Broccoli sprouts activate the AMPK pathway, Part 4

Today someone viewed the 2020 Part 3 of Broccoli sprouts activate the AMPK pathway which lacked citations at the time. Checking again, here are three citing 2022 papers, starting with a review:

“Nrf2 is an important transcription factor that regulates expression of a large number of genes in healthy and disease states. Nrf2 regulates expression of several key components of oxidative stress, mitochondrial biogenesis, mitophagy, autophagy, and mitochondrial function in all organs of the human body, and in the peripheral and central nervous systems.

Overall, therapeutic drugs including sulforaphane that target Nrf2 expression and Nrf2/ARE pathway are promising. This article proposes additional research in Nrf2’s role within Parkinson’s disease, Huntington’s disease, and ischemic stroke in preclinical mouse models and humans with age-related neurodegenerative diseases.”

https://www.sciencedirect.com/science/article/pii/S1568163722001982 “Role of Nrf2 in aging, Alzheimer’s and other neurodegenerative diseases” (not freely available) Thanks to Dr. P. Hemachandra Reddy for providing a copy.


One of the Part 3 study’s coauthors contributed to this very detailed review:

“Due to observed overlapping cellular responses upon AMPK or NRF2 activation and common stressors impinging on both AMPK and NRF2 signaling, it is plausible to assume that AMPK and NRF2 signaling may interdepend and cooperate to readjust cellular homeostasis.

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The outcome and underlying signaling events of AMPK-NRF2 crosstalk may diverge between:

  1. in vitro and in vivo studies (one cell type in isolation vs inter-organ crosstalk in living organisms);
  2. Different cell types/organs/organisms of different cultivation conditions, genetic background, age or sex;
  3. Different stress-regimens (chronic vs acute, nature of stress (lipotoxicity, redox stress, xenobiotic, starvation, etc));
  4. Different modes of Nrf2 or AMPK activation and inhibition (genetic vs pharmacological, constitutive vs transient/intermittent, systemic vs organ-specific, electrophilic vs PPI, allosteric vs covalent, or pan vs subtype-specific);
  5. Different target genes with distinct promoter and enhancer structure; or
  6. Different timing of activation.

The latter should deserve increased attention as Nrf2 is one of the most cycling genes under control of the circadian clock. Feeding behavior, metabolism and hence AMPK activity follow and substantiate the biological clock, indicating an entangled circadian regulation of metabolic and redox homeostasis.”

https://www.sciencedirect.com/science/article/pii/S089158492200497X “AMPK and NRF2: Interactive players in the same team for cellular homeostasis?”


A third citing paper was a study of lens cells that provided an example of similar metformin effects noted in Part 2 of Broccoli sprouts activate the AMPK pathway:

“Loss of Nrf2 and Nrf2 antioxidant genes expression and activity in aging cells leads to an array of oxidative-induced deleterious responses, impaired function, and aging pathologies. This deterioration is proposed to be the primary risk factor for age-related diseases such as cataracts.

AMPK regulates energy at physiological levels during metabolic imbalance and stress. AMPK is a redox sensing molecule, and can be activated under cellular accumulation of reactive oxygen species, which are endogenously produced due to loss of antioxidant enzymes.

The therapeutic potential of AMPK activation has context-dependent beneficial effects, from cell survival to cell death. AMPK activation was a requisite for Bmal1/Nrf2-antioxidants-mediated defense, as pharmacologically inactivating AMPK impeded metformin’s effect.

Using lens epithelial cell lines (LECs) of human or mouse aging primary LECs along with lenses as model systems, we demonstrated that metformin could correct deteriorated Bmal1/Nrf2/ARE pathway by reviving AMPK-activation and transcriptional activities of Bmal1/Nrf2, resulting in increased antioxidants enzymatic activity and expression of Phase II enzymes. Results uncovered crosstalk between AMPK and Bmal1/Nrf2/antioxidants mediated by metformin for blunting oxidative/aging-linked pathobiology.”

https://www.mdpi.com/2073-4409/11/19/3021/htm “Obligatory Role of AMPK Activation and Antioxidant Defense Pathway in the Regulatory Effects of Metformin on Cellular Protection and Prevention of Lens Opacity”


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If you were given a lens to see clearly, would you accept it?

Two papers, starting with a 2022 rodent study of maternal behaviors’ effects on offspring physiologies:

Early life adversity (ELA) is a major risk factor for development of pathology. Predictability of parental care may be a distinguishing feature of different forms of ELA.

We tested the hypothesis that changes in maternal behavior in mice would be contingent on the type of ELA experienced, directly comparing predictability of care in the limited bedding and nesting (LBN) and maternal separation (MS) paradigms. We then tested whether predictability of the ELA environment altered expression of corticotropin-releasing hormone (Crh), a sexually-dimorphic neuropeptide that regulates threat-related learning.

MS was associated with increased expression of Crh-related genes in males, but not females. LBN primarily increased expression of these genes in females, but not males.”

https://www.sciencedirect.com/science/article/pii/S2352289522000595 “Resource scarcity but not maternal separation provokes unpredictable maternal care sequences in mice and both upregulate Crh-associated gene expression in the amygdala”


I came across this first study by it citing a republished version of 2005 epigenetic research from McGill University:

“Early experience permanently alters behavior and physiology. A critical question concerns the mechanism of these environmental programming effects.

We propose that epigenomic changes serve as an intermediate process that imprints dynamic environmental experiences on the fixed genome resulting in stable alterations in phenotype. These findings demonstrate that structural modifications of DNA can be established through environmental programming and that, in spite of the inherent stability of this epigenomic marker, it is dynamic and potentially reversible.”

https://www.tandfonline.com/doi/full/10.31887/DCNS.2005.7.2/mmeaney “Environmental programming of stress responses through DNA methylation: life at the interface between a dynamic environment and a fixed genome”


This post commemorates the five-year anniversary of Dr. Arthur Janov’s death. Its title is taken from my reaction to his comment on Beyond Belief: Symptoms of hopelessness. Search his blog for mentions of the second paper’s coauthors, Drs. Meaney and Szyf.

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All about walnuts’ effects

Five 2022 papers focusing on walnuts, starting with a comparison of eight tree nuts:

“The aim of the present study was to examine 8 different popular nuts – pecan, pine, hazelnuts, pistachio, almonds, cashew, walnuts, and macadamia. Total content of phenolic compounds in nuts ranged from 5.9 (pistachio) to 432.9 (walnuts) mg/100 g.

Walnuts had the highest content of polymeric procyanidins, which are of great interest as important compounds in nutrition and biological activity, as they exhibit antioxidant, anti-inflammatory, antimicrobial, cardio- and neuroprotective action. Walnuts are good sources of fatty acids, especially omega-3 and omega-6.”

https://www.sciencedirect.com/science/article/pii/S2590157522002164 “Nuts as functional foods: Variation of nutritional and phytochemical profiles and their in vitro bioactive properties”


A second study compared the same eight tree nuts plus Brazil nuts and peanuts:

“The highest total content of all analyzed flavonoids was determined in walnuts (114.861 µg/g) with epicatechin the most abundant, while the lowest was in almonds (1.717 µg/g). Epicatechin has antioxidant, anti-inflammatory, antitumor, and anti-diabetic properties. Epicatechin has beneficial effects on the nervous system, enhances muscle performance, and improves cardiac function.”

https://www.mdpi.com/1420-3049/27/14/4326/htm “The Content of Phenolic Compounds and Mineral Elements in Edible Nuts”


Next, two systematic reviews and meta-analyses of human studies:

“We carried out a systematic review of cohort studies and randomized controlled trials (RCTs) investigating walnut consumption, compared with no or lower walnut consumption, including those with subjects from within the general population and those with existing health conditions, published from 2017 to 5 May 2021.

  • Evidence published since 2017 is consistent with previous research suggesting that walnut consumption improves lipid profiles and is associated with reduced CVD risk.
  • Evidence pointing to effects on blood pressure, inflammation, hemostatic markers, and glucose metabolism remains conflicting.
  • Evidence from human studies showing that walnut consumption may benefit cognitive health, which is needed to corroborate findings from animal studies, is now beginning to accumulate.”

https://academic.oup.com/nutritionreviews/advance-article/doi/10.1093/nutrit/nuac040/6651942 “Walnut consumption and health outcomes with public health relevance – a systematic review of cohort studies and randomized controlled trials published from 2017 to present”


“We aimed to perform a systematic review and meta-analysis of RCTs to thoroughly assess data concerning effects of walnut intake on selected markers of inflammation and metabolic syndrome in mature adults. Our findings showed that:

  • Walnut-enriched diets significantly decreased TG, TC, and LDL-C concentrations, while HDL-C levels were not significantly affected.
  • No significant changes were noticed on anthropometric, cardiometabolic, and glycemic indices after higher walnut consumption.
  • Inflammatory biomarkers did not record statistically significant results.”

https://www.mdpi.com/2076-3921/11/7/1412/htm “Walnut Intake Interventions Targeting Biomarkers of Metabolic Syndrome and Inflammation in Middle-Aged and Older Adults: A Systematic Review and Meta-Analysis of Randomized Controlled Trials”


Finishing with a rodent study that gave subjects diabetes with a high-fat diet, then mixed two concentrations of walnut extract in with the treatment groups’ chow:

“This study was conducted to evaluate the protective effect of Gimcheon 1ho cultivar walnut (GC) on cerebral disorder by insulin resistance, oxidative stress, and inflammation in HFD-induced diabetic disorder mice. After HFD feed was supplied for 12 weeks, samples were orally ingested for 4 weeks to GC20 and GC50 groups (20 and 50 mg/kg of body weight, respectively).

  • Administration of GC improved mitochondrial membrane potential function, and suppressed oxidative stress in the brain.
  • GC inhibited hepatic and cerebral lipid peroxidation and the formation of serum AGEs, and increased serum antioxidant activity to improve HFD-induced oxidative stress.
  • The HFD group showed significant memory impairment in behavioral tests. On the other hand, administration of GC showed improvement in spatial learning and memory function.

walnut brain effects

Based on these physiological activities, GC showed protective effects against HFD-induced diabetic dysfunctions through complex and diverse pathways.”

https://www.mdpi.com/1420-3049/27/16/5316/htm “Walnut Prevents Cognitive Impairment by Regulating the Synaptic and Mitochondrial Dysfunction via JNK Signaling and Apoptosis Pathway in High-Fat Diet-Induced C57BL/6 Mice”


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Minds of their own

It’s the weekend, so it’s time for: Running errands? Watching sports? Other conditioned behavior?

Or maybe broadening our cognitive ability with Dr. Michael Levin’s follow-ups to his 2021 Basal cognition paper and 2020 Electroceuticals presentation with a 2022 paper and presentation starting around the 13:30 mark:

Michael Levin - Cell Intelligence in Physiological and Morphological Spaces

“A homeostatic feedback is usually thought of as a single variable such as temperature or pH. The set point has been found to be a large-scale geometry, a descriptor of a complex data structure.”


His 2022 paper Technological Approach to Mind Everywhere: An Experimentally-Grounded Framework for Understanding Diverse Bodies and Minds:

“It is proposed that the traditional problem-solving behavior we see in standard animals in 3D space is just a variant of evolutionarily more ancient capacity to solve problems in metabolic, physiological, transcriptional, and morphogenetic spaces (as one possible sequential timeline along which evolution pivoted some of the same strategies to solve problems in new spaces).

Developmental bioelectricity works alongside other modalities such as gene-regulatory networks, biomechanics, and biochemical systems. Developmental bioelectricity provides a bridge between the early problem-solving of body anatomy and the more recent complexity of behavioral sophistication via brains.

This unification of two disciplines suggests a number of hypotheses about the evolutionary path that pivoted morphogenetic control mechanisms into cognitive capacities of behavior, and sheds light on how Selves arise and expand.

While being very careful with powerful advances, it must also be kept in mind that existing balance was not achieved by optimizing happiness or any other quality commensurate with modern values. It is the result of dynamical systems properties shaped by meanderings of the evolutionary process and the harsh process of selection for survival capacity.”


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Don’t bother eating broccoli sprouts if you’re old?

I try to not curate research that wastes resources. Couldn’t help but present this 2022 rodent study:

“We aimed to evaluate if sulforaphane (SFN) long-term treatment was able to prevent age-associated cognitive decline in adult (15-month-old) and old (21-month-old) female and male rats.

Our results showed that SFN restored redox homeostasis in brain cortex and hippocampus of adult rats, preventing cognitive decline in both sexes. However, redox responses were not the same in males and females.

Old rats were not able to recover their redox state as adults did, but they had a mild improvement. These results suggest that SFN mainly prevents rather than reverts neural damage; though, there might also be a range of opportunities to use hormetins like SFN, to improve redox modulation in old animals.”

https://link.springer.com/article/10.1007/s10522-022-09984-9 “Long-term sulforaphane-treatment restores redox homeostasis and prevents cognitive decline in middleaged female and male rats, but cannot revert previous damage in old animals” (not freely available)


These researchers cited Sulforaphane in the Goldilocks zone for hormetic effects of sulforaphane, so I asked:

“Did you develop any preliminary dose/response data for stating ‘there might also be a range of opportunities to use hormetins like SFN to improve redox modulation in old animals’?”

They cited Broccoli sprouts activate the AMPK pathway for long-term effects of a small sulforaphane dose, so I asked:

“Also, the three studies cited for ‘0.5 mg/Kg, i.e. 2.82 μmol/Kg BW for 3 months’ were all mouse studies. Since this was a rat study, wouldn’t there be increased dose and duration equivalencies?”

I’ll update this blog post in the event either of my questions to these researchers are answered.

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Non-patentable boron benefits

To follow up Is boron important to health? I’ll highlight a 2022 review of boron intake:

“Boron is essential for activity of several metabolic enzymes, hormones, and micronutrients. It is important for growth and maintenance of bone, reduction in inflammatory biomarkers, and increasing levels of antioxidant enzymes.

The average person’s daily diet contains 1.5 to 3 milligrams of boron. Boron intakes of 1–3 mg/day have been shown to improve bone and brain health in adults when compared to intakes of 0.25–0.50 mg/day.

One week of 10 mg/d boron supplementation resulted in a 20% reduction in inflammatory biomarkers TNF-α, as well as significant reductions (nearly 50%) in plasma concentrations of hs-CRP and IL-6. Calcium fructoborate, a naturally occurring, plant-based boron-carbohydrate complex, had beneficial effects on osteoarthritis (OA) symptoms. A double-blind study in middle-aged patients with primary OA found that all groups except the placebo group saw a reduction in inflammatory biomarkers after 15 days of food supplementation with calcium fructoborate.

Dietary boron intake significantly improves brain function and cognitive functioning in humans. Electroencephalograms showed that boron pharmacological intervention after boron deficiency improved functioning in older men and women, such as less drowsiness and mental alertness, better psychomotor skills (for example, motor speed and dexterity), and better cognitive processing (e.g., attention and short-term memory). Boron compounds can help with both impaired recognition and spatial memory problems.

We discussed the role of boron-based diet in memory, boron and microbiome relation, boron as anti-inflammatory agents, and boron in neurodegenerative diseases. Boron reagents will play a significant role to improve dysbiosis.”

https://www.mdpi.com/1420-3049/27/11/3402/htm “The Role of Microbiome in Brain Development and Neurodegenerative Diseases”


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Broccoli sprouts and your brain

A 2022 review of Nrf2 signaling hilariously avoided mentioning sulforaphane, although of ~4,000 sulforaphane published articles, two were cited. I’ll curate it anyway to highlight referenced brain effects.

“A good stability of NRF2 activity is crucial to maintain redox balance and therefore brain homeostasis. In this review, we have gathered recent data about the contribution of the NRF2 pathway in the healthy brain as well as during metabolic diseases, ageing, and ageing-related neurodegenerative diseases.

A functional NRF2 system is important to regulate both neuroinflammation, i.e., activation of microglia and astrocytes, and oxidative stress in the brain. NRF2 and NF-κB transcription factors regulate cellular responses to inflammation and oxidative stress in order to maintain brain homeostasis. Both pathways have been described to inhibit each other.

Nrf2 brain aging

Future challenges will be to establish novel therapies to:

  • Increase NRF2 activation in specific cell types and/or brain regions; and
  • Modulate NRF2 pathway in senescent cells.

Modulation of NRF2 signalling pathway by using specific food products [like unmentioned broccoli sprouts] and phytochemicals [like unmentioned sulforaphane], dietary supplements [like unmentioned Vitamin D3], drugs, and epigenetic modifiers, alone or in combination, will help to limit inflammatory diseases, ageing process, and subsequently ageing-related diseases.”

https://www.mdpi.com/2076-3921/11/8/1426/htm “Normal and Pathological NRF2 Signalling in the Central Nervous System”


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Is boron important to health?

Three papers on boron, starting with a 2022 review:

“Boron-containing compounds (BCC) have effects in the metabolism of living organisms. Information regarding effects and interaction of these compounds was compiled, and potential applications for treating human metabolic disorders was suggested.

Dietary boron supplementation affects metabolism of calcium, magnesium, triglycerides, glucose, amino acids, reactive oxygen, nitrogen species, and hormones such as 17β-estradiol, calcitonin, and 25-hydroxy-cholecalciferol. When food is boron-deprived, there are adverse effects like depressed growth, reduced serum steroid hormone concentrations, changes in plasma and organ calcium and magnesium concentrations, plasma alkaline phosphatase, and bone calcification on animal biological functions.

boron effects

Exploration of basic BCC as metabolism regulators is expanding. Although mechanisms of action are uncertain, limitation of damage induced by reactive species, inflammatory modulation, or activities on some enzymes and membrane transporters are often related to reported effects.

An increasing number of new BCC are emerging as potential tools for prevention, diagnosis, and therapy of metabolism maladies such as diabetes, metabolic syndrome, osteoporosis, cardiovascular, and liver diseases. For those innovative BCC, mechanisms of action are often clear.”

https://link.springer.com/article/10.1007/s12011-022-03346-9 “Boron‑Containing Compounds for Prevention, Diagnosis, and Treatment of Human Metabolic Disorders” (not freely available) Thanks to Dr. Marvin A Soriano-Ursúa for providing a link to a freely available document.


A second paper was a 2021 human study:

“In our elderly population-based sample, a boron-rich diet appeared to be characterized by high intakes of plant foods presumed to be healthy, low intakes of plant foods presumed to be less healthy, and low intakes of all kinds of animal foods.

Higher plasma boron concentrations were related to lower BMI and circulating concentrations of CRP. Plasma boron concentrations were associated with age, phosphate, and plasma lipid metabolism, and showed seasonal variations.

Human intervention studies are warranted to derive causal relationships of circulating and dietary boron with human health and metabolism. Robust databases on boron content of foods are needed to facilitate investigation of dietary boron intake in human studies.

Clarification of the non-/essentiality of trace element boron for human health will form the basis to derive recommendations for a dietary boron intake being sufficient to exert boron’s proposed beneficial physiological roles.”

https://link.springer.com/article/10.1007/s00394-021-02730-w “Plasma boron concentrations in the general population: a cross-sectional analysis of cardio-metabolic and dietary correlates”

As noted in this study, public agencies don’t consider dietary boron content important enough to include in public databases. My daily boron dietary intake estimated from published private databases is:

  • Walnuts, 1.63 mg x (28.3 g / 100 g) = .5 mg
  • Red kidney beans, 1.4 mg x (12 g / 100 g) = .2 mg
  • Chickpeas, 0.71 mg x (40 g / 100 g) = .3 mg
  • Celery, 0.5 mg x (72 g / 100 g) = .4 mg
  • Carrots, 0.3 mg x ( 76 g / 100 g) = .3 mg
  • Coffee .07 mg x 3 cups = .2 mg

2 mg boron daily dietary total


A third paper was a 2022 rodent study:

“Sodium pentaborate pentahydrate (NaB) 1 and 2 mg elemental B/kg supplementation induces the anagen phase in rats via Wnt-1, β-catenin, VEGF, PDGF, and TGF-β1 signaling pathways, which are important molecular mechanisms involved in hair growth.

NaB 4 mg B/kg suppresses these pathways and adversely affects hair growth.”

https://www.sciencedirect.com/science/article/abs/pii/S0946672X22000876 “Sodium pentaborate pentahydrate promotes hair growth through the Wnt/β-catenin pathway and growth factors” (not freely available)

A human equivalent of this study’s rat 1 mg elemental boron intake is (1 mg x .162) x 70 kg = 11 mg.


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The goddess of rainbows

Two 2022 papers, starting with a review of irisin:

“This article is an overview of irisin generation, secretion, and tissue distribution. Its targeting of tissues or organs for prevention and treatment of chronic diseases is systematically summarized, with discussion of underlying molecular mechanisms.

Irisin is an exercise-induced myokine expressed as a bioactive peptide in multiple tissues and organs. Exercise and cold exposure are major inducers for its secretion.

Mechanistic studies confirm that irisin is closely correlated with lipid metabolism, insulin resistance, inflammation, ROS, endocrine, neurotrophic factors, cell regeneration and repairing, and central nervous system regulation. Irisin decreases with age, and is closely associated with a wide range of aging-related diseases.

A number of studies in elderly humans and animal models have shown that exercise can promote the body’s circulation and increase irisin levels in some tissues and organs. Resistance, aerobic, or combined exercise seem to play a positive role. However, exercise could not change serum irisin in some reported studies.

irisin human studies

There are large individual differences in exercise training in the elderly population. Since the half-life of irisin in the body is less than 1 h, it is necessary to pay attention to the time of blood sampling after a single exercise intervention. Some factors that impede detection of irisin levels in vivo include the half-life of irisin protein, sampling time, different tissues, and different health statuses before and after intervention.

It is worth noting that high-intensity exercise shows higher irisin levels even with the same energy expenditure during exercise. Precision studies of irisin in elderly subjects following exercise intervention need to be further clarified.”

https://www.sciencedirect.com/science/article/pii/S1568163722001222 “Irisin, An Exercise-induced Bioactive Peptide Beneficial for Health Promotion During Aging Process” (not freely available) Thanks to Dr. Ning Chen for providing a copy.


A second paper was a human study too recent to be cited by the first paper. I’ll highlight its irisin findings:

“We investigated the complex relationship among DNAm based biomarkers of aging, including DNAmFitAge, a variety of physiological functioning variables, blood serum measures including cholesterol, irisin level, and redox balance, and the microbiome on 303 healthy individuals aged between 33 and 88 years with a diverse level of physical fitness. Regular exercise was associated with younger biological age, better memory, and more protective blood serum levels.

Our research intends to show that regular physical exercise is related to microbiota and methylation differences which are both beneficial to aging and measurable. Our research provides the first investigation between microbiome derived metabolic pathways and DNAm based aging biomarkers.

Irisin levels decrease with age (0.23 average decrease for every 1 year older). We found age-related decreases in irisin levels were attenuated by exercise training. The link between irisin to GrimAge Acceleration and FitAge Acceleration is a novel observation.

HDL is positively associated with irisin. HDL and irisin have complex roles in physiology, and the positive relationship we observe between physical exercise and HDL and irisin align with protective effects seen between HDL and irisin with glucose homeostasis.

This work further supports the biological importance of irisin to the aging process. It is possible our research motivates interventions to boost irisin, like through physical exercise, as possible anti-aging therapies.”

https://www.medrxiv.org/content/10.1101/2022.07.22.22277842v1 “DNA methylation clock DNAmFitAge shows regular exercise is associated with slower aging and systemic adaptation


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Non-CpG methylation

Three 2022 papers on methylation epigenetic modifiers, starting with a human study focused on mitochondrial DNA non-CpG methylation involving nucleobases other than guanine (arginine, cytosine, or thymine):

“We collected brain tissue in the nucleus accumbens and prefrontal cortex from deceased individuals without (n = 39) and with (n = 14) drug use, and used whole-genome bisulfite sequencing to cover cytosine sites in the mitochondrial genome. Epigenetic clocks in illicit drug users, especially in ketamine users, were accelerated in both brain regions by comparison with nonusers.

Unlike the predominance of CpG over non-CpG methylation in the nuclear genome, the average CpG and non-CpG methylation levels in the mitochondrial genome were almost equal. The utility of non-CpG methylation was further illustrated by the three indices constructed in this study with non-CpG sites having better distinction between brain areas, age groups, and the presence or absence of drug use than indices consisting of CpG sites only. Results of previous studies on the mitochondrial genome that were solely based on CpG sites should be interpreted cautiously.

The epigenetic clock made up of age-related cytosine sites in mtDNA of the control group was consistently replicated in these two brain regions. One possibility for the correlation is the cycle theory that involves mitochondrial activity, mitochondrial DNA methylation, and alpha-ketoglutarate.

As mitochondrial activity fades with aging, mitochondria gradually lose the ability to eliminate methylation on cytosines through alpha-ketoglutarate. Further investigation of the underlying mechanisms is warranted.

To our knowledge, this is the first report that ketamine might change the mitochondrial epigenetic clock in human brain tissues. We believe this is the first report to elucidate comprehensively the importance of mitochondrial DNA methylation in human brain.”

https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-022-01300-z “Mitochondrial DNA methylation profiling of the human prefrontal cortex and nucleus accumbens: correlations with aging and drug use”


A second rodent study focused on RNA methylation:

“We investigated the role of RNA N6-methyladenosine (m6A) in improved resilience against chronic restraint stress. A combination of molecular, behavioral, and in vivo recording data demonstrates exercise-mediated restoration of m6A in the mouse medial prefrontal cortex, whose activity is potentiated to exert anxiolytic effects. To provide molecular explanations, it is worth noting that epigenetic regulation, such as histone modification, microRNA, and DNA methylation all participate in mental and cognitive rehabilitation following exercise.

To generalize these rodent data to humans, we recruited a small group of patients with major depressive disorder with prominent anxiety disorders. Compared to age- and sex-matched healthy individuals, patients displayed decreased circulating methyl donor S-adenosyl methionine (SAM) levels. Serum SAM levels were found to be inversely correlated with the Hamilton Anxiety Scale, suggesting the potential value of SAM as a biomarker for depression or anxiety disorders.

Hepatic biosynthesis of methyl donors is necessary for exercise to improve brain RNA m6A to counteract environmental stress. The dependence on hepatic-brain axis suggests the ineffectiveness of exercise training on people with hepatic dysfunctions.

This novel liver-brain axis provides an explanation for brain network changes upon exercise training, and provides new insights into diagnosis and treatment of anxiety disorders. Exercise-induced anxiolysis might be potentiated by further replenishment of RNA methylation donors, providing a strategy of exercise plus diet supplement in preventing anxiety disorders.”

https://onlinelibrary.wiley.com/doi/10.1002/advs.202105731 “Physical Exercise Prevented Stress-Induced Anxiety via Improving Brain RNA Methylation”


A third paper was a review of mitochondrial-to-nuclear epigenetic regulation. I’ll highlight one mitochondrial metabolite, alpha-ketoglutarate (α-KG):

“Apart from established roles in bioenergetics and biosynthesis, mitochondria are signaling organelles that communicate their fitness to the nucleus, triggering transcriptional programs to adapt homeostasis stress that is essential for organismal health and aging. Emerging studies revealed that mitochondrial-to-nuclear communication via altered levels of mitochondrial metabolites or stress signals causes various epigenetic changes, facilitating efforts to maintain homeostasis and affect aging.

Metabolites generated by the tricarboxylic acid (TCA) cycle, the electron transport chain (ETC), or one-carbon cycle within mitochondria can act as substrates or cofactors to control epigenetic modification, especially histone acetylation and methylation and DNA methylation. α-KG produced in the TCA cycle serves as an essential cofactor for the chromatin-modifying Jumonji C (JmjC) domain-containing lysine demethylases (JMJDs) and ten-eleven translocation (TETs) DNA demethylases. Changes in α-KG levels are capable of driving nuclear gene expression by affecting DNA and histone methylation profiles.

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α-KG deficiency in progenitor stem cells increases with age. For example, the level of α-KG is reduced in follicle fluids of aged humans, and supplementation with α-KG preserves ovarian function in mice.

α-KG extends lifespan in Drosophila by activating AMPK signaling and inhibiting the mTOR pathway. Supplementing α-KG in the form of a calcium salt promoted a longer and healthier life associated with decreased levels of inflammatory cytokines in old mice.

A human study showed a nearly 8-year reversal in DNA methylation clock biological ages of 42 individuals taking an α-KG based formulation for 4–10 months. α-KG supplementation leads to both demethylation and hypermethylation of some CpG sites in the genome, suggesting that α-KG may have a broader effect on methylation-based aging, such as metabolic functions.

Outstanding questions:

  1. How is production of mitochondrial metabolites regulated both spatially and temporally to elicit epigenetic changes in response to mitochondrial dysfunction?
  2. What are specific epigenetic factors involved in mitochondrial-to-nuclear communications, and how do they cooperate with transcription factors in response to various external and internal stimuli?
  3. Do various mitochondrial metabolites act alone or in concert on the epigenome to regulate the aging process?
  4. Are some organs or tissues more at risk than others in maintaining mitochondrial-to-nuclear communication during aging?
  5. Can intervention of mitochondrial-to-nuclear communications mimic beneficial epigenetic changes to delay aging or alleviate age-onset diseases?”

https://www.sciencedirect.com/science/article/pii/S0968000422000676 “Mitochondrial-to-nuclear communication in aging: an epigenetic perspective”


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Blanching broccoli sprouts

Three 2022 papers of interest cited Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease. Let’s start with a fairly straightforward analysis of blanching broccoli sprouts to produce sulforaphane:

“We investigated the effect of blanching conditions to determine the optimal treatment that maximizes sulforaphane (SFN) content in broccoli sprouts. Broccoli seeds grown under controlled conditions were harvested after 11 days from germination and subjected to different blanching conditions based on a central composite design with temperature and time as experimental factors.

Optimum conditions were blanching at 61 °C for 4.8 min, resulting in 54.3 ± 0.20 µmol SFN/g dry weight, representing a 3.3-fold increase with respect to untreated sprouts. This is the highest SFN content reported for sprouts subjected to any treatment so far.

sfn heat response curve

Broccoli sprouts (20 g) were put in plastic bags, which were vacuum-sealed, and then subjected to time (3.4–11 min)–temperature (32–88 °C) combinations.

  • Blanching at 60 °C for less than 8 min resulted in the highest SFN content.
  • Above this temperature, SFN content decreases.
  • The exceptionally high values obtained in this work may be related to treatment, but also to broccoli cultivar and culture conditions.

Different broccoli tissues and developmental stages express different myrosinase isoforms, and catalytic properties of the enzyme may vary among different tissues. Myrosinase found in broccoli florets has an optimal temperature of around 40 °C, and considering myrosinases from other sources, this temperature may vary between 30 and 70 °C.”

https://www.mdpi.com/2304-8158/11/13/1906/htm “Maximization of Sulforaphane Content in Broccoli Sprouts by Blanching”

This first study used heat-only techniques similar to the uncited Enhancing sulforaphane content. It similarly found a 60°C (140°F) myrosinase cliff as have many other uncited studies.


A second paper was a rodent study:

“We investigated the role of sulforaphane, a well-known NRF2 activator, on age-related mitochondrial and kidney dysfunction. Young (2–4 month) and aged (20–24 month) male Fischer 344 rats were treated with sulforaphane (15 mg/kg body wt/day) in drinking water for four weeks.

Sulforaphane significantly improved mitochondrial function and ameliorated kidney injury by increasing cortical NRF2 expression and activity and decreasing protein expression of KEAP1, a NRF2 repressor. Sulforaphane treatment did not affect renal NRF2 expression or activity and mitochondrial function in young rats.”

https://www.mdpi.com/2076-3921/11/1/156/htm “Age-Related Mitochondrial Impairment and Renal Injury Is Ameliorated by Sulforaphane via Activation of Transcription Factor NRF2”

A human equivalent to this second study’s daily dose was intolerable at (.162 x 15 mg) x 70 kg = 170 mg. I curated this study anyway just to show an example of negligible treatment effects in young animals even when a dose is too high for humans.


A third paper was a review that focused on sulforaphane and its analogs’ chemistry:

“Analysis of the Web of Science database shows that, since 1992, about 3,890 articles have been published on SFN, and over 5,600 on isothiocyanates. Its natural analogs include iberin, alyssin, iberverin, erucin, berteroin, cheirolin, and erysolin.

SFN is a biologically active, natural isothiocyanate found in cruciferous vegetables, and is non-toxic. It has been selected for phase I and II clinical trials, where it is administered in the form of an extract or broccoli sprouts. There are no differences in biological activity between SFN and its natural analogs, such as erucin or alyssin.

No synthetic analogs of SFN described in this review qualified for clinical trials. This is likely due to the toxicity of these compounds in higher doses.”

https://www.mdpi.com/1420-3049/27/5/1750/htm “Sulforaphane and Its Bifunctional Analogs: Synthesis and Biological Activity”


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Taurine week #7: Brain

Finishing a week’s worth of 2022 taurine research with two reviews of taurine’s brain effects:

“We provide a overview of brain taurine homeostasis, and review mechanisms by which taurine can afford neuroprotection in individuals with obesity and diabetes. Alterations to taurine homeostasis can impact a number of biological processes such as osmolarity control, calcium homeostasis, and inhibitory neurotransmission, and have been reported in both metabolic and neurodegenerative disorders.

Models of neurodegenerative disorders show reduced brain taurine concentrations. On the other hand, models of insulin-dependent diabetes, insulin resistance, and diet-induced obesity display taurine accumulation in the hippocampus. Given cytoprotective actions of taurine, such accumulation of taurine might constitute a compensatory mechanism that attempts to prevent neurodegeneration.

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Taurine release is mainly mediated by volume-regulated anion channels (VRAC) that are activated by hypo-osmotic conditions and electrical activity. They can be stimulated via glutamate metabotropic (mGluR) and ionotropic receptors (mainly NMDA and AMPA), adenosine A1 receptors (A1R), and metabotropic ATP receptors (P2Y).

Taurine mediates its neuromodulatory effects by binding to GABAA, GABAB, and glycine receptors. While taurine binding to GABAA and GABAB is weaker than to GABA, taurine is a rather potent ligand of the glycine receptor. Reuptake of taurine occurs via taurine transporter TauT.

Cytoprotective actions of taurine contribute to brain health improvements in subjects with obesity and diabetes through various mechanisms that improve neuronal function, such as:

  • Modulating inhibitory neurotransmission, which promotes an excitatory–inhibitory balance;
  • Stimulating antioxidant systems; and
  • Stabilizing mitochondria energy production and Ca2+ homeostasis.”

https://www.mdpi.com/2072-6643/14/6/1292/htm “Taurine Supplementation as a Neuroprotective Strategy upon Brain Dysfunction in Metabolic Syndrome and Diabetes”


A second review focused on taurine’s secondary bile acids produced by gut microbiota:

“Most neurodegenerative disorders are diseases of protein homeostasis, with misfolded aggregates accumulating. The neurodegenerative process is mediated by numerous metabolic pathways, most of which lead to apoptosis. Hydrophilic bile acids, particularly tauroursodeoxycholic acid (TUDCA), have shown important anti-apoptotic and neuroprotective activities, with numerous experimental and clinical evidence suggesting their possible therapeutic use as disease-modifiers in neurodegenerative diseases.

Biliary acids may influence each of the following three mechanisms through which interactions within the brain-gut-microbiota axis take place: neurological, immunological, and neuroendocrine. These microbial metabolites can act as direct neurotransmitters or neuromodulators, serving as key modulators of the brain-gut interactions.

The gut microbial community, through their capacity to produce bile acid metabolites distinct from the liver, can be thought of as an endocrine organ with potential to alter host physiology, perhaps to their own favour. Hydrophilic bile acids, currently regarded as important hormones, exert modulatory effects on gut microbiota composition to produce secondary bile acids which seem to bind a number of receptors with a higher affinity than primary biliary acids, expressed on many different cells.

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TUDCA regulates expression of genes involved in cell cycle regulation and apoptotic pathways, promoting neuronal survival. TUDCA:

  • Improves protein folding capacity through its chaperoning activity, in turn reducing protein aggregation and deposition;
  • Reduces reactive oxygen species production, leading to protection against mitochondrial dysfunction;
  • Ameliorates endoplasmic reticulum stress; and
  • Inhibits expression of pro-inflammatory cytokines, exerting an anti-neuroinflammatory effect.

Although Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and cerebral ischemia have different disease progressions, they share similar pathways which can be targeted by TUDCA. This makes this bile acid a potentially strong therapeutic option to be tested in human diseases. Clinical evidence collected so far has reported comprehensive data on ALS only.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9166453/ “Tauroursodeoxycholic acid: a potential therapeutic tool in neurodegenerative diseases”

Taurine week #6: Stress

Two 2022 rodent studies of taurine’s associations with long-term stress, starting with a chronic restraint stress model:

“We show that chronic restraint stress can lead to hyperalgesia accompanied by changes in gut microbiota that have significant gender differences. Corresponding changes of bacteria can further induce hyperalgesia and affect different serum metabolism in mice of the corresponding sex.

Different serum metabolites between pseudo-germ-free mice receiving fecal microbiota transplantation from the chronic restraint stress group and those from the control group were mainly involved in bile secretion and steroid hormone biosynthesis for male mice, and in taurine and hypotaurine metabolism and tryptophan metabolism for female mice.

Effects of gut microbiota transplantation on serum metabolomics of female host: Taurine and hypotaurine metabolism, tryptophan metabolism, serotonergic synapse, arachidonic acid metabolism, and choline metabolism in cancer were the five identified pathways in which these different metabolites were enriched.

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Taurine and hypotaurine play essential roles in anti-inflammation, anti-hypertension, anti-hyperglycemia, and analgesia. Taurine can be used as a diagnostic index for fibromyalgia syndrome and neuropathic pain.

These findings improve our understanding of sexual dimorphism in gut microbiota in stress-induced hyperalgesia and the effect of gut microbiota on blood metabolic traits. Follow-up research will investigate causal relationships between them.”

https://www.sciencedirect.com/science/article/pii/S1043661822000743 “Gut microbiota and its role in stress-induced hyperalgesia: Gender-specific responses linked to different changes in serum metabolites”

Human equivalents:

  • A 7-8 month-old mouse would be a 38-42 year-old human.
  • A 14-day stress period is about two years for humans.

A second study used a chronic social defeat stress model:

“The level of taurine in extracellular fluid of the cerebral medial prefrontal cortex (mPFC) was significantly reduced in mice with chronic social defeat stress (CSDS)-induced depression. We found that taurine supplementation effectively rescued immobility time during a tail suspension assay and improved social avoidance behaviors in CSDS mice.

Male C57BL/6 J mice (∼ 23 g) and male CD-1 mice aged 7–8 months (∼ 45 g) were used. CD-1 mice were screened for aggressive behavior during social interactions for three consecutive days before the start of the social defeat sessions. Experimental C57BL/6 J mice were subjected to physical interactions with a novel CD-1 mouse for 10 min once per day over 10 consecutive days.

We found significant reductions in taurine and betaine levels in mPFC interstitial fluid of CSDS mice compared with control mice.

csds taurine betaine

We additionally investigated levels of interstitial taurine in chronic restraint stress (CRS) mice, another depressive animal model. After 14 days of CRS treatment, mice showed typical depression-like behaviors, including decreased sucrose preference and increased immobility time. mPFC levels of interstitial taurine were also significantly decreased in CRS mice.

Taurine treatment protected CSDS mice from impairments in dendritic complexity, spine density, and proportions of different types of spines. Expression of N-methyl D-aspartate receptor subunit 2A, an important synaptic receptor, was largely restored in the mPFC of these mice after taurine supplementation.

These results demonstrated that taurine exerted an antidepressive effect by protecting cortical neurons from dendritic spine loss and synaptic protein deficits.”

https://link.springer.com/article/10.1007/s10571-022-01218-3 “Taurine Alleviates Chronic Social Defeat Stress-Induced Depression by Protecting Cortical Neurons from Dendritic Spine Loss”

Human equivalents:

  • A 7-8 month-old mouse would be a 38-42 year-old human.
  • A 500 mg/kg taurine dose injected intraperitoneally is (.081 x 500 mg) x 70KG = 2.835 g.
  • A 10-day stress period is about a year and a half for humans.

Don’t think aggressive humans would have to be twice as large to stress those around them. There may be choices other than enduring a year and a half of that.

Taurine week #2: Bile acids

Two papers investigated taurine’s integration into bile acids, starting with a review:

“Bile acids (BAs) are produced from cholesterol in the liver and are termed primary BAs. Primary BAs are conjugated with glycine and taurine in the liver, and stored in the gallbladder. BAs are released from the gallbladder into the small intestine via food intake to facilitate digestion and absorption of lipids and lipophilic vitamins by forming micelles in the small intestine.

After deconjugation by the gut microbiome, primary BAs are converted into secondary BAs. Most BAs in the intestine are reabsorbed and transported to the liver, where both primary and secondary BAs are conjugated with glycine or taurine and rereleased into the intestine.

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Some BAs reabsorbed from the intestine spill into systemic circulation, where they bind to a variety of nuclear and cell-surface receptors in tissues. Some BAs are not reabsorbed and bind to receptors in the terminal ileum.

BAs can affect cell-surface and intracellular membranes, including those of mitochondria and the endoplasmic reticulum. BAs are also hormones or signaling molecules, and can regulate BA, glucose, and lipid metabolism in various tissues, including the liver, pancreas, and both brown and white adipose tissue. BAs also affect the immune system.

BAs can affect the nervous system. More than 20 BAs have been detected in the brain of humans and rodents. The brain communicates with the gut and gut microbiome through BAs.”

https://www.mdpi.com/2076-2607/10/1/68/htm “Physiological Role of Bile Acids Modified by the Gut Microbiome”


Reference 56 was a human study:

“Centenarians (individuals aged 100 years and older) have a decreased susceptibility to ageing-associated illnesses, chronic inflammation, and infectious diseases. Centenarians have a distinct gut microbiome enriched in microorganisms that are capable of generating unique secondary bile acids.

We identified centenarian-specific gut microbiota signatures and defined bacterial species as well as genes and/or pathways that promote generation of isoLCA, 3-oxoLCA, 3-oxoalloLCA, and isoalloLCA. To our knowledge, isoalloLCA is one of the most potent antimicrobial agents that is selective against Gram-positive microorganisms, including multidrug-resistant pathogens, suggesting that it may contribute to maintenance of intestinal homeostasis by enhancing colonization-resistance mechanisms.”

https://www.nature.com/articles/s41586-021-03832-5 “Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians” (not freely available)


A few more papers will be coming on taurine and bile acids. I haven’t seen one investigate both taurine and glycine treatments to aid bile acid in achieving therapeutic results.

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The oligosaccharide stachyose

Two 2022 stachyose papers to follow on to Don’t take Beano if you’re stressed, which studied raffinose. Stachyose is in the raffinose oligosaccharide group with similar characteristics, and its content is usually larger in legumes. First is a rodent study:

“Stress can activate the hypothalamic–pituitary–adrenal (HPA) axis and elevate glucocorticoids in the body (cortisol in humans and corticosterone in rodents). Glucocorticoid receptors are abundant in the hippocampus, and play an important role in stress-induced cognition alteration.

Corticosterone is often used to model cognitive impairment induced by stress. Long-term potentiation (LTP) deficit and cognitive impairment always coexist in stress models, and LTP impairment is often considered as one mechanism for stress-induced cognitive deficits.

N-methyl-D-aspartate (NMDA) receptors play critical roles both in normal synaptic functions and excitotoxicity in the central nervous system. D-serine, a coactivator of NMDA receptors, plays an important role in brain function.

In this study, we focused on effects of stachyose, on LTP impairment by corticosterone, gut flora, and the D-serine pathway.

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Data in this study showed that 7-consecutive-day intragastric (i.g.) administration of stachyose had protective effect. There was little effect via intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration.

To disturb gut flora, a combination of non-absorbable antibiotics (ATB) were applied. Results showed that ATB canceled the protective effect of stachyose without affecting LTP in control and corticosterone-treated mice, suggesting that stachyose may display its protective effects against LTP impairment by corticosterone via gut flora.

Further study is needed to uncover the relation between gut flora and the D-serine metabolic pathway.”

https://www.frontiersin.org/articles/10.3389/fphar.2022.799244/full “Stachyose Alleviates Corticosterone-Induced Long-Term Potentiation Impairment via the Gut–Brain Axis”

One of this study’s references was Eat oats and regain cognitive normalcy.


A stachyose clinical trial is expected to complete this month:

“In the stachyose intervention group, each person took 5 g of stachyose daily before breakfast. Administration method was 100 ml of drinking water dissolved and taken orally for two months. Each person in the placebo control group took the same amount of maltodextrin daily. Stool samples of the 36 subjects were collected weekly.

Primary outcome measures:

  1. Expression of microRNA; and
  2. Structure of gut microbiota.”

https://clinicaltrials.gov/ct2/show/NCT05392348 “Regulatory Effect of Stachyose on Gut Microbiota and microRNA Expression in Human”


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