Rhythmicity

This 2021 review subject was circadian signaling in the digestive system:

“The circadian system controls diurnal rhythms in gastrointestinal digestion, absorption, motility, hormones, barrier function, and gut microbiota. The master clock, located in the suprachiasmatic nucleus (SCN) region of the hypothalamus, is synchronized or entrained by the light–dark cycle and, in turn, synchronizes clocks present in peripheral tissues and organs.

Rhythmic clock gene expression can be observed in almost every cell outside the SCN. These rhythms persist in culture, indicating that these cells also contain an endogenous circadian clock system.

Processes in the gastrointestinal tract and its accessory digestive organs display 24-hour rhythmicity:

Clock disruption has been associated with disturbances in gut motility. In an 8-day randomized crossover study, in which 14 healthy young adults were subjected to simulated day-shift or night-shift sleeping schedules, circadian misalignment increased postprandial hunger hormone ghrelin levels by 10.4%.

Leptin, a satiety hormone produced by white adipose tissue, peaks at night in human plasma. A volunteer ate and slept at all phases of the circadian cycle by scheduling seven recurring 28-hour ‘days’ in dim light and eating four isocaloric meals every ‘day’. Plasma leptin levels followed the forced 28-hour behavioural cycle, while their endogenous 24-hour rhythm was lost. However, since meal timing can entrain the circadian system, this forced desynchrony study could not exclude a potential role of the circadian system.

Another constant routine protocol study with 20 healthy participants showed that rhythms in plasma lipids differed substantially between individuals, suggesting the existence of different circadian metabolic phenotypes.

Composition, function, and absolute abundance of gut microbiota oscillate diurnally. For example, microbial pathways involved in cell growth, DNA repair and energy metabolism peaked during the dark phase, while detoxification, environmental sensing and motility peaked during the day.

It is unclear how phase information is communicated to gut microbiota. However, human commensal bacterium Enterobacter aerogenes showed an endogenous, temperature-compensated 24-hour pattern of swarming and motility in response to melatonin, suggesting that the host circadian system might regulate microbiota by entraining bacterial clocks.

With increasing popularity of time-restricted eating as a dietary intervention, which entrains peripheral clocks of the gastrointestinal tract, studies investigating circadian clocks in the human digestive system are highly needed. Additionally, further research is needed to comprehend shifts in temporal relationships between different gut hormones during chronodisruption.”

https://www.nature.com/articles/s41575-020-00401-5 “Circadian clocks in the digestive system” (not freely available). Thanks to Dr. Inge Depoortere for providing a copy.


This review included many more human examples. I mainly quoted gut interactions.

A long time ago I was successively stationed on four submarines. An 18-hour schedule while underway for weeks and months wiped out my circadian rhythms.

The U.S. Navy got around to studying 18-hour schedule effects this century. In 2014, submarine Commanding Officers were reportedly authorized to switch their crews to a 24-hour schedule.

Adaptive and innate immunity

Two 2021 reviews presented aspects of human immune systems:

“The adaptive immune system’s challenge is to protect the host through generation and differentiation of pathogen‐specific short‐lived effector T cells, while in parallel developing long‐lived memory cells to control future encounters with the same pathogen.

The system highly relies on self‐renewal of naïve and memory T cells, which is robust, but eventually fails. Genetic and epigenetic modifications contribute to functional differences in responsiveness and differentiation potential.

Less than 20% of nascent T cells are produced from the thymus in young adults, which dwindles to less than 1% after the age of 50 years. Even in young adults, the majority of T cells are produced in the periphery. A pickup in proliferation has been described in late life, possibly as a consequence of increased cell death and evolving lymphopenia.

One challenge of the aging process is to replenish cells while keeping integrity of the organ. The dynamic lymphoid system employs a vast number of T cells (>1011) and maintains a balance between cell production, death, and differentiation.

Enormous TCR ( T cell receptor) diversity is required to be able to respond to the universe of possible peptides (>209). Only T cell generation in the thymus can add new TCR specificities. Homoeostatic proliferation at best maintains diversity, >108 unique TCRs in a given adult.

Antigen-specific memory T cells adopt several fates with age:

  • Decrease in stem-like memory T cells;
  • Increase in NK (natural killer) cell-like TEMRA (terminally differentiated effector T cells);
  • Increase in exhausted T cells;
  • Increase in short-lived effector memory T cells; and
  • Decrease in tissue-residing T memory cells.

Virtual memory T cells without prior experience of antigen encounter also increase with age.”

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/febs.15770 “Hallmarks of the aging T cell system”


“Trained immunity is characterized by long‐term functional reprogramming of innate immune cells following challenge with pathogens or microbial ligands during infection or vaccination. This cellular reprogramming leads to increased responsiveness upon re‐stimulation, and is mediated through epigenetic and metabolic modifications.

Trained immunity has been shown to last for at least 3 months and up to 1 year, while heterologous protection against infections can last for at least 5 years. These long-term effects are mediated through reprogramming of myeloid progenitor cells in bone marrow, which in turn generate myeloid cells with a trained immunity phenotype.

Molecular mechanisms underlying trained immunity, for example induced by β‐glucan or Bacille Calmette‐Guérin (BCG) vaccination, can be investigated by using and integrating different layers of information, including genome, epigenome, transcriptome, proteome, metabolome, microbiome, immune cell phenotyping and function. Interplay between epigenetic and metabolic reprogramming is necessary for induction of trained immunity, as certain metabolites have a direct effect on enzymes involved in epigenetic remodeling.

High-throughput methods allow researchers to use an unbiased approach examining many potential genes or markers in relation to health and disease, rather than examining a limited number of candidate genes or markers.

One strength of integrating multiple levels of data is an increased power to identify key regulatory molecular networks driving trained immunity. For example, results obtained from one level (i.e. genes) can be used to reduce the number of traits to test in a second level (i.e. proteins), thereby increasing power.

One important pitfall when it comes to designing effective omics studies, is sample size. With a large number of markers measured, and the relatively small contributing effect size of individual analytes, the risks of both type 1 and 2 errors are high without sufficient sample sizes for both discovery and validation cohorts.”

https://onlinelibrary.wiley.com/doi/pdf/10.1002/eji.202048882 “Resolving trained immunity with systems biology”

Eat broccoli sprouts to prevent lung infections

A 2021 rodent study investigated lung infections:

“Mycobacterium avium complex (MAC) is the most common cause of pulmonary nontuberculous mycobacteria disease worldwide. It is thought that both environmental exposure and host susceptibility are required for the establishment of pulmonary MAC disease, because pulmonary MAC diseases are most commonly observed in slender, postmenopausal women without a clearly recognized immunodeficiency.

Host factors that regulate MAC susceptibility have not been elucidated until now. The Nrf2 system is activated in alveolar macrophages, the most important cells during MAC infection, as both the main reservoir of infection and bacillus-killing cells.

Treatment with sulforaphane (SFN) decreases Mycobacterium growth upregulating the expression of Nramp1 (natural resistance-associated macrophage protein 1, a susceptibility gene for pulmonary nontuberculous mycobacteria disease) and HO-1 (heme oxygenase 1). Mycobacterial counts in the lung, liver, and spleen were reduced after SFN treatment.

These results indicate that Nramp1 and HO-1, regulated by Nrf2, are essential in defending against MAC infection due to the promotion of phagolysosome fusion and granuloma formation, respectively. Nrf2 is thought to be a critical determinant of host resistance to MAC infection.”

https://mbio.asm.org/content/12/1/e01947-20 “Nrf2 Regulates Granuloma Formation and Macrophage Activation during Mycobacterium avium Infection via Mediating Nramp1 and HO-1 Expressions”


Don’t brew oat sprouts – eat them!

This 2020 study chemically analyzed four grains and their brew-processing products:

“Side-stream products of malting, particularly rootlet, are currently treated as animal feed. Instead of ending up in final products (e.g., malt and beer), a substantial portion of phytochemicals end up in side streams.

Rootlets are being increasingly investigated to overcome their bitter taste and to unleash their potential. Adding the fact that side-stream products produced in high quantity are also rich in protein, their nutritional value may be too high to justify usage as feed rather than food.

Grains were steeped for 26 to 30 h with a wet–dry–wet steeping program. Oats were wet steeped for 4 h at 13 °C before and after 18 h of dry steeping at 15 °C.

All grains were germinated for 6 days at 15 °C, after which they were dried with a gentle kilning program to a final temperature of 83 °C and moisture of 4%. Rootlets were separated from malt after drying.

Statistically significant changes occurred in abundance of all 285 annotated phytochemicals during malting, when comparing whole grain with malted grain or rootlet. In oats, cumulative levels of avenanthramides increased by 2.6-fold in the malted grain compared to intact whole grain. Up to 25-fold increase has been reported previously after a slightly longer germination.

Phenolamides cumulative levels in oats increased in both malted grain (11-fold) and rootlet (50-fold). Cumulative flavonoid levels were nearly 3-fold higher in malted grain and rootlet compared to whole grain.

Avenanthramides and phenolamides had much lower extractability into the water extract and wort.

To our knowledge, this is the first time avenanthramides are reported from any other species than oats, suggesting that the synthesis pathway for avenanthramides evolved before oats diverged from the other cereals. Furthermore, benzoxazinoids are herein reported for the first time in oats.

Several previously uncharacterized saponins were found in oats in addition to the previously known avenacins and avenacosides. However, because of limited reference data currently available, their identity could not be determined beyond compound class and molecular formula in this study.

Plants can synthetize up to hundreds of thousands of secondary metabolites, and current spectral databases only contain a fraction of them to allow identification. Compounds found in this study do not represent the complete range of phytochemicals existing in cereals.”

https://www.nature.com/articles/s41538-020-00081-0 “Side-stream products of malting: a neglected source of phytochemicals”


Twice a day for six weeks I’ve eaten oat sprouts 3-to-6-days old from two species and three varieties. I’ve never noticed any “bitter taste” of rootlets mentioned.

Maybe “a final temperature of 83 °C and moisture of 4%” had something to do with it? Oat sprouts I ate never got above 25°C, and I doubt their moisture content was < 80%.

Maybe “Oats were wet steeped for 4 h at 13 °C before and after 18 h of dry steeping at 15 °C” gave oat sprouts a bitter taste? I process oat sprout batches the same way I do broccoli sprout batches. A new batch soaks to start germination every 12 hours, then is rinsed three times every 24 hours on a 6 hours – 6 hours – 12 hours cycle. Temperature in my kitchen is 21°C (70°F) because it’s winter outside.

The above graphic is a heat map of 29 studied C-type avenanthramides. Don’t know why 26 known A-type avenanthramides described in Eat oats today! weren’t analyzed. The second study of Sprouting oats stated:

“There is a higher concentration of A-type AVAs [avenanthramides] than C-type AVAs in sprouted oats.”

Reference 33’s “up to 25-fold increase” is curated in Eat oat sprouts for AVAs.

Treat your gut microbiota as one of your organs

Two 2021 reviews covered gut microbiota. The first was gut microbial origins of metabolites produced from our diets, and mutual effects:

“Gut microbiota has emerged as a virtual endocrine organ, producing multiple compounds that maintain homeostasis and influence function of the human body. Host diets regulate composition of gut microbiota and microbiota-derived metabolites, which causes a crosstalk between host and microbiome.

There are bacteria with different functions in the intestinal tract, and they perform their own duties. Some of them provide specialized support for other functional bacteria or intestinal cells.

Short-chain fatty acids (SCFAs) are metabolites of dietary fibers metabolized by intestinal microorganisms. Acetate, propionate, and butyrate are the most abundant (≥95%) SCFAs. They are present in an approximate molar ratio of 3 : 1 : 1 in the colon.

95% of produced SCFAs are rapidly absorbed by colonocytes. SCFAs are not distributed evenly; they are decreased from proximal to distal colon.

Changing the distribution of intestinal flora and thus distribution of metabolites may have a great effect in treatment of diseases because there is a concentration threshold for acetate’s different impacts on the host. Butyrate has a particularly important role as the preferred energy source for the colonic epithelium, and a proposed role in providing protection against colon cancer and colitis.

There is a connection between acetate and butyrate distinctly, which suggests significance of this metabolite transformation for microbiota survival. The significance may even play an important role in disease development.

  • SCFAs can modulate progression of inflammatory diseases by inhibiting HDAC activity.
  • They decrease cytokines such as IL-6 and TNF-α.
  • Their inhibition of HDAC may work through modulating NF-κB activity via controlling DNA transcription.”

https://www.hindawi.com/journals/cjidmm/2021/6658674/ “Gut Microbiota-Derived Metabolites in the Development of Diseases”


A second paper provided more details about SCFAs:

“SCFAs not only have an essential role in intestinal health, but also enter systemic circulation as signaling molecules affecting host metabolism. We summarize effects of SCFAs on glucose and energy homeostasis, and mechanisms through which SCFAs regulate function of metabolically active organs.

Butyrate is the primary energy source for colonocytes, and propionate is a gluconeogenic substrate. After being absorbed by colonocytes, SCFAs are used as substrates in mitochondrial β-oxidation and the citric acid cycle to generate energy. SCFAs that are not metabolized in colonocytes are transported to the liver.

  • Uptake of propionate and butyrate in the liver is significant, whereas acetate uptake in the liver is negligible.
  • Only 40%, 10%, and 5% of microbial acetate, propionate, and butyrate, respectively, reach systemic circulation.
  • In the brain, acetate is used as an important energy source for astrocytes.

Butyrate-mediated inhibition of HDAC increases Nrf2 expression, which has been shown to lead to an increase of its downstream targets to protect against oxidative stress and inflammation. Deacetylase inhibition induced by butyrate also enhances mitochondrial activity.

SCFAs affect the gut-brain axis by regulating secretion of metabolic hormones, induction of intestinal gluconeogenesis (IGN), stimulation of vagal afferent neurons, and regulation of the central nervous system. The hunger-curbing effect of the portal glucose signal induced by IGN involves activation of afferents from the spinal cord and specific neurons in the parabrachial nucleus, rather than afferents from vagal nerves.

Clinical studies have indicated a causal role for SCFAs in metabolic health. A novel targeting method for colonic delivery of SCFAs should be developed to achieve more consistent and reliable dosing.

The gut-host signal axis may be more resistant to such intervention by microbial SCFAs, so this method should be tested for ≥3 months. In addition, due to inter-individual variability in microbiota and metabolism, factors that may directly affect host substrate and energy metabolism, such as diet and physical activity, should be standardized or at least assessed.”

https://www.hindawi.com/journals/cjidmm/2021/6632266/ “Modulation of Short-Chain Fatty Acids as Potential Therapy Method for Type 2 Diabetes Mellitus”


Eat broccoli sprouts for your kidneys

Starting Year 7 of curating research with a 2021 review of kidney disease and sulforaphane:

“Many chronic kidney disease (CKD) patients progress to end-stage kidney disease – the ultimate in failed prevention. While increased oxidative stress is a major molecular underpinning of CKD progression, no treatment modality specifically targeting oxidative stress has been established clinically.

Pathophysiologic effects occur when there is an imbalance between oxidation and reduction – an altered redox state in which excess free radicals react with other molecules, including lipids, proteins, and nuclear DNA. Mitochondrial DNA is also susceptible to oxidative damage.

All mechanisms discussed above have been shown to be present in CKD. When levels of antioxidant agents such as SOD, CAT, GPx/glutathione, and NRF2 are reduced, harmful effects of oxidation and generation of ROS cannot be appropriately mitigated.

Data suggest continued SFN [sulforaphane] administration is needed to maintain activation of the NRF2 pathway to confer protection against oxidative damage of diabetes. Renal protective effect of SFN has been demonstrated in many other models of kidney injury.

SFN may have therapeutic potential in kidney disease by stimulating the NRF2 pathway.”

https://www.mdpi.com/2072-6643/13/1/266/htm “Eat Your Broccoli: Oxidative Stress, NRF2, and Sulforaphane in Chronic Kidney Disease”


Didn’t see where these researchers intended to perform a suggested “clinical study to assess the effect of SFN in CKD.” Keep reading before experimentally treating patients. Targets they missed included:

  • Myrosinase hydrolization of glucoraphanin;
  • “Consumption of broccoli strains with more glucoraphanin leads to higher plasma levels of SFN” and
  • “It follows that SFN could also pose similar adverse effects, particularly if taken in an isolated preparation.”

Also missing from this kidney review were connections to broccoli sprouts’ effectiveness in preventing bladder disease. Not coincidentally, isothiocyanate metabolites accumulate in the bladder.

I came across this paper from it citing Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease. I curated it due to informatively citing Microwave broccoli to increase sulforaphane levels.

Harnessing endogenous defenses with broccoli sprouts

This 2019 article was by the author of Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease. It isn’t widely available, so I’ll quote liberally:

“Demand for solutions to digestive health issues is accelerating, especially since both scientific literature and popular press dedicate significant resources to promoting awareness of what has come to be known as ‘gut health’. In considering available therapies and the possibility that a somewhat different approach may more comprehensively optimise function of the gut ecosystem, a number of questions which do not yet have satisfactory answers are ponderable dilemmas:

  1. If diet alone can dramatically shift composition of the microbiome within 24 hours, what do we expect of a probiotic supplement?
  2. Even though probiotics as food or supplements demonstrate favourable clinical outcomes, they typically don’t colonise the gut. How do we expect them to restore diversity and lost species to the gut microbiome after antibiotics? If no trace of an administered probiotic organism can be found a few weeks later, is there any sustained benefit?
  3. Presence of obesity and other diseases is indirectly proportional to diversity of microbial organisms inhabiting the human gut. What can we expect of a few selected probiotic strains in helping to solve this problem?
  4. No antimicrobial approach selectively destroys a pathogen without impacting commensals to some degree. If we select a tool to eradicate gut pathogens, pathobionts or rogue commensals, how do we avoid damaging protective commensals with which we live symbiotically?
  5. The value of using a probiotic supplement after antibiotic therapy to recolonise the gut is uncertain. A 2018 multi-centre study showed that probiotic supplementation after antibiotics delayed gut microbiome reconstitution by around five months.
  6. If the gut can harbour around 1,000 different species, why do we expect a probiotic supplement harbouring just a few species to favourably modify a human microbiome?
  7. If Lactobacilli make up <0.1% of total microbes, why do we so readily choose them as probiotic supplements?
  8. If L-glutamine is a preferred energy source for the small intestine and not the colon, why is it used almost universally in gut repair programmes regardless of the affected region?

Removal of gluten and administration of probiotics have lesser impact than endogenous factors like elevated HbA1c:

Shift emphasis closer to optimising colonocyte metabolism as the primary driver of dysbiosis in the colon. Since these mechanisms within the human gut ecosystem already exist, intervene at this level, as distinct from using antimicrobials and exogenous probiotic strains to influence host cell function.

Phytonutrients that potently activate these core processes have been identified and are sufficiently bioavailable to achieve this end. Restoring homeostasis to the intestinal epithelial cells can be readily justified as a key initial step.

Sulforaphane is a potent inducer of hundreds of genes associated with cellular defences mechanisms. In this context, these genes include those that code for antioxidant and phase II detoxification enzymes, glutathione and metallothionein.

Sulforaphane exhibits other more specific gut and immune-related effects. As the most potent single food-derived activator of Nrf2, sulforaphane is capable of upregulating protective genes in colonocytes and other cells.

A growing body of work has identified the colonocyte as the driver of dysbiosis. Targeting colonocyte function provides an alternative to targeting microbes for remediation of dysbiosis.”

https://www.researchgate.net/publication/336578800_Restoring_Gut_Ecology_Harnessing_the_Inbuilt_Defence_Mechanisms_of_the_Gut_Epithelium “Restoring Gut Ecology: Harnessing the Inbuilt Defence Mechanisms of the Gut Epithelium” (registration required)


If you can’t access this paper, read The future of your brain is in your gut right now. If you can’t access that paper, listen to Switch on your Nrf2 signaling pathway.

Sprouting hulless oats

I finished a 3-lb. bag of hulled Avena sativa oats used in Sprouting hulled oats after starting 20 gram batches twice a day. Amazon said that Montana farmer’s products were “Currently unavailable. We don’t know when or if this item will be back in stock.” I went to their website and emailed an inquiry.

Turns out it’s Amazon’s problem in restocking pallets that are already received! I placed an order directly with the farmer.

In the meantime, I’m trying another oat species, Avena nuda, from an Illinois farmer. I’ll reuse Degree of oat sprouting as the model, since it was also an Avena nuda oat variety.

  • Oat seed size was 7-9 mm x 2-3 mm. The model used “huskless oat ‘Gehl’” which may be a different variety.
  • 100 seeds weighed 2.9 grams. There were close to 700 seeds per 20 g batches.
  • Oat sprout batches were processed the same way I do broccoli sprout batches. A new batch started soaking to start germination every 12 hours, then was rinsed three times every 24 hours on a 6 hours – 6 hours – 12 hours cycle.
  • Temperature in my kitchen was 21°C (70°F) because it’s snowing outside. The model findings included “Temperatures between 20° and 25°C yielded the most dramatic changes in properties of sprouted oats.”

I evaluated germination results per the model’s Degree of Sprouting finding:

“Length of the coleoptile [shoot] was selected as a criterion of categorization of degree of sprouting. Grains of degree 0 do not show any radicle [root] or coleoptile growth. Degree:

  1. Has visible embryos (small white point), while radicles and coleoptile are not visible;
  2. Shows a developed embryo emerging from the seed coat;
  3. Coleoptile lengths of at least half the oat grain length;
  4. Coleoptile lengths between half and a full grain length; and
  5. Coleoptile longer than a full grain length.”

Here’s what this hulless oat variety’s seeds and 3-day-old sprouts looked like:

The tedious part was evaluating degrees of sprouting. I took as large a bottom-to-top sample as I could tolerate sorting (160 seeds / sprouts, about 23%), with these results:

A 91% germination rate. 🙂 Average weight of 3-day-old batches was 42.5 grams, for a 213% weight gain. That wasn’t as much as 3-day-old hulled oats’ 97% germination rate and 260% weight gain.

For degree-of-sprouting comparisons, here are my eyeball estimates of the model study’s 3-day-old hulless oats:

These 3-day-old hulless oat sprouts taste starchier with less enzyme aftertaste than 3-day-old hulled oat sprouts. Will extending their growth to four days increase degree-of-sprouting categories 4 and 5, and change their taste?

An extra day from 5 to 6 didn’t make a difference in Sprouting whole oats germination rate. I don’t expect non-germinated percentages to change from 3 to 4 days, but we’ll see.

I expect similar overall increases in antioxidants, GABA, phenolic compounds, protein, amino acids, β-glucan, and polyunsaturated fatty acids as hulled oat sprouts.

Update: Four-day-old hulless oat sprouts have a little more sweetness and enzyme aftertaste. Their degree-of-sprouting and germination rate didn’t change much, though. I’ll stick with four days for this variety.

Broccoli sprouts activate the AMPK pathway

I’ll curate this 2020 rodent study through its summary graphic and caption:

“Type 2 diabetes exhibits elevated levels of circulating fatty acids and CD36. This results in excessive fatty acids binding with CD36 to suppress AMPK [adenosine 5′ monophosphate-activated protein kinase, a key player in regulating energy metabolism].

Inactivation of AMPK breaks homeostasis in lipid metabolism and the antioxidative system, and subsequently induces cardiac oxidative stress, inflammation, and fibrosis. These damages contribute to diabetic cardiomyopathy.

SFN [sulforaphane] treatment significantly induces AMPK activation, which:

  • Enhances mitochondrial fatty acids oxidation via PPARα/CPT-1B and PGC1-α pathways; and
  • Inhibits SCD-1 to down-regulate lipid synthesis.

This greatly alleviates cardiac lipid accumulation.

NRF2-mediated antioxidative effects can be activated via AMPK/AKT/GSK3β pathway, developing another pathway to confront cardiac oxidative damage.

AMPK is indispensable in SFN-mediated cardiac prevention against T2D.”

https://www.metabolismjournal.com/article/S0026-0495(19)30217-3/fulltext “Protective effects of sulforaphane on type 2 diabetes-induced cardiomyopathy via AMPK-mediated activation of lipid metabolic pathways and NRF2 function” (not freely available)


1. A human-mouse relative age perspective:

  • Experiments started with subjects at 2-months-old, equivalent to 20 human years. Treatment subjects ate a high-fat diet.
  • Sulforaphane was injected subcutaneously at 0.5 mg/kg every working day. It didn’t have significant effects on cardiac lipid accumulation at 5 months (a 30-year-old human), but did at 8 months (a 42-year-old human).

2. This study demonstrated that for sulforaphane to produce evidenced Nrf2 pathway effects, it first activated the AMPK/AKT/GSK3β pathway. For 5 days a week, over periods of human-equivalent decades.

3. CPT-1B pictured above is carnitine palmitoyltransferase-1B, an enzyme in the outer membrane of mitochondria. It controls transfer of long-chain fatty acyl CoA into mitochondria to convert fat into energy.

AMPK pathway activation also subsequently activates “PPARα/CPT-1B and PGC1-α pathways.” See A case for carnitine supplementation for a review.


It’s the fiber, not the fat

I came across this 2020 fiber-vs-fat rodent study from its citation in Gut microbiota and aging:

“Dietary intervention studies largely revolve around altering fat content. Little consideration has been given to amount of fiber and whether or not it is soluble.

We examined age- and sex-specific effects of a refined high-fat/low soluble fiber diet (rHFD) on body weight and gut microbiota composition relative to mice fed a refined low-fat diet (rLFD) that is nutritionally and compositionally matched to rHFD.

Chow diet supplied energy as 13.4% fat, 28% protein, 57.9% carbohydrates, and 15% dietary fiber (range of total dietary fiber between 15 and 25% with 15–20% insoluble and 2–5% soluble fiber).

Two refined diets were used: rLFD supplying energy as 12% fat, 21% protein, and 67% carbohydrates; and rHFD supplying energy as 45% fat, 20% protein, and 35% carbohydrates. [Both rLFD and rHFD contained] 5% fiber in the form of insoluble cellulose.

Young adult animals consumed chow diet for 17 weeks, and 1-year aged animals consumed chow diet for 60 weeks. We included a 1-week transition period wherein all mice were fed rLFD. For the following 4 weeks, half of the animals remained on rLFD while the other half consumed rHFD.

After 4 weeks, young adult female mice showed resistance to weight gain to rHFD, consistent with previous reports. Aged females fed rHFD showed rapid body weight gain relative to rLFD-fed aged females.

Young adult and 1-year aged males showed a significant gain in body weight that was independent of refined diet formulation, suggesting that other components of the refined diet contribute to body weight gain that is independent of dietary fat.

Transition from chow diet to rLFD resulted in changes to microbiota community structure and composition in all groups, regardless of sex and age. This dietary transition was characterized by a loss within phylum Bacteroidetes and a concomitant bloom of Clostridia and Proteobacteria in a sex- and age-specific manner.

No changes to gut microbiota community structure and composition were observed between mice consuming either rLFD or rHFD, suggesting that transition to rLFD that lacks soluble fiber is the primary driver of gut microbiota alterations, with limited additional impact of dietary fat on gut microbiota.”

https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-0791-6 “It’s the fiber, not the fat: significant effects of dietary challenge on the gut microbiome”


It’s alright for researchers in the Abstract and Introduction section to interpret how their rodent study may apply to humans. I appreciate when they confine their statements elsewhere to what they actually measured and found.

This study didn’t measure inflammation, behaviors, neurobiologics, metabolic parameters, immune biomarkers, or hormones. They can qualify statements with “may” all they want, but there wasn’t direct evidence for either:

“Age-specific vulnerability to diet-induced body weight gain in females may be related to aging-related changes to estrogens.”

or

“The lack of differences between rLFD- and rHFD-fed mice may indicate that gut microbiota structure and composition can be dissociated from body weight and systemic inflammation.”

Papers that cite this study can’t rely on its Abstract for “regulating metabolic, immune, behavioral, and neurobiological outcomes” because its experiments didn’t directly measure such outcomes.

Removing 2-5% soluble fiber from subjects’ diet had large effects. I look forward to reading human studies that are informed by this study.

Gut microbiota and aging

This 2020 review explored the title subject:

“The human body contains 1013 human cells and 1014 commensal microbiota. Gut microbiota play vital roles in human development, physiology, immunity, and nutrition.

Human lifespan was thought to be determined by the combined influence of genetic, epigenetic, and environmental factors including lifestyle-associated factors such as exercise or diet. The role of symbiotic microorganisms has been ignored.

Age-associated alterations in composition, diversity, and functional features of gut microbiota are closely correlated with an age-related decline in immune system functioning (immunosenescence) and low-grade chronic inflammation (inflammaging). Immunosenescence and inflammaging do not have a unidirectional relationship. They exist in a mutually maintained state where immunosenescence is induced by inflammaging and vice versa.

Immunosenescence changes result in both quantitative and qualitative modifications of specific cellular subpopulations such as T cells, macrophages and natural killer cells as opposed to a global deterioration of the immune system. Neutrophils and macrophages from aged hosts are less active with diminished phagocytosing capability.

Gut microbiota transform environmental signals and dietary molecules into signaling metabolites to communicate with different organs and tissues in the host, mediating inflammation. Gut microbiota modulations via dietary or probiotics are useful anti-inflammaging and immunosenescence interventions.

The presence of microbiomic clocks in the human body makes noninvasive, accurate lifespan prediction possible. Prior to occurrence of aging-related diseases [shown above], bidirectional interactions between the gut and extraenteric tissue will change.

Correction of accelerated aging-associated gut dysbiosis is beneficial, suggesting a link between aging and gut microbiota that provides a rationale for microbiota-targeted interventions against age-related diseases. However, it is still unclear whether gut microbiota alterations are the cause or consequence of aging, and when and how to modulate gut microbiota to have anti-aging effects remain to be determined.”

https://www.tandfonline.com/doi/abs/10.1080/10408398.2020.1867054 “Gut microbiota and aging” (not freely available; thanks to Dr. Zongxin Ling for providing a copy)


1. The “Stable phase” predecessor to this review’s subject deserved its own paper:

“After initial exposure and critical transitional windows within 3 years after birth, it is generally agreed that human gut microbiota develops into the typical adult structure and composition that is relatively stable in adults.

gut microbiota by age phenotype

However, the Human Microbiome Project revealed that various factors such as food modernization, vaccines, antibiotics, and taking extreme hygiene measures will reduce human exposure to microbial symbionts and led to shrinkage of the core microbiome, while the reduction in microbiome biodiversity can compromise the human immune system and predispose individuals to several modern diseases.”

2. I looked for the ten germ-free references in the “How germ-free animals help elucidate the mechanisms” section of The gut microbiome: its role in brain health in this review, but didn’t find them cited. Likewise, the five germ-free references in this review weren’t cited in that paper. Good to see a variety of relevant research.

There were a few overlapping research groups with this review’s “Gut-brain axis aging” section, although it covered only AD and PD research.

3. Inflammaging is well-documented, but is chronic inflammation a condition of chronological age?

A twenty-something today who ate highly-processed food all their life could have gut microbiota roughly equivalent to their great-great grandparents’ at advanced ages. Except their ancestors’ conditions may have been byproducts of “an unintended consequence of both developmental programmes and maintenance programmes.

Would gut microbiota be a measure of such a twenty-something’s biological age? Do we wait until they’re 60, and explain their conditions by demographics? What could they do to reset themself back to a chronological-age-appropriate phenotype?


The future of your brain is in your gut right now

A 2020 paper by the author of Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease:

“The gut and brain communicate bidirectionally via several pathways which include:

  1. Neural via the vagus nerve;
  2. Endocrine via the HPA axis;
  3. Neurotransmitters, some of which are synthesized by microbes;
  4. Immune via cytokines; and
  5. Metabolic via microbially generated short-chain fatty acids.

How does nature maintain the gut-microbiome-brain axis? Mechanisms to maintain homeostasis of intestinal epithelial cells and their underlying cells are a key consideration.

The symbiotic relationship that exists between microbiota and the human host is evident when considering nutrient requirements of each. The host provides food for microbes, which consume that food to produce metabolites necessary for health of the host.

Consider function of the human nervous system, not in isolation but in integration with the gastrointestinal ecosystem of the host, in expectation of a favorable impact on human health and behavior.”

https://www.sciencedirect.com/science/article/pii/B9780128205938000148 “Chapter 14 – The gut microbiome: its role in brain health” (not freely available)


Always more questions:

  • What did you put into your gut today?
  • What type of internal environment did it support?
  • What “favorable impact on human health and behavior” do you expect from today’s intake?
  • How will you feel?
  • Will you let evidence guide feeding your gut environment?

See Switch on your Nrf2 signaling pathway for an interview with the author.

How will you feel?

Consider this a partial repost of Moral Fiber:

“We are all self-reproducing bioreactors. We provide an environment for trillions of microbes, most of which cannot survive for long without the food, shelter and a place to breed that we provide.

They inhabit us so thoroughly that not a single tissue in our body is sterile. Our microbiome affects our development, character, mood and health, and we affect it via our diet, medications and mood states.

The microbiome:

  • Affects our thinking and our mood;
  • Influences how we develop;
  • Molds our personalities;
  • Our sociability;
  • Our responses to fear and pain;
  • Our proneness to brain disease; and
  • May be as or more important in these respects than our genetic makeup.

Dysbiosis has become prevalent due to removal of prebiotic fibers from today’s ultra-processed foods. I believe that dietary shift has created a generation of humans less able to sustain or receive love.

They suffer from reduced motivation and lower impulse control. They are more anxious, more depressed, more selfish, more polarized, and therefore more susceptible to the corrosive politics of identity.


Other recent blog posts by Dr. Paul Clayton and team include Skin in The Game and Kenosha Kids.

Image from Thomas Cole : The Consummation, The Course of the Empire (1836) Canvas Gallery Wrapped Giclee Wall Art Print (D4060)

Problematic rodent sulforaphane studies

I was asked to give an example of Human relevance of rodent sulforaphane studies. I’ll use the 2020 Sulforaphane Diminishes the Formation of Mammary Tumors in Rats Exposed to 17β-Estradiol.

1. This study’s sulforaphane dose was “100 μmol/kg SFN..gavage regimen on Monday, Wednesday and Friday for 56 weeks.” From the October 2019 Broccoli or Sulforaphane: Is It the Source or Dose That Matters? “Allometric scaling uses the correction factor for rat doses 0.162” and this graphic:

Interpreting the human-relevant range:

Interpreting that 8% of the rodent studies were clinically relevant to human sulforaphane doses as a lower boundary (1.43 µmol / kg) and tolerable to humans as an upper boundary (4 µmol / kg):

A human equivalent of this study’s dose is (100 μmol/kg x .162) = 16.2 µmol / kg. See the original blog post for a study showing that a majority of both treatment and control group subjects will refuse and stop with sulforaphane doses less than half of this study’s human equivalent.

2. From Week 28, “The maximum lifespan for rats and humans were set to 3.8 years and 122.5 years, respectively.” A human-equivalent multiplication factor that can be applied to a rat post-development time period is 122.5 / 3.8 = 32.2.

Assuming this study’s subjects could achieve maximum lifespan, a human equivalent of 56 weeks is (56 x 32.2) ≈ 1,803 weeks, or ≈ 34.7 years.

3. Let’s assert that the main purpose of animal studies is to help humans.

Was it possible for this study to achieve this goal when it used intolerable human-equivalent sulforaphane doses for a period equivalent to over three decades of our lives?

Yet its Discussion section proposed that it’s useful for human guidance on:

  • Obesity;
  • Breast cancer in premenopausal and postmenopausal women;
  • Hormone status;
  • Phospholipids for cellular health and homeostasis;
  • Serum free fatty acids and triglycerides; and
  • Lipid metabolism and DNA damage.

4. These researchers definitely knew what this study was going to do. A coauthor of the above referenced 2019 paper was also a coauthor of this study, who “conceived the original study design and supervised the project.”

Why did they do it? The coauthor’s shared apology – published in the October 2019 paper – for these types of studies was:

“Animal studies have not delivered all that might be expected of them. Pre-clinical experimentalists have not thought carefully about the selection of dose (or route) and its relevance to clinical utility.

Authors of this review have contributed to this dose skewing.”

This study was published in July 2020.

It wasn’t just a waste of resources. It detracted from science because people won’t recognize that its findings are inapplicable to humans.

Part 2 of Switch on your Nrf2 signaling pathway

To follow up topics of Part 1‘s interview:

1. “We each have a unique microbial signature in the gut. Metabolites that you produce might not be the same ones that I produce. This makes clinical studies very difficult because you don’t have a level playing field.”

This description of inter-individual variability could inform researchers’ investigations prior to receiving experimental results such as:

Post-experimental analysis with statistical packages of these types of results is apparently required. But it doesn’t produce meaningful explanations for such individual effects.

Analysis of individual differences in metabolism can better inform explanations, because it would investigate causes for widely-variable effects. Better predictive hypotheses could be a result.

2. Today I’m starting my 40th week of eating a clinically-relevant amount of microwaved 3-day-old broccoli sprouts every day. To encourage sulforaphane’s main effect of Nrf2 signaling pathway activation, I won’t combine broccoli sprouts with anything else either during or an hour before or after.

I had been taking supplements at the same time. This interview got me thinking about the 616,645 possible combinations of my 19 supplements and broccoli sprouts.

That’s way too many to be adequately investigated by humans. Especially because contexts for each combination’s synergistic, antagonistic, or additive activities may be influenced by other combinations’ results.

I’ll just eat food and take supplements outside of this sulforaphane window. Two that I’ve started to further research because of this interview are:

A. I’ve taken 750 mg fructo-oligosaccharides (FOS) twice a day for sixteen years. I’ve considered it as my only prebiotic. Hadn’t thought of either of these points:

  • “Polyphenols are now considered to be a prebiotic food for microflora in the gut. They tend to focus on producing additional amounts of lesser known species like Akkermansia muciniphila, and have a direct prebiotic effect. Microbiota break these big, bulky molecules down into smaller metabolites, which clearly are absorbed. Some beneficial effects that come from polyphenols are not from the original molecule itself, but from a variety of metabolites produced in the gut.
  • We use a prebiotic, actually called an immunobiotic, which is a dead lactobacillus plantarum cell optimised for its cell wall content of lipoteichoic acid. Lipoteichoic acid attaches to toll-like receptor 2, and that sets off a whole host of immune-modulating processes, which tend to enhance infection control and downregulate inflammation and downregulate allergenicity.”

B. Every day I take a 400 mg capsule of 1/3, 1/6 yeast β-glucan to train my innate immune system. β-glucan also works with toll-like receptor 2, but differently than does lipoteichoic acid. Have a dozen browser tabs open on the subject.

3. “Quinone reductase is critical because it is the final enzyme in the phase two detox pathway that stops DNA being mutated or prevents deformation of DNA adducts which are mutagenic. I want to look at genes that govern redox balance, inflammation, detoxification processes, cellular energetics, and methylation.”

Gene functional group classifications are apparently required in studies, to accompany meaningless statistics. When I’ve read papers attaching significance to gene functional groups, it often seemed like hypothesis-seeking efforts to overcome limited findings.

I’ll start looking closer when study findings include Nrf2 signaling pathway targets quinone reductase, DNA damage marker 8-hydroxydeoxyguanosine, and enzymes glutathione peroxidase and glutathione S-transferase.

4. I bolded “unregulated inflammation” in Part 1 because it’s a phrase I’d ask to be defined if that site enabled comments. Thinking on inflammation seems to come from:

“We focus on the intestinal epithelial cell as a key player because if you enhance function of that cell, and Nrf2 is part of that story, once you get those cells working as they should, they are modulating this whole underlying immune network.”

An environmental signaling paradigm of aging and Reevaluate findings in another paradigm have a different focus. That paradigm looks at inflammation in the context of aging:

“A link between inflammation and aging is the finding that inflammatory and stress responses activate NF-κB in the hypothalamus and induce a signaling pathway that reduces production of gonadotropin-releasing hormone (GnRH) by neurons.

The case is particularly interesting when we realize that the aging phenotype can only be maintained by continuous activation of NF-κB. So here we have a multi-level interaction:

  1. Activation of NF-κB leads to
  2. Cellular aging, leading to
  3. Diminished production of GnRH, which then
  4. Acts (through the cells with a receptor for it, or indirectly as a result of changes to GnRH-receptor-possessing cells) to decrease lifespan.

Cell energetics is not the solution, and will never lead to a solution because it makes the assumption that cells age. Cells take on the age-phenotype the body gives them.

Aging is not a defect – it’s a programmed progressive process, a continuation of development with the body doing more to kill itself with advancing years. Progressive life-states where each succeeding life-stage has a higher mortality (there are rare exceptions).

Cellular aging is externally controlled (cell non-autonomous). None of those remedies that slow ‘cell aging’ (basically all anti-aging medicines) can significantly extend anything but old age.

For change at the epigenomic/cellular level to travel up the biological hierarchy from cells to organ systems seems to take time. But the process can be repeated indefinitely (so far as we know).”