During physiological ketosis, KB can supply almost 60% of the brain's energy requirement. Plasma KB pass the BBB through monocarboxylic acid transporters (MCT) in a gradient-dependent manner and are readily available for neurons and astrocytes (which are also ketogenic (1)). In fact, the brain is happy without glucose and using ketones (2). Besides being neuroprotective and metabolically more efficient, the main KB, bOHB (3-HB), could enhance memory and learning.Zou et al (3) tested this hypothesis in mice:"This study sought to investigate the effect of 3-HB and derivatives on neuroglial cell metabolic activity and gap junctional intercellular communication of hippocampal neurons, to evaluate the hippocampal expression of PUMA-G and proteins related to memory following treatment with 3-HB, and to determine whether 3-hydroxybutyrate methyl ester (3-HBME) improves learning and memory in the normal mouse."
Interneuronal communication can occur indirectly and directly. In the former, chemical synapses are involved, in which transmitters are released into the extracellular space and bind the postsynaptic cell membrane. In the latter, electrical synapses mediate communication. The most prevalent group of electrical synapses, neuronal gap junctions, connect directly the intracellular space of two cells by gap junction channels. Connexin proteins are the structural components of gap junction channels in the nervous system. Specifically, connexin 36 has been involved in learning and memory (4).3-HB is the endogenous ligand for PUMA-G (5). Its activation in adipose tissue is also produced by nicotinic acid, inhibiting lipolysis and controlling the rate of ketogenesis. This represents an homeostatic mechanism by which 3-HB controls its own production, preventing ketoacidosis. Researchers used the Morris water maze for measuring learning and memory, and analyzed hippocampal neuron exposure to 3-HB and derivatives in vitro. All of the metabolites stimulated metabolic activity in neuroglial cells. 3-HMBE increased gap junction intercellular communication, as well as connexin 36 expression by 30% (compared to 12% in mice treated with acetyl-L-carnitine) and pERK2 levels (phosphorylated ERK2, necessary for connexin assembly). Moreover, PUMA-G mRNA was found in the hippocampus, subthalamic nucleus, temporal cortex and frontal cortex, and 3-HBME enhanced the transcription of PUMA-G in the hippocampus.So this translates to improved memory and learning?"The escape latency of mice in all groups decreased with time. Overall treatment comparisons indicated that statistically significant differences existed among the groups. Notably, the 30 mM 3-HBME groups took less time (p < 0.05) than the control groups on days 1 and 3–5 to find the platform (Fig. 5B). All treatment groups were faster (p <0.05) than the control group on day 5."
Treatment groups include mice treated with either 3-HBME (20, 30 or 40mg/kg/d) or acetyl-L-carnitine. Control group was water. "Paths taken to the platform area on the fifth day of spatial training by mice in the 30 mg/kg/d 3-HBME group were more direct than those taken by mice in the control group, which took more circuitous paths."
Ketotic mice knew exactly what they wanted. This causes less total swimming distance (ie. increased efficiency):"Similar to the escape latency results, the total swimming distance of the 30 mM 3-HBME groups was shorter (p <0.05) than the control groups on days 1 and 3–5. (...) Moreover, the total swimming distance of mice in the 30 mg/kg/d 3-HBME group was shorter (p <0.05) than that of the control or other treatment groups at days 3–5."
A probe test* then was performed to evaluate memory. Three parameteres were measured: the number of crossings of the exact place where the platform had been located, the swimming distance in the quadrant of the former platform position, and the swimming path in the pool. Number of times that mice crossed the former position of the hidden platform within 60s: Swimming distance in the platform quadrant:Swimming path:Finally, thigmotaxis was lowest in the 30mg/kg/d 3-HMBE group. A retention test was performed two days after the probe test: These test showed that the 30mg/kg/d 3-HMBE group:a. Crossed more times the exact place where the platform had been located,b. Had the larger swimming distance in such quadrant,c. Found the platform faster than the other groups, d. Were the most calmed (less thigmotaxis), ande. Found the platform faster than the other groups during the retention test These results suggest that 3-HMBE, given at 30mg/kg/d, enhanced learning and memory**.Researchers used polyhydroxybutyrate (PHB) (a polyhydroxyalkanoate) to produce 3-HB and derivatives. Can we extrapolate these results to physiologically produced 3-HB? The finding that hippocampal neurons expressed PUMA-G receptors is encouraging. As mentioned, 3-HB is the endogenous ligand for PUMA-G, but there seems to be a desensitization of the receptor when exposed to large amounts of 3-HB compared to 3-HBME and 3-HBEE. As PUMA-G interaction with several ligands is not well understood, we cannot draw many conclusions. Some studies have evaluated the potential benefit of 3-HB in neurological disorders which compromise memory and learning, such as Alzheimer's disease (AD). For example, in some patients with AD and mild cognitive impairment, MCT oil produces an improvement on cognitive testing and paragraph recall (6), correlated with the increase in plasma ketones. This neuroprotective effect has been explained by the increase in metabolic efficiency associated with 3-HB (7) and inhibition of apoptosis (8), but it might also act through PUMA-G and connexin 36 dependent mechanisms. Further studies should help discovering the molecular pathways involved. In the meantime, maybe schools should start giving students coconut oil shots instead of skim milk. * The platform was removed from the pool and mice were challenged to a single search trial for 60s. ** Note that this group performed best than 20mg/kg/d and 40mg/kg/d.
Zou XH, Li HM, Wang S, Leski M, Yao YC, Yang XD, Huang QJ, & Chen GQ (2009). The effect of 3-hydroxybutyrate methyl ester on learning and memory in mice. Biomaterials, 30 (8), 1532-41 PMID: 19111894
When trying to understand human metabolism and physiology, one must consider an organism as a whole. Because of our human nature, we tend to synthesize most of the available information to the point of applying the Occam's razor principle incorrectly. Living organisms are open systems in which every metabolic pathway is interrelated, maintaining a dynamic steady state. This is one of the main issues of studying cells in vitro versus in vivo.As I pointed out in my introductory post, every human biological process has its purpose. There are not "bad" molecules or "good" ones. Glycolysis is not bad, excessive glycolisis is bad. Lipolysis is not bad, excessive lipolysis is bad. And so on. Considering this principle is essential for understanding modern diseases, treating and preventing them. Modern medicine has made the huge mistake of applying the bad/good concept for trying to understand diseases. We should not try to understand diseases only by proximate causes, but by evolutionary causes as well (for an in depth review, see Harris and Malyango, 2005). Lately, IR is one hot topic in the scientific community as well as in the blogosphere. Because of its implication in almost every modern disease, many apply the "morality principle", by which IR is bad and IS is good. If you ask the regular health reader the question "Is IR bad?" you will probably hear an unanimous YES!. On the contrary, my answer would be "it depends". To undestand why something completely normal like IR goes pathological we have to look at the Randle Cycle. In a nutshell, every substrate promotes its own oxidation. If you eat more glucose, you burn more glucose; if you eat more fat, you burn more fat*. The human body has adapted to use both sources of fuel as energy. Early humans evolved in highly different ecological niches, some with an increased amount of sugar/starch and others with less or none. Mechanisms should have been developed for coupling both energy substrates. When the main source of fuel is glucose, the expression of key enzymes involved in glycolisis increases. Glut-4 translocation increases for clearing glucose more efficiently from the bloodstream. Insulin reduces the rate of lipolysis by downregulating HSL, so less FFA are used for energy. Glucose itself promotes its storage, both as glycogen or fat, directly or indirectly (activation of hepatic/adipose DNL, ChREBP and several lipogenic genes, etc.). On the contrary, when you eat more fat, you will use more fat. Fat ingestion produces an increase in lipolysis and beta-oxidation. CPTI, UCP, CD36/FAT are all increased. Because the insulin response to fat is null, HSL is not supressed and lipolysis serves to deliver the necessary energy for the tissues. If more FFA than needed are released, re-esterification occurs. Excess flux of FFA to the liver produces KB to reduce the need for glucose and to control the rate of lipolysis. There are some cells that can only use glucose. GnG is the mechanism by which we evolved to supply this demand in a coordinate way: there is never going to be "too much" glucose produced by GnG. When carbohydrate intake is reduced drastically, we supply the exact amount to these cells by this mechanism. We rely less on glycogenolisis and more on GnG for controlling glycemia. Peripheral IR develops to redistribute the produced glucose to the glucose-strict using cells. The muscle functions as well or better with FFA and KB, and has its own glucose reserovir. There is no evolutionary logic on relying on glucose when you have more efficient fuels readily avilable. Palmitate is the key metabolic mediator in this process. It serves as an intercellular signal that integrates energy metabolism, reducing the utilization of glucose and increasing fat oxidation. Peter from Hyperlipid has written about this before in his Physiological Insulin Resistance series. In a normal physiological scenario the body is adapted to handle increased amounts of glucose or FFA in plasma. Is abnormal to have both substrates high at the same time. If this happens it means that you have a dysregulated metabolism. And here is where the main problems of interpretation arise when evaluating pathological IR. Some say the culprit is high glucose. Others say its lipotoxicity. This last mechanism has gotten much attention lately because of the rise of carb conscious bloggers who dismiss the insulin/carbohydrate hypothesis. Lipotoxicity is the mechanism by which high plasma FFA concentrations produce deleterious metabolic effects. Lets think for a second. We store energy as fat. We use energy as fat. We store fat mainly as palmitate (the principal muscle IR agent and responsible for modern diseases). When we need energy, we hydrolize TG and free palmitate into plasma. High palmitate and high FFA produces lipotoxicity and IR, so then, are we designed to kill ourselves? The answer is obviously no. Lipotoxicity only occurs if there is a mismatch between lipolysis and beta-oxidation. For instance, there is evidence that saturated FA trigger a specific inflammatory response in coronary artery endothelial cells (1), lipoapoptosis (2) and endothelial dysfunction (3). When skeletal muscle cells are exposed to increased levels of palmitate, we see that the deleterious effects build-up dose dependently (4). This means that while myocites can handle a physiological increase in palmitate, they start to develop defense mechanisms when levels rise to pathological. This only occurs in abnormal or broken** metabolisms, like in T2DM. Having endothelial cells chronically exposed to very high FFA is bad, so muscle cells try to reduce this exposure by storing lipids as IMTG. Accordingly, IR has been proposed as a defense mechanism for controlling body fat distribution (5). NEFAs have also shown to impair insulin secretion, possibly as a preventive mechanism (6). So for lipotoxicity to occur, there must be a metabolic dysregulation by which the rate of lipolysis is not controlled. As we all know, insulin is the key enzyme controlling HSL. It has been proposed that adipose tissue IR (ATIR) is the starting point in the pathogenesis of pathological IR (7). Without insulin, lipolysis is unregulated. Because this is not due to a physiological need***, oxidation is not correlated with lipolysis and FFA start to rise in plasma and accumulate in extra-adipose tissues. But pathological IR is not characterized only by ATIR. Loss of control of GnG also occurs because of hepatic IR, producing hyperglycemia. Now the body has two potential fuels in excess and each one promotes its own oxidation. To compensate, hyperinsulinemia occurs and aggravates the situation. Chronic hyperinsulinemia and hyperglicemia, without an excess of FFA, have shown to impair insulin sensitivity and insulin secretion (8) by a different mechanism (impaired non-oxidative glucose disposal). This, combined with high plasma FFA is a recipe for disaster. When talking about lipotoxicity one must be careful with the evidence. As I stated, we cannot make conclusions based only on in vitro studies. When you eat no carbohydrates (or at least not intentionally) FFA will and should rise in plasma. Its completely normal, you use fat as fuel, you need it available. Just like ketosis. But if you eat carbohydrates, you shouldn't have elevated FFA nor ketones. This is when lipotoxicity occurs. Bottomline: Each metabolic fuel controls its own oxidation. We cannot use isolated mechanisms or variables to try to understand physiology and metabolism as the body is a highly regulated and integrated system. Arguing that FFA in plasma cause lipotoxicity is as misleading and wrong as saying that insulin by itself causes insulin resistance. * Not necessarily your own body fat. ** As per the definition of Dr. Harris.*** You need energy = you burn more fat (increased rate of lipolysis). FFA are supplied according to the energy demand and oxidized.
