Superoxide Causes Insulin Resistance, Type 2 Diabetes

Intracellular Nutrient Excess Produces Mitochondrial Electron Accumulation
 (Article referenced below was brought to may attention by Cristian Stremiz - thanks)

Insulin resistance blocks insulin-based transport of glucose into cells that are already overloaded with nutrients. The spilling-over of excess high energy electrons in the mitrochondrial electron transport chain onto oxygen produces superoxide. Superoxide is the trigger to block the import of still more glucose. Thus, insulin resistance is a cellular defense against sudden death by superoxide and other reactive oxygen species (ROS).

High Energy Electrons of Glucose Are Used to Make ATP

Cells are biochemical machines that turn on genes to produce enzymes to convert the high energy electrons on the carbon and hydrogen atoms of glucose into ATP energy and molecular components of the cell. The high energy electrons are systematically depleted of energy, protons are pumped to produce a proton gradient across the inner mitochondrial membrane, ATP is made using the proton gradient and the low energy electron are passed off to oxygen molecules to make water. That is a quick summary of cytoplasmic glycolysis, the tricarboxylic acid cycle (mitochondrial matrix) and the mitochondrial electron transport chain. The final step of transferring the depleted electrons to oxygen to make water is how oxygen is consumed in respiration. Note that if everything works well, the high energy electrons of glucose, which could suddenly release all of their energy directly interacting with oxygen and start a fire, just produce water. Another bad alternative would be for the high energy electrons to bind to molecular oxygen making superoxide.

Cells Adjust their Glucose Individually to Match ATP Use

If the supply of ATP from the mitochondrial electron transport chain of a cell gets low, this triggers the migration of vesicles with glucose transport proteins to the cytoplasmic membrane. Since the number of transport proteins determines the rate of import of glucose, then more transporters means an increase in glucose and more ATP. Type 2 diabetes and insulin resistance represents the others extreme, i.e. what happens when cells get too much glucose, max out their capacity to make ATP and high energy electrons build up in the electron transport chain.

High Blood Sugar Triggers Insulin Production to Import the Glucose into Cells

Cells can also participate in body-wide metabolism coordinated by hormones, such as insulin. A sudden increase in blood glucose concentration triggers the pancreas islet cells to release insulin into the blood. The insulin binds to insulin receptor proteins on the surface of cells and that signal brings more glucose transport proteins to the cytoplasmic membrane. The cells import additional glucose and their metabolism increases and more ATP is produces. This lowers the blood glucose level. Some cells can continue to accumulate glucose in the form of glycogen or fat droplets, but other cells do not have this storage capacity. If glucose is supplied beyond the capacity of the cell to use it, then the mitochondrial electron transport chain begins to produce superoxide.

Superoxide Is a Reactive Oxygen Species (ROS)

Oxidation stress is the reason that plant antioxidants, vitamin C and N-acetyl-cysteine are recommended to avoid inflammation. One of the major sources of oxidation stress is the production of superoxide. Cells produce an enzymes, superoxide dismutase, to convert superoxide into hydrogen peroxide, and catalase to convert hydrogen peroxide into oxygen and water. Superoxide can also interact with nitric oxide to produce the nitric oxide radical. Unfortunately, superoxide can also produce hydroxyl radicals that can react with unsaturated lipids to produce lipid peroxides. Thus, superoxides can contribute to the production of many ROS, cause oxidation damage and trigger inflammation.

Many Different Processes that Produce Insulin Resistance all Produce Superoxide

The trigger for insulin resistance appears to be mitochondrial superoxide accumulation. A recent article used numerous mouse models of insulin resistance that mimic the typical human risk factors for insulin resistance and type 2 diabetes, e.g. excess nutrition, physical inactivity, pregnancy, polycystic ovarian syndrome, metabolic syndrome, inflammation, oxidative stress, anti-inflammatory corticosteroids, etc. and demonstrated that in each case mitochondrial superoxide accumulated. Moreover, mutant mice with lowered superoxide dismutase were more susceptible to insulin resistance and mutants producing an overabundance of superoxide dismutase were resistant to insulin resistance.

Insulin Resistance Is a Natural Defense Against Energy Excess

Superoxide sensing and insulin resistance protect cells against too much energy input and oxidative stress, but without the ability to reduce blood sugar, hyperglycemia leads to the suite of degenerative reactions that provide the symptoms of type 2 diabetes.

reference
Hoehn KL, Salmon AB, Hohnen-Behrens C, Turner N, Hoy AJ, Maghzal GJ, Stocker R, Van Remmen H, Kraegen EW, Cooney GJ, Richardson AR, James DE.Insulin resistance is a cellular antioxidant defense mechanism.Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17787-92. Epub 2009 Sep 30.

Bacterial Amyloid (Curli Fibers) Forms Biofilms

E. coli Curli Stacks in Congo Red Staining Fibers

We can’t cure diseases, because we don’t understand basic chemistry (what is hydrophobic) and biology (which came first the biofilm or the bacterial cell wall?)  Let’s look at a fundamental biological process, how bacteria form biofilms.

Biofilm Formation from Secreted Proteins and Polysaccharides

Investigators passed some E. coli through a special slide chamber so they could watch at high magnification as a single bacterium attached to the surface, divided to produce a colony of a few bacteria and then began to secrete proteins (curli fibers) and polysaccharides (colanic acid and cellulose) to make the biofilm matrix.  The matrix stained red with Congo Red.

Congo Red Stains Amyloids, Cellulose and Rare LPS



Staining with Congo Red shows that the spacing of hydrophobic patches on the surface of the biofilm matrix matches the flat hydrophobic, aromatic rings of the dye, Congo Red.  This particular dye is important, because Congo Red also specifically stains amyloid, e.g. beta amyloid of Alzheimer’s disease.  But Congo Red also binds to cellulose, a linear beta 1,4-glucan polysaccharide.  This seems paradoxical, because we are taught that the sugars of which a polysaccharide are made are hydrophilic.  That turns out to be a half-truth. 

Faces of Sugars Are Hydrophobic

The hydrogen bonding hydroxyl groups that make sugars water soluble and hydrophilic, radiate from a ring of carbons, and the faces of that ring cannot make hydrogen bonds.  The faces of sugars are hydrophobic and in most cases will bind to hydrophobic surfaces, such as aromatic amino acids, e.g. tryptophan, tyrosine and phenylalanine.  Thus, carbohydrate binding enzymes, such as shown in the figure bind cellulose (in grey and red) in a groove lined with aromatic amino acids (yellow and orange) so that each sugar orients over and sometimes sandwiched between aromatic amino acid residues.  This also explains why Congo Red binds to cellulose, since the aromatic rings of the dye bind to neighboring glucose residues along the relatively flat cellulose strand.  Most other polysaccharides and smaller sugars lack this spacing of sugars and they don’t stain red with Congo Red.

Basic Amino Acids Bind Hydrophobically

Another misperception is that basic amino acids, positively charged lysine and arginine, are hydrophilic.  The nitrogen atoms that make these amino acids positively charged, can form hydrogen bonds, but the hydrocarbon tails that have these nitrogenous tips, are hydrophobic.  Thus, basic amino acids and aromatic amino acids can stack to form tryptophan/arginine ladders in which they alternate.  A prominent example of these interdigitations are the way that nuclear localization signals, a quartet of basic amino acids, bind to importin via its projecting, spaced tryptophans and drag proteins through pores into the nucleus.  Similarly, the basic amino acids of heparin-binding domains extend across the hydrophobic faces of the sugars of heparin and hydrogen bond with their tips to the sulfates of the heparin.  In each of these binding examples the binding is primarily hydrophobic.

Amyloid Binds Congo Red by Stacked Heparin-Binding Domains

Amyloids are proteins that stack together like stacking chairs, so that each protein is oriented in the same way all along the stack.  In the case of the beta amyloid that makes up the toxic plaque in Alzheimer’s disease, each amyloid peptide is stacked like a hair pin on top of the next to make a fiber.  At the bend in beta amyloid, is a basic amino acid and the amyloid fiber has a band of basic amino acids along its length.  The spacing between the basic amino acids in an amyloid stack is just spanned by Congo Red, so amyloids are diagnostically stained red.  This same spacing of basic amino acids fits the sugars in heparin.  Thus, heparin can catalyze amyloid formation and is abundant in amyloid plaques in Alzheimer’s

Bacterial Biofilms Form from Amyloids and Polysaccharides

The E. coli cells that formed the biofilms that started this article secrete a protein, curli, that stacks as an amyloid into fibers.  These fibers stained by Congo Red and bind to the cellulose that is also produced by the E. coli.  It should not be surprising that biofilm formation is catalyzed by heparin and biofilm formation is a major problem in catheter infection, since heparin is used to coat catheters to keep them from forming blood clots.  Amyloids are also formed from stacked seminal acid phosphatase proteins that form fibers in the presence of heparin and facilitate HIV infection.

Do Biofilms Foment Amyloid Production?

Basic amino acids, sugars, aromatic amino acids and plant phytochemicals all bind each other via their hydrophobic surfaces.  It would not be surprising that bacteria that produce proteins and acidic polysaccharides that interact hydrophobically would also interact with host molecules with a similar spacing of hydrophobic surfaces, which are common in heparin-binding interactions and nucleic acid interactions.  The bacteria in biofilms produce both proteins and polysaccharides that may catalyze amyloid production.  The acidic biofilm polysaccharide, colanic acid, may replace heparan sulfate and curli should bind to heparin.

Berberine Binds Heparin and Blocks Amyloids and Biofilms

Just as bacterial products may compete for host heparin and heparin-binding domains, phytochemicals that interact with heparin, such as the phytochemical berberine, should disrupt heparin mediated molecular interactions, and by extension also biofilms.  There is experimental evidence for berberine both disrupts amyloid formation and biofilm assembly.

Psoriasis, IL-17, Cathelicidin, TLRs, NFkB, Inflammation and Heparin Therapy

Host DNA Released by Keratinocyte Apoptosis Binds LL-37 and Activates Dendrocytes

Psoriasis is an inflammation of the skin that leads to overproduction of keratinocytes resulting in a thick crust.  Skin inflammation, in this case, is considered a result of autoimmunity, but an autoantigen has not been identified.  It is more likely that psoriasis results from an autoinflammatory condition, in which inflammation produces a complex of self molecules that mimic bacterial DNA and trigger TLR/NFkB inflammation signaling.  And of course, if this is going to be interesting, it has to involve heparin.

Vitamin D Binds to a Transcription Factor Receptor that Controls Antimicrobial Peptides
A significant component of the innate immune system is a group of antimicrobial peptide  (defensins, cathelicidins, e.g. LL-37).  These short polypeptides owe their natural antibiotic activity to numerous basic (positively charged, arginine and lysine) amino acids.  The transcription factor that controls the expression of these peptides is the vitamin D receptor.  Thus, various forms of vitamin D influence the amount of antimicrobial peptides produced in the mouth, skin and crypts of the intestinal villi.  Oral vitamin D3 would be expected to directly improve defensin production in the gut and LL-37 production in the skin.

IL-17 Stimulates Skin Inflammation and LL-37 Production
A specific group of lymphocytes, called T helper 17 cells, produce IL-17.  These Th17 cells accumulate in some sites of inflammation, such as psoriasis and their secretion of IL-17 is associated with ongoing inflammation and may contribute to LL-37 production, as well as apoptosis of keratinocytes in the thickening skin of psoriasis plaques.
http://www.ncbi.nlm.nih.gov/pubmed/19623255?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_SingleItemSupl.Pubmed_Discovery_PMC&linkpos=2&log$=citedinpmcarticles&logdbfrom=pubmed

Th17 Cells Are Produced in the Gut in Response to Segmented Bacteria
One of my readers brought to my attention an article that shows that one of the hundreds of species of gut bacteria, segmented filamentous baceria, stimulates the gut to develop T helper 17 cells that subsequently migrate to sites of inflammation.
http://www.medpagetoday.com/Gastroenterology/InflammatoryBowelDisease/16472
This emphasizes the link between the gut and inflammatory diseases and parallels other examples of gut influence on disease, such as the ability of Helicobacter pylori to affect asthma or parasitic worms to tame Crohn’s disease, allergies and asthma.

Inflammation Lowers Heparan Sulfate Production and Spreads LL-37
One of my students induced inflammation in cells in vitro and showed by quantitative PCR that genes involved in heparan sulfate proteoglycan production are selectively silenced.  This observation explains in part the loss of heparan sulfate in kidneys and intestines that contributes to the leakiness of these organs in response to inflammation and the partial repair of these organs by heparin treatment.  Decrease of heparan sulfate that normally coats cells and binds antimicrobial peptides, such as LL-37, would explain the enhanced movement of LL-37 in psoriatic skin.

LL-37 Binds to Host DNA and Triggers Toll-Like Receptors
DNA is released from keratinocytes in psoriatic skin and this host DNA binds the antimicrobial peptide cathelicidin LL-37.  The LL-37/DNA complex mimics bacterial DNA and triggers the Toll-like receptors (TLR) on the surface of immune cells, dendrocytes, to activate NFkB, the transcription factor controlling inflammation.
http://www.ncbi.nlm.nih.gov/pubmed/19050268?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_SingleItemSupl.Pubmed_Discovery_RA&linkpos=1&log$=relatedarticles&logdbfrom=pubmed

Heparin Treats Psoriasis
It seemed obvious to me that the heparin binding domains (Look at all the basic amino acids in blue in the illustration of LL-37.) of LL-37 were involved in DNA binding and the reason the LL-37 was binding to host DNA, was that heparan sulfate had been depleted as a result of local inflammation.  It also seemed obvious that topical heparin should eliminate psoriasis plaques.  So I did a Google search of psoriasis + topical heparin and got a hit on a 1991 patent application that claims a broad applicability for heparin use in curing symptoms of a wide variety of diseases, including psoriasis.
http://www.patentstorm.us/patents/5037810/description.html
The only topical form of heparin that I know of is Lipactin (available in Canada and Europe?), a treatment for coldsores, which makes sense because herpes viruses use heparan sulfate to infect cells.

Biofilms as Human Gut Mycorrhizals

Are Biofilms Healthy Extensions of Intestinal Villi?

If soil is the stomach of the earth, then plant roots and mycorrizal fungi must be the intestines.

Mycorrhizal fungal hyphae extend from root hairs of plants into surrounding soil and enhance the uptake of phosphate and other nutritents.  Many plants cannot colonize new soil without taking their fungal partners with them.  It would seem obvious that the highly adapted human gut flora would include bacteria and fungi that actively communicate with intestinal epithelial cells.  Perhaps that communication includes both nutrients, e.g. hydrogen, ammonia, etc., vitamins and bacterial wall components, e.g. LPS.

Plants Sit and Mine Soil, Humans Mine Nutrients Passed through Their Gut

I want to try to give a plant’s view of human digestion.  Plants elaborate roots that branch repeatedly and the final extensions sprout hairs from individual epithelial cells.  Mycorrhizal fungal hyphae further extend the reach of the plant into the soil for nutrients. 

I think that a plant would look at us and see us stuffing soil/food into our mouths and watch it come out the other end.  It would then try to figure out where are roots are, i.e. how we absorb the water and minerals from our moving internal stream of soil.  The villi of the small intestines would look like root hairs, but where are the mycorrhizal fungi?  Another problem is that the soil keeps moving past the root hairs and would break off fungal hyphae extending into the soil.  Still another problem is the constant shedding of epithelial cells from the tips of the villi.  The plant would be perplexed, but closer inspection would reveal that biofilms could solve the problems.

Biofilms Coat the Intestinal Villi

Biofilms coating and perhaps spanning the villi of the small intestines may enhance the transport of nutrients into the villi.  This may be controversial and the biofilms may be more commonly limited to the smoother surface of the colon.  The point here is that biofilms may enhance the intestinal uptake of nutrients from food.  Biofilms may, therefore, be essential for health and extend the reach of the intestinal epithelial cells.

Bacterial Community Composition May Be Determined by Diet

A biofilm is composed of some type of linear polymer, such as DNA, heparan sulfate or bacterial acidic polysaccharides, with bacteria that bind to the polymer and to the intestinal epithelium.  Diet determines the bacterial composition of the biofilm.  Thus, the newborn starts without biofilms, gut development is finished by growth hormones in milk and a single species of Bifidobacteria excludes biofilm production, until solid food or formula initiates adult biofilms.  The bacteria in the biofilm depend on diet, so the biofilms can be either beneficial or pathogenic.

Communication within Biofilms and with the Intestines

The bacteria respond to the presence of other bacteria by quorum sensing, which involves release of small molecules that alter the gene expression of other bacteria in the community.  As a consequence, genes, e.g. antibiotic resistance, are exchanged and metabolism is altered.  This is how Klebsiella nitrogenase and hydrogen production is controlled.  The biofilm bacteria also produce compounds, e.g. vitamin D (?), that alter the behavior of the intestinal epithelial cells.  The intestines can respond with inflammation to recognized pathogen components or by triggering development of cells of the immune system.  The intestines are the home of most of the body’s immune cells.

Stimulation of Tregs

Helicobacter pylori adhering to the stomach lining increases the stomach’s quota of regulatory T cells that are involved in immunological tolerance.  Presumably, the supply of Tregs in the intestines is also regulated by biofilms.  Disruption of this system by chronic inflammation can deplete Tregs and lead to unrestrained immune attack that is observed as inflammatory bowel disease.  Thus, Crohn’s disease and ulcerative colitis may be triggered by damaged biofilms.