in ATP production by mitochondria. ATP is a universal fuel for all body processes. When mammalian cells encounter lower oxygen levels – experiencing hypoxia (0.3–3% O2) – reactions occur to increase oxygen intake while reducing oxygen use. Many of these adaptive responses to low-oxygen conditions are triggered or enhanced by mitochondrial ROS.

A family of hypoxia-induced factors (HIF: HIF-1 and HIF-2) regulates the response to hypoxia-promotes the production of:

  • the hormone erythropoietin (EPO) to increase the production of red blood cells;
  • Vascular endothelial growth factor (VEGF) to stimulate the formation of new blood vessels;
  • glycolytic enzymes to maintain ATP levels.

Although an increase in ROS is vital for HIF activation, sustained growth in ROS production can be detrimental. Interestingly, cells lacking HIF-1 maintain elevated ROS levels in chronic hypoxia and eventually die. It suggests that HIF-1 provides feedback and reduces ROS levels as an adaptive mechanism for cell survival in chronic hypoxia.

Thus, the cells use the dramatic increase in mitochondrial ROS to stabilize HIF under hypoxic conditions and subsequently restrain ROS production under chronic hypoxic conditions to avoid cell damage.

In mammals, the response to hypoxia involves the contraction of the pulmonary vasculature to divert blood from the hypoxic regions of the lungs, the release of neurotransmitters by the carotid body to increase respiratory rate, and a decrease in the use of ATP.

Hypoxia stimulates the contraction of the pulmonary vasculature, causing an influx of calcium into the smooth muscle cells of the pulmonary artery (PASMC) – and calcium activates cell contraction. Some studies suggest that reducing PASMC under hypoxic conditions requires ROS. This is the primary evidence that mitochondrial inhibitors and mitochondrial antioxidants block hypoxic pulmonary vasoconstriction and PASMC contraction under hypoxic conditions.

Mitochondrial ROS regulate autophagy

Autophagy is how cells absorb and destroy intracellular proteins and organelles in the lysosome and redirect components for new biosynthesis. Under normal conditions, this happens all the time to remove and recycle damaged proteins and organelles.

Cells lacking the ability to undergo autophagy accumulate dysfunctional mitochondria, which are usually removed by mitochondrial autophagy, called mitophagy. These cells accumulate ROS due to both an increase in the total number of mitochondria and an increase in the number of damaged mitochondria that secrete higher ROS levels. Thus, mitophagy is a mechanism for limiting the level of ROS.

In addition to its role in maintaining homeostasis, autophagy is also an essential response to cellular stress, including starvation, ischemia, and infection. In fasting, the function of autophagy is to recycle intracellular molecules when external nutrients are limited.

Mitochondrial ROS are essential for triggering autophagy during fasting. ROS and mitophagy can form a feedback loop through which ROS triggers mitophagy, limiting further ROS production by reducing the number of mitochondria.

Mitochondrial ROS regulate stem cell differentiation

Embryonic stem cells give rise to all the cells of a multicellular organism, and adult stem cells replenish the cells throughout the life of the body and after injuries. Stem cells are characterized by their ability to self-renew to maintain a pool of stem cells and differentiate to form fresh specialized tissue. The molecular mechanisms that drive pool renewal and differentiation are not fully understood, but recent studies show that mitochondrial ROS plays an important role.

Studies have shown that ROS are essential for stem cell differentiation:

  • Removing ROS from multipotent Drosophila hematopoietic progenitor cells slows down differentiation into mature blood cells. Conversely, an increase in ROS accelerates differentiation.
  • Mitochondrial-directed antioxidants inhibit the differentiation of human multipotent stem cells into mammalian adipose tissue cells-adipocytes. This fact confirms that ROS are necessary for the differentiation of mammalian cells.
  • Mitochondrial ROS are important for human stem cell differentiation. Human pluripotent stem cells (hPSCs) inhibit the release of the UCP2 protein during differentiation. A decrease in the release of UCP2 usually increases the production of ROS.

Future studies should investigate whether mitochondrial ROS is a common requirement for multiple differentiation systems and whether they are necessary for in vivo differentiation.

Mitochondrial ROS regulate aging

The cause of aging remains a matter of debate. Some argue that aging is pre-programmed, while others suggest that it is simply the effect of accumulating damage to proteins, lipids, and DNA.

In the 1950s, Denham Harman proposed the free radical theory of aging as a molecular explanation for why aging occurs. The idea is that free radicals, as byproducts of oxidative metabolism, cause cumulative cell damage, which over time leads to a general loss of the body’s viability. If this theory is correct, then increasing the antioxidant capacity should increase the body’s life span. However, over the past two decades, there has been no consensus on whether this is happening or not, and conflicting reports and reviews show that ROS is both a cause and just a property of the aging process.

Since ROS is essential for maintaining normal physiology, an increase in antioxidant capacity will limit oxidative damage and normal adaptation to stress.

Recent evidence suggests that low ROS levels activate stress responses that are beneficial to the body and extend life expectancy:

  • In yeast, inhibition of TOR (the target of rapamycin) increases life expectancy by increasing ROS.
  • Calorie restriction increases the life span of yeast due to the release of hydrogen peroxide H2O2 – a product of ROS.
  • Both TOR inhibition and caloric restriction increase life expectancy in mammals, although no association with ROS has been established.
  • In Drosophila, ROS levels increase with age but do not change with interventions that increase life expectancy.
  • In elegans, glucose restriction increases ROS, increasing life expectancy.
  • In mice, there is a correlation between increased ROS production and increased life expectancy.

Data on the regulation of human life expectancy are scarce. Replicative aging of human cells in vitro is used as a surrogate to study human life expectancy.

Interestingly, an early pillar of the free radical theory is based on the observation that human diploid fibroblasts have an increased replicative lifespan under hypoxic conditions. It has been suggested that a decrease in oxygen content reduces ROS and the accumulation of oxidative damage, thereby increasing the duration of replicative life. However, as noted above, hypoxia paradoxically increases ROS. Also, ROS is necessary to increase the replicative lifespan of human fibroblasts under hypoxic conditions.

As a result of numerous studies of lower organisms, the theory has emerged that low levels of mitochondrial ROS slow down aging.

Mitochondrial ROS can serve as an alarm signal to notify the cell of a change in the extracellular environment. ROS production is tightly regulated: high levels of ROS cause damage and subsequent cell death, while moderate levels cause the body to adapt and promote cell survival. Inhibition of ROS by antioxidants does not have a predictable outcome for cell function, as the role of ROS varies under different environmental conditions.

Mitochondrial ROS regulate immunity

To destroy pathogens, immune cells use an oxidative explosion – a quick release of ROS. However, a wide range of innate immune functions, including antiviral, antibacterial, and antiparasitic responses, require more subtle changes in the intracellular redox state regulated by ROS.

More than a decade ago, it was discovered that mice with a knockout of the UCP2 protein had increased immunity to Toxoplasma gondii and Salmonella typhimurium. Notably, while all the control mice died from T. gondii infection, the knockout mice were protected. The less the UCP2 protein is produced, the mitochondria release the more ROS. In fact, isolated UCP2 knockout macrophages generate more ROS and eliminate tachyzoites more effectively T.gondii in vitro. This effect is eliminated by treatment with antioxidants. LPS endotoxin has also been found to inhibit UCP2 in wild-type macrophages, increase ROS production to activate the MAPK signaling pathway and enhance the oxidative burst to kill pathogens.

Several proofs point to a role for mitochondrial ROS in acquired and innate immune cell function. Treatment of primary T cells with antioxidants suppresses the proliferation and production of the proinflammatory cytokine protein IL-2 following stimulation of the T cell receptor / CD28. This fact indicates that ROS are required in the early stages of T cell activation. Besides, treating mice with oral antioxidants reduces T cell growth following viral infection. Thus, ROS are necessary for the functioning of T cells in vivo.

Mitochondrial ROS regulate interferon type I responses in plasmacytoid dendritic cells

ROS are involved in a variety of signaling pathways, including antiviral responses.

Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. They produce the signal protein interferon type I (IFN) 200-1000 times more than other white blood cells or tissue cells in response to viruses. The sustainable production of pDC IFN-I is critical to the elimination of some acute viral infections. This is evidenced when the pDC is depleted, the secretion of IFN-I is disrupted, and the viral infection becomes chronic.

Viruses are very heterogeneous in structure, genomic composition and replication strategy. Therefore, several receptors are required to detect their presence in different parts of the cell.

The researchers proposed a model in which the TLR receptor organizes the first phase of IFN-I production, whereas the RIG-I receptor participates only in the late stage of antiviral responses, enhancing IFN-I production in the pDC:

  • The initial recognition of viral nucleic acids and the first wave of IFN-I production is mainly due to endosomal Toll-like receptors (TLR) 7 and 9 in the pDC, which are selectively expressed in the pDC. In the early stage of viral infection, PDCs circulating in the blood or localized in secondary lymphoid tissues absorb non-infectious viral particles or parts of dead infected cells and produce a large amount of type I interferon depend on the internal replication of the virus in the pDC.
  • Later, when PDCs leave the bloodstream and accumulate at the site of infection, they can become infected with viruses, which requires the detection of virus replication intermediates by cytosolic receptors such as RIG-I. Unlike conventional dendritic cells and macrophages, RIG-I is not expressed in resting pDC, but its expression is rapidly enhanced by endosomal TLR stimulation.
  • The researchers investigated the regulatory role of ROS in antiviral signaling pathways initiated by receptors located in different parts of the cell. They studied the potential effect of elevated ROS levels on the early phase of IFN-I production mediated by endosomal TLRs and the late phase of IFN-I secretion induced by the cytosolic RIG-I receptor in the pDC.

Research progress:

  • To cause increased ROS formation, the cells were treated with antimycin-A (AMA).
  • The TLR9 receptor was stimulated with CpG-A.
  • To induce RIG-I expression, the cells were incubated with CpG-A for 16 hours.

Results of the study:

  • AMA treatment increases ROS production in cells, which can be prevented by pretreatment with an antioxidant
  • Elevated ROS levels do not significantly change the cell phenotype but reduce the release of proinflammatory cytokines and chemokines
  • Mitochondrial ROS inhibits CpG-A-induced production of IFN-α
  • Elevated ROS levels reduce CpG-A-induced RIG-I expression
  • Increased ROS increases IFN-I production in cells activated by RIG-I, whereas it decreases it in cells re-stimulated by CpG-A
  • High ROS levels enhance the expression and phosphorylation of key signaling molecules in the RIG-I signaling cascade

Conclusions

Various exogenous and endogenous stimuli, such as hypoxia, growth factor stimulation, infection, fasting, and glucose restriction, can increase mitochondrial ROS levels. Too high ROS levels cause tissue damage, while moderate levels increase the body’s resistance to stressors and increase life expectancy.

ROS are essential for healthy cell function. They regulate adaptation to hypoxia, autophagy, immunity, stem cell differentiation, and longevity.

Over the past decades, many studies have demonstrated the critical role of ROS in cellular signaling pathways and innate immune responses, including antiviral and antibacterial immunity. The immune system is susceptible to ROS. Endosomal TLR-mediated antiviral responses of human PDCs are reduced when exposed to exogenous/endogenous ROS sources.

It was previously thought that pDC, unlike conventional dendritic cells or macrophages, preferentially use the TLR system rather than RIG-I-like receptors (RLRs) to detect viral infections. However, scientists have recently discovered that RIG-I promotes virus recognition and subsequent antiviral immune responses by the pDC. In resting human PDCs, RIG-I is expressed at a superficial level, but its expression is significantly increased by stimulating the TLR7 or TLR9 cellular receptors. Thus, in recognizing viral nucleic acids, the early response of IFN-I depends on TLR-mediated signals, whereas the transmission of RLR signals can mediate the second wave of IFN-I production.

Plasmacytoid DCS are the primary sources of type I interferons in viral infections. However, excessive production of IFN-I is not always beneficial, as it can lead to uncontrolled inflammation and destruction of healthy tissues. Therefore, the control of TLR-mediated systemic IFN responses by circulating PDCs during the early phase of viral infection can prevent excessive tissue damage.

In the later stages of infection, the pDC is redistributed into the infected peripheral tissues. They may be exposed to elevated levels of ROS produced by inflammatory cells and be infected with replicating viruses. It leads to cytosolic recognition of viral RNA by RIG-I and causes a second wave of IFN-I production in the pDC. The second phase of IFN production mediated by RIG-I is much weaker than the first phase initiated by TLR. However, the second phase is enhanced by ROS, which is confirmed by the study results.

Further study of how ROS regulates signaling pathways will help improve drugs targeting ROS-dependent molecules to treat inflammatory diseases and prolong human life.

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