The Durk Pearson & Sandy Shaw®
Life Extension NewsTM
Volume 17 No. 5 • June 2014


Rapid New Advances in the Understanding of Senescence Leading To Practical Methods of Deterring Aging Such As Decreasing Loss of Muscle

Extending the Replicative Lifespan of WI-38 Human Fibroblasts with Selenium

Selenium Increases Population Doublings, Reduces Rate of Telomere Shortening, and Decreases Rate of Age-Associated Senescence in Human WI-38 Cells

Human chromosomes (gray)
capped by telomeres (white)
The WI-38 human fibroblast is the cell line that Leonard Hayflick used in the discovery of the famous Hayflick limit, where he demonstrated that cells would replicate only a limited number of times before becoming terminally senescent.AA Senescent cells don’t immediately die but they no longer replicate and they undergo many changes that distinguish them from non-senescent cells. It has been learned, for example, that senescent cells are not generally beneficial to have around, other than the fact that their exit from the cell cycle acts as a protective mechanism against those cells becoming cancerous. A growing population of senescent cells with age has been found to have a deleterious effect on the functioning of non-senescent cells in their neighborhood by, for example, sustaining chronic inflammation and extracellular matrix remodeling.A The Hayflick limit was one of the earliest studies in which the processes that impair the function of aging cells started to be discovered.

Today, we report the publication of a new study (published today, Feb. 28, 2014 in the Journal of Biological Chemistry.1) describing how selenium depletion (Dpl) impairs the proliferative capacity of WI-38 cells and accelerates their conversion to senescence and how the cells could undergo additional doublings by being supplemented with selenium (Sup) beyond the amount of selenium considered adequate (Ctrl). The form of selenium used was sodium selenite, the form we recommend and take ourselves. [See “If You Take Selenium To Help Reduce Your Risk of Cancer …” in the October 2010 issue of Life Enhancement.]

The researchers first note that selenium levels decline with age and describe two independent studies that suggest a link between lifespan and selenium, finding that selenium levels in the blood of elderly people was a predictor of longevity2,3 The results of their new work1 showed that “the change in selenium concentrations in young cells triggers rapid changes in replicative senescence-associated markers and signaling pathways and regulates the entry into replicative senescence.”1

Consistent with prior studies, the researchers found that their cells grown in Ctrl (Control) conditions maintained their proliferative capacity until the level of population doubling #51 (CPD 51). At that time they showed signs of early senescence (presenescence) and they became senescent at CPD 57. The researchers separated cells that had reached CPD 36 (late middle aged) and were still actively dividing and exposed them to the different concentrations of selenium, finding that in two passages (two divisions) the effect of different concentrations of selenium was already apparent. Hence, these changes can take place fast at a late stage of replicative lifespan under the influence of either deficient selenium or selenium supplementation. In all cases, they reported, “selenium supplementation led to an extended replicative lifetime.”

The scientists examined changes in markers of senescence in young proliferating WI-38 fibroblasts in Dpl, Ctrl, and Sup media for two passages and found a nearly threefold increase in one such marker, SAHF, between the cells grown under Dpl conditions as compared to those grown in the Ctrl condition. Again, these changes were in response to different media selenium concentrations. The researchers propose that, “the entry into replicative senescence of cultured human diploid fibroblasts is due to an irreversible cell cycle arrest mainly controlled by the p53-p21 and p-16-pRb signaling pathways.”1 Selenium supplementation was found to significantly reduce (by more than 3 times) the expression levels of p16, p21, p53, and pRb as compared with cells grown under Ctrl conditions.

Young WI-38 Cells Grown Under Conditions of Selenium Deficiency (Dpl) Look Like Senescent Cells

Very interestingly, the researchers found that the young WI-38 cells, after growing in Dpl media for two passages “present several characteristics very similar to those of senescent cells, which include morphological changes, increased number of positive cells for SAHF and SABG [biomarkers of senescence], and telomere length shortening.”1

Similarities in Senescent Cells and Cells Exposed to Chronically Elevated Levels of Oxygen

Another paper1B reports finding similar gene expression in senescent and chronic mild hyperoxic cells (cells exposed to higher than normal oxygen), suggesting that hyperoxia could be considered a model of accelerated senescence and, therefore, another good reason to avoid chronic oxidative stress as much as possible.

PREVENTING MUSCLE LOSS WITH AGING

Can Selenium Supplementation Prevent Age-Associated Sarcopenia?

A very recent paper4 reported that p16INK4a, a p16 tumor suppressor protein (part of the p16 signaling pathway), was responsible for inducing senescence in the regenerative capacity of muscle stem cells. These researchers found that p16INK4a, thought to be a master regulator of cellular senescence, is (as part of the p16INK4a/Rb/E2F-signaling axis) responsible for a senescent-like state that may be a key part of the process of sarcopenia, the age-associated loss of muscle. In the new study,4 the scientists found that genetically silencing p16INK4a expression restored self-renewal and proliferation in aged skeletal muscle satellite cells.

We wonder: Would selenium supplementation tend to reduce the expression of p16INK4a as it did the expression of p16 in the WI-38 fibroblast study discussed above? If so, selenium supplementation could be a practical means of protecting against sarcopenia.

Magnesium Deficiency Accelerates Cellular Senescence in Cultured Human Fibroblasts

According to this 2008 paper,4B more than half the population of the U.S. ingests an inadequate amount of dietary magnesium. The researchers cultured human diploid lung fibroblasts (IMR-90) in media containing various amounts of magnesium. Cells cultured in media with 50% of the magnesium contained in standard media were shown, in four independent lifespan studies, to lose 2.5 population doublings as compared with cells grown in media with the full amount of magnesium. Cells cultured in 13% of normal magnesium concentration lost 4.5 population doublings. [See “Durk Pearson & Sandy Shaw Ring the Bell for … Magnesium Aspartate” in the August 1998 issue of Life Enhancement.]

Is It Beneficial To Remove Senescent Cells?

Removing p16INK4a-positive senescent cells from experimental animals (mice) has already been done in order to see whether that might be beneficial (or not).6 In that study, mice with a progeroid genetic background were treated with an experimental drug to eliminate p16INK4a-positive senescent cells. The researchers reported that, “In tissues—such as adipose tissue, skeletal muscle, and eye—in which p16INK4a contributes to the acquisition of age-related pathologies, lifelong removal of p16INK4a-expressing cells delayed onset of these phenotypes. Furthermore, late life clearance attenuated progression of already established age-related disorders.” Hence, at least in this model, it would appear that senescent cells can be removed with beneficial effects.

p16INK4a Positive Cells in Human Skin Found to Reflect Biological Age

A powerful link between the p16INK4a senescence marker and biological age was reported in another new paper,7 when researchers found that a younger biological age associates with lower levels of p16INK4a positive cells in human skin. (The researchers were referring to chronological age as biological age, presumably due to the lack of a standardized way of distinguishing between them.) They observed that increased numbers of senescent cells were associated with age-related pathologies such as atherosclerosis, diabetes, and kidney disease. This makes it possible for a test to be developed in which one could find out how well he or she is doing in terms of aging through the detection of this senescence marker in their skin cells. (NOTE: The FDA is likely to try to block this information from being made available directly to the public. However, under the First Amendment, they do not have the authority to do so.)

Osteoarthritis Characterized by Accumulation of Senescent p16INK4a Positive Cells

A very common age-associated disease that has been linked to cellular senescence is osteoarthritis, which can be a source of considerable pain and disability. In a 2014 paper,A researchers reported that “[o]steoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP) including the matrix remodelling metallo-proteases MMP1/MMP13 and pro-inflammatory cytokines IL-8, and IL-6.” The researchers found that p16INK4a is induced by treatment with the pro-inflammatory cytokine IL-1beta and also during in vitro chondrogenesis.

Senescence Can Be Transmitted to Non-Senescent Neighboring Cells

It is interesting to note that recent studies cited in another paper9 have shown that “some SASP factors, including IL-1beta, can induce senescence in normal cells. Thus, senescence can be transmitted to untransformed neighboring cells through the paracrine activity of the SASP.”9 There goes the neighborhood! (Seriously, this is another reason to try to prevent the buildup of senescent cells during aging.)

Metformin May Be Another Anti-Senescence Treatment

In addition to selenium, magnesium, and zinc, there is also evidence that metformin, a drug used widely in the treatment of diabetes and symptoms of metabolic syndrome, such as insulin resistance, may also protect against the premature senescence that accompanies high glucose conditions.10 In a study of kidney cells (primary rat glomerular mesangial cells) in culture, exposed to high glucose conditions, researchers found that the cells initially and transiently entered a proliferative phase, but was followed by cell cycle arrest along with changes seen in senescent cells (for example, increased expression of cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1)). In addition, connexin43, a gap junction that importantly allows cells to communicate with each other, is less expressed in cells exposed to high glucose conditions and in older cells. This decreased intercellular communication is also observed in senescent cells.10

AMPK, an important regulator of energy metabolism (increased under conditions of reduced energy availability, decreased when energy supplies are plentiful) is reported to maintain the expression of connexin43 under high glucose conditions. Incubation of the rat glomerular mesangial kidney cells with metformin in this study10 significantly increased p-AMPK (phosphorylated AMPK, the active form) and up-regulated connexin43 compared to cells exposed to high glucose conditions but not treated with metformin. Metformin treatment also decreased the expression of the senescence markers p27(kip1) and p21(cip1).

Too Much Zinc May Worsen Osteoarthritis

A caution concerning zinc supplementation: A 2014 Cell paper11 reported that ZIP8, a zinc transporting enzyme, is overexpressed in chondrocytes (cartilage cells) of humans with osteoarthritis. This caused increased intracellular levels of zinc and of zinc-dependent metalloprotease matrix-degrading enzymes that have been identified as causative factors in osteoarthritis. The question is: could zinc supplementation in those with osteoarthritis have an exacerbating effect on the disease? The authors11 speculate that local depletion of zinc, inhibition of ZIP8 or of the metalloproteases involved in osteoarthritis might be effective therapies for osteoarthritis. Also, we note, reducing IL-1beta (which induces ZIP8) is another possible therapeutic approach. (IL-1beta is an important part of the human wound-healing response.11A)

How old would you be if you didn’t know how old you are?
— Leroy “Satchel” Paige

References

AA. Hayflick. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res. 37:614-36 (1965).
A. Philipot et al. p16INK4a and its regulator miR-24 link senescence and chrondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis. Arthritis Res Ther. 16:R58 (2014).
1. Legrain et al. Interplay between selenium levels, selenoprotein expression, and replicative senescence in WI-38 human fibroblasts. J Biol Chem. 289:6299-310 (2014).
1B. Saretzki et al. Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol. 53A(6):B438-42 (1998).
2. Ray et al. Low serum selenium and total carotenoids predict mortality among older women living in the community. The women’s health and aging studies. J Nutr. 136:172-6 (2006).
3. Akbaraly et al. Selenium and mortality in the elderly. Results from the EVA study. Clin Chem. 51:2117-23 (2006).
4. Sousa-Victor et al. Geriatric muscle stem cells switch reversible quiescence into senescence. Nature. 506:316-21 (2014).
4B. Killilea and Ames. Magnesium deficiency accelerates cellular senescence in cultured human fibroblasts. Proc Natl Acad Sci USA. 105(15):5768-73 (2008).
5. Li and Belmonte. Genetic rejuvenation of old muscle. Nature. 506:304-5 (2014). This is the commentary on paper #2.
6. Baker et al. Clearance of p16INK4a-positive senescent cells delays ageing-associated disorders. Nature. 479:232-6 (2011).
7. Waaijer et al. The number of p16INK4a positive cells in human skin reflects biological age. Aging Cell. 114:722-5 (2012).
9. Salama et al. Cellular senescence and its effector programs. Genes Dev. 28:99-114 (2014).
10. Guo et al. AMPK-mediated downregulation of connexin43 and premature senescence of mesangial cells under high-glucose conditions. Exp Gerontol. 51:71-81 (2014).
11. Kim et al. Regulation of the catabolic cascade in osteoarthritis by the zinc-ZIP8-MTF1 axis. Cell. 156:730-43 (2014).
11A. Kraus. The zinc link. Nature. 507:441-2 (2014).

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