2. Homeostatic regulation of food intake
In this page we deal with the organs, tissues, brain regions and messengers (hormones and neurotransmitters) that are involved in the homeostatic regulation of food intake. We deal with the communication between the digestive tract, pancreas and adipose tissue, on the one hand and the arcuate nucleus in the brain on the other (see figure 1). We focus on the arcuate nucleus because, with respect to regulation of food intake, it is considered the major entry point of hormonal signals. Another important entry point, but this time for neurotransmitters, is the nucleus of the solitary tract (NTS). We describe how the digestive system senses both emptiness and fullness, and sends, respectively, appetite or satiety messages to the brain. We also describe how elevated blood-glucose levels, via the intermediate of the pancreas, as well as elevated levels of fat in adipose tissue, lead to the emission of satiety messages (see figure 1a and table 1). Collectively, these messages provide the brain with a symbolic representation of the feeding status of the organism. The neurons in the arcuate nucleus then send signals towards other regions of the brain giving rise to either a food seeking (orexic) or fasting (anorexic) behaviour. We present a simplified version of what really happens but this description of events should provide a trunk to which new branches can be added.
From Greek: óμος, hómos, "similar"; and ιστημι, histemi, "standing still"; coined by Walter Bradford Cannon, is the property of a system, either open or closed, that regulates its internal environment and tends to maintain a stable, constant condition. Typically used to refer to a living organism. Multiple dynamic equilibrium adjustment and regulation mechanisms make homeostasis possible. Source; Wikepedia
Figure 1 (a) Homeostatic regulation; messengers from the digestive tract (ghrelin, PYY, CCK, leptin), from the pancreas (insulin) and from adipose tissue (leptin) directly converge upon the arcuate nucleus in the hypothalamus or they send messages via the intermediate of afferent neurons of the vagus nerve (which carry receptors for PYY, CKK, leptin and ghrelin). Collectively they provide a symbolic representation of the feeding status of the organism. These messages are then translated into either a food-seeking (orexic) or a fasting (anorexic) behaviour. We ignore the role of the spinal nerves in these webpages. (b) Anatomic location of the arcuate nucleus and the nucleus of the solitary tract (nucleus tractus solitarius or NTS).
Table 1 Examples of peripheral mediators (messengers) that regulate food intake. Note that only ghrelin and anandamide act as orexigenic substances, all the others provide an anorexigenic signal. Ghrelin, insulin and leptin are treated extensively in this page and in the page of team 4. We have added the SwissProt entry codes so that you can learn as much as you like about these mediators and their receptors “Active learning, any time, any place and anywhere”.
Next we will describe the sources of mediators involved in the regulation of food intake and then we describe their target, the arcuate nucleus and its nerve cells (neurons) that control feeding or fasting behaviour.
Ghrelin is an appetite-inducing peptide hormone. It is secreted by entero-endocrine cells in the fundus region of the stomach. What exactly drives its release from these cells is not known. Some suspect mechanoreceptors that sense the filling state of the stomach. Stretching of the wall may turn off hormone release. It is also produced in small quantities in other parts of the digestive tract, the pancreas and importantly in ghrelin-neurons in the hypothalamus (see below). Ghrelin levels in blood were found to be at their peak just before and at their lowest just after a meal (“post prandial” dip). Important evidence for its role in control of appetite came from the observation that mice lacking either ghrelin or its receptor (GSHR) are protected from diet-induced obesity (although feeding behaviour does not differ from control mice under normal feeding conditions).
Long term regulation of ghrelin may be influenced by the adiposity state of the organism or other unknown factors. Ghrelin is generally low in obese persons (inhibition by elevated fat storage) and high in people with anorexia nervosa (empty stomach and empty fat stores). It is also unusually high in (obese) people with the Prader-Will syndrome?
Ghrelin is a peptide hormone, comprising 28 amino-acids (figure 2). It is obtained from a 94 amino-acid precursor named proghrelin. Other products of the prohormone are; des-Gln14-ghrelin (or 27 ghrelin), C-ghrelin and obestatin. About 20% of ghrelin is modified (on Ser-3) by n-octanoic acid (process referred to as octanoylation), through the action of a membrane bound “ghrelin O-acylgransferase” (GOAT). This occurs in the rough-endoplasmic reticulum. Octanoylation is essential for binding to the ghrelin receptor and thus for the induction of appetite (and other functions).
Figure 2 (a) An important source of ghrelin is the fundus region of the stomach. The oxyntic mucous contains entero-endocrine cells of different types, of which X/A cells stain most positive for ghrelin. (b) The molecular composition of n-octanoyl-ghrelin. The octanoic acid tail is vital for receptor binding and thus for biological activity of ghrelin.
The discovery of ghrelin
First there was the receptor, discovered as the binding site of synthetic compounds that caused the immediate secretion of growth hormone (GH) from somatotrophic cells of the anterior pituitary. These compounds were developed as potential medicaments aiming to restore body growth (by boosting the production of GH). The “orphan” receptor, lacking a physiological ligand, was named growth hormone-secretagogue receptor (GSHR, of which two splicevariants exist; GSHR1a (full length) and GSHR1b (truncated).
Then there was the ligand which, surprise, was isolated from extracts from the stomach and not, as expected, from the pituitary gland or hypothalamus! Because the newly identified physiological ligand controlled secretion of growth hormone, it was named ghrelin, after ghre, the proto-indo-european root of the word “grow”. Strangely enough, mice lacking either ghrelin or its receptor grow normally.
Only later it was discovered that, when injected in the bloodstream or into cerebral ventricles, it stimulates food intake in rodents. The attentions shifted entirely from studying its role in growth to studying its role in appetite control!
Ghrelin diffuses into the tissues and into the blood. Ghrelin is also detected in the hypothalamus but how it actually reaches this brain region remains unclear. There are two reasons that plead against the idea that stomach-produced ghrelin diffuses into the hypothalamus. Firstly, ghrelin does not easily cross the endothelial cells in the central nervous system because these are tightly associated (tight junctions) and surrounded by pericytes and astrocytes which make passive transport sheer impossible (together these qualities make up the blood-brain-barrier) (figure 3). Active transport has been detected but in the direction of brain-to-blood and not the other way round. Secondly, the appetite-inducing effect of ghrelin is abrogated when the nervus vagus is cut (vagotomy).
Figure 3 The blood brain barrier (endothelial cells + pericytes + astrocyte foot processes) and a lack of active transport prevents diffusion of stomach-produced ghrelin into the hypothalamus.
The current line of thinking is that the ghrelin produced in the stomach acts on feeding behaviour by inhibiting the activity of the vagus nerve (reducing the discharge of neurotransmitters in the brain). Ghrelin receptors (GHSR) are present on afferent neurons of the vagus nerve. This in turn may cause the local release of ghrelin in the hypothalamus (see figure 13). In the arcuate nucleus, ghrelin stimulates appetite by increasing the activity of orexigenic neurons (the Npy/AgRP/GABA containing neurons) (see figure 13). More information about its mode of action is provided in the web page of team 4.
Sleep loss and weight gain
Sleep restriction in healthy humans is linked to elevated ghrelin and reduced leptin hormone levels, concomitant with an increase in appetite. Suppression of the slow wave sleep (decreasing the “quality” of sleep) leads to a decreased insulin sensitivity and decreased glucose tolerance (diabetes type II symptoms). In other words, sleep, or lack of it, affects the metabolic state of humans and the bottom line is that a good night rest keeps you slim.
In the nervous system, afferent neurons (otherwise known as sensory or receptor neurons), carry nerve impulses from receptors or sense organs toward the central nervous system. This term can also be used to describe relative connections between structures. Afferent neurons communicate with specialized interneurons. The opposite activity of direction or flow is efferent. In the nervous system there is a "closed loop" system of sensation, decision, and reactions. This process is carried out through the activity of afferent neurons, interneurons, and efferent neurons. Source; wikipedia
The discovery of leptin (from leptos, thin) started with the discovery of very plump young mice in the V-stock of the Jackson Memorial Laboratory way back in 1949. From breeding data it was concluded that their obese status was due to a recessive gene which they designated by the symbol ob. The recessive gene caused sterility in the homozygote, but there seemed to be no indication of any affect on the life span of the animals during a period of twelve months. Like human obese, the mice develop type II diabetes (see figure 4, images adapted from Ingalls AM, Dickie MM, Snell GD. Obese, a new mutation in the house mouse. J Hered. 1950;41(12):317-318).
Figure 4 (a) Growth curve of control, “yellow” and obese mice from the V-stock of the Jackson Memorial Laboratory. (b) Control and obese mice at 21 days of age and (c) after 10 months. Images adapted from Ingalls et al. J Hered.1950;41(12):317-318). Much later it was shown that the obese mice lack leptin and thus lack a satiety signal. The “yellow” mice produce an excess of a mutated agouti protein. This normally controls coat colour but, due to aberrant expression, seems to be capable of blocking the α-MSH-mediated satiety signal in the hypothalamus (see below, figure 13).
When, in 1994, the ob gene was cloned (meaning the gene locus identified and DNA-sequence determined) it was shown that the obese mice carry a nonsense mutation in codon 105 (a nonsense mutation results in a premature stop codon (a nonsense codon), leading to a truncated and usually nonfunctional protein). From this and other data it was concluded that “the ob gene product (the protein) may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot”. A year later it was shown that the protein encoded by the obese gene had weight-reducing effects and was subsequently named leptin.
Progress in the obesity field owes much to fat mice. These were either obtained through natural mutations or through created mutations (targeted mutations) in the laboratory. The genes carrying the mutations, such yellow (ay), obese (ob), diabetes (db), fatty (fa) and tubby (tub), have been cloned and revealed important components of the signalling pathways that regulate food intake (Liebel RL, Chung WK, Chua SC. The molecular genetics of rodent single gene obesities. J Biol Chem 1997:272:31937-31940).
Leptin is a peptide hormone comprising 167 amino acids. It structure resembles that of long-chain helical cytokines (figure 5) such as granulocyte colony-stimulating factor (G-CSF), leukocyte inhibitory factor (LIF), interleukin-6 (IL-6) or human growth hormone (hGH).
Figure 5 Structure of leptin. Highly conserved amino acids are coloured purple (present in human, gorilla, chimpanzee, orangutan, rhesus monkey, dog, cow, pig, rat and mouse). Pdb entry 1ax8
Leptin circulates at levels proportional to body fat and white adipose tissue is its main source (figure 6). It enters the central nervous system in proportion to its plasma concentration. Its receptors are found in brain areas involved in regulating food intake and in areas that control energy expenditure. In addition to adipose tissue, it is also detected in brown adipose tissue, placenta (syncytiotrophoblastes), ovaries, skeletal muscle, stomach (in the lower part of fundus glands where ghrelin is produced), mammary epithelial cells, bone marrow, pituitary gland and the liver. How the storage of triglycerides in adipocytes (adiposity) regulates levels of circulating leptin remains unclear.
Adipose tissue; white versus brown
Adipose tissue is an anatomical term for loose connective tissue composed of fat storing cells (adipocytes) (figure 6).
Figure 6 White adipose tissue and a zoom-up of an adipocyte adjacent to a blood capillary (note the size of the adipocytes which equals 70 micrometers, compared to 7 micrometers of the red blood cell and 10 to 20 micrometers for an ordinary body cell).
Leptin inhibits appetite by decreasing the activity of orexigenic neurons (Npy/AgRP/GABA containing neurons) and increasing the activity of the anorexigenic neurons (POMC/CART containing neurons) in the hypothalamus (see figure 13). Both populations express the leptin receptor.
The leptin receptor is highly expressed in the hypothalamus and it has the characteristics of a cytokine receptor. It is discovered in “diabetic” mice (db), which are fat mice that develop type II diabetes. In short they suffer from a syndrome that resembles morbid human obesity. The mutation in diabetic mice causes abnormal splicing of the mRNA, giving rise to a protein that lacks its cytoplasmic segment. The receptor can bind leptin but cannot signal into the cell (no transduction of the signal from outside to inside). More information about its mode of action is provided in the web page of team 4.
Insulin is a peptide hormone of 51 amino acids derived from a 110 amino acid precursor. The prohormone is cleaved, giving rise to two chains; chain-a and chain-b. These are connected by two disulphide bonds. Insulin is produced by the β-cells of the islets of Langerhans in the pancreas (figure 7).
Figure 7 (a) Composition of pro-insulin and mature insulin. Processing occurs in the Golgi. (b) Insulin is produced by β-cells that reside in the Islets of Langerhans of the pancreas. Glucagon, also involved in glucose metabolism, is produced by α-cells.
Blood levels of insulin are regulated by the blood glucose concentration (also known as glycemia). Gastric and intestinal absorption of carbohydrates raises blood glucose levels above a steady 70-100 mg/dl. This leads to an increased uptake of glucose in β-cells of the pancreas, which, in reply, release insulin (figure 8). Insulin has a general anabolic effect; it assures removal of glucose from the blood and stimulates its conversion into glycogen (a glucose polymer). When glycogen reaches its saturation point (not more than 300 grams in an adult) insulin contributes to the storage of glucose in adipose tissue in the form of triglycerides (unlimited storage). Insulin in the blood can be regarded as a messenger of plentiness.
When blood glucose levels drop, insulin secretion seizes. Regulation of blood-glucose levels is taken over by glucagon, also produced by the pancreas. It stimulates the conversion of glycogen in the liver into glucose and causes its release into the blood stream (gluconeogenesis). (NB the glucose in the skeletal muscles cannot enter the bloodstream and are for muscle-use only. The breakdown of muscle glycogen is under control of adrenaline).
Glucose and the brain
Stored glucose, in the form of glycogen, can serve many purposes but an important role is to serve as fuel necessary for the production of ATP by the mitochondria (something that can also very effectively be achieved by fatty acids in case the glycogen stores are depleted). The brain is particularly sensitive to glucose levels as it does not easily use fatty acids for the production of ATP. Blood concentrations at 30 mg/dl or below (hypoglycemia) therefore cause confusion, convulsions and unconsciousness (coma). This is one of the problems diabetics have to deal with because they do not stock the glucose effectively and therefore risk very low levels in between meals.
Insulin enters the hypothalamus and its receptors are expressed on neurons in the arcuate nucleus. It provides a satiety signal by stimulating the anorexigenic POMC/CART neurons and by inhibiting the orexigenic Npy/AgRP/GABA neurons (see figure 13). Amplifying the insulin signal makes mice resistant to obesity. An excess of adipose tissue renders liver and muscle cells insensitive to insulin (weak intracellular signal) and this may also be true for the neurons in the arcuate nucleus. This leads to a weak satiety signal and may augment food intake. The molecular mechanism by which insulin regulates neurotransmitter release is treated in the web page of team 4.
Figure 8 Insulin maturation in secretory vesicles of the trans-Golgi network followed by glucose-mediated release from a β-cell in the pancreas. Increased glucose levels in the blood are translated in the β-cells by an increased production of ATP by the mitochondria (more substrate available). Elevated levels of ATP cause the closure of K+ channels and this leads to depolarization of the plasma membrane. As a consequence the Ca2+ conductance increases and the ensuing elevated level of intracellular Ca2+ signals the fusion of secretion vesicles, loaded with insulin, with the plasma membrane. Insulin diffuses into the blood. Important targets are skeletal muscles and the liver. Insulin promotes removal of glucose from the blood (through induction of membrane expression of glucose transporters) and its storage in the form of glycogen. Insulin also reaches the arcuate nucleus and contributes to a fasting behaviour.
Diabetes means siphon (also spelled as syphon), describing the excessive production of urine and the subsequent thirst this provokes. Medically one can siphon in two ways: one where the urine tastes sweet, honey-like, and is thus referred to as diabetes mellitus, the other where the urine is tasteless, named diabetes insipidness. “Sweet siphoning” is the consequence of glucose in the urine shortly after a meal. Due to a lack of insulin (type I diabetes) or due to resistance to insulin (type II diabetes), the glucose that arrives in the blood is not properly stored inside body cells. The excess of glucose spills over in the kidneys and because glucose attracts water, it impedes efficient water retentions from the filtrate, hence an excess of urine. Normally, glucose is reabsorbed in the kidney, before it reaches the bladder, but with such an excess, the transport system simply saturates and the urine gets sweet. In the old days the doctor would taste the urine for diagnosis purpose; nowadays a little glucose indicator stick will tell the bad news.
Cholecystokinin is a peptide hormone of 95 amino acids, derived from a 115 precursor (preprocholecystokinin). Ten variants of different sizes are excised from the pro-hormone, giving rise to CCK-58, -39, -33 etc. Which of these 10 variants are important for the regulation of food intake is not clear to us. CCK is produced by I-cells in the mucosal epithelium of the small intestine in response to long fatty acid chains (more than 12 carbons). It is released into the blood at the level of the duodenum. CCK stimulates bile production by the liver, causes release of bile from the gallbladder (hence its name cholecystokinin = move the bile-sac), it stimulates release of enzymes from the (exocrine) pancreas and it decreases the rate of gastric emptying (control of gastric sphincter). Collectively these actions allow optimal digestion of fat and protein in the small intestine.
CCK also provides a satiety message by having a stimulatory effect on the vagus nerve (increasing the discharge of neurotransmitters in the brain). Vagal afferent neurons (going from the gut to the brain) carry receptors for CCK (CCK1-R). Its stimulatory action is thought to oppose the effect of ghrelin, which inhibits the nervus vagus (see above). Moreover, CCK induces the release of leptin from the stomach and this may enhance the short term satiety signal. We will not return to CCK in the following web pages.
Peptide tyrosine tyrosine (PYY) is composed of 36 amino-acids, which are cleaved from a 94 amino-acid precursor. It belongs to the family of “tyrosine (Y) peptides”, including neuropeptide Y (NPY), pancreatic polypeptide (PP) and petide Y (found only in fish). Both NPY and PYY have five tyrosines while PP has 4 or 5. It is produced in the ileum (distal part of the small intestine) and in the colon (large intestine) but what precisely causes its secretion remains to be discovered. Levels increase before arrival of the food bolus in the ileum.
PYY acts as a satiety messenger. PYY readily passes the blood-brain barrier and its receptors (type Y2 and Y4) have been demonstrated in the arcuate nucleus. PPY also act indirectly, through the intermediate of the vagus nerve, with which it interacts at the level of the dorsal vagal complex in the medulla oblongata (area postrema and nucleus of the solitary tract). Like CKK, it has a stimulatory effect, meaning it increases the discharge of neurotransmitters and these somehow have an anorexigenic effect.
Cases of severe obesity in male Pima Indians (Arizona, New Mexico) are associated with a mutation in PYY, where glutamine-62 is replaced by proline (Q62P). In anorexic humans, levels of PYY are increased fourfold. Agonists of PPY are currently being tested as anti-obesity agents.
The arcuate nucleus target of ghrelin, leptin and insulin
The arcuate nucleus (in humans also known as the infundibular nucleus) is situated in the mediobasal hypothalamic area of the brain (figure 9 and 10). It constitutes an aggregation of neural cell bodies (or soma) which are visible as dense dots after staining of brain slices with for instance Cresyl violet. These cell bodies are of course connected with other parts of the brain through their dendrites (upstream) and axons (downstream). The cell bodies contain the nucleus of the neuron.
From arcus (latin), bow or curved, thus translated as “nucleus in the shape of a bow”. In figures often shortened as “Arc”.
Infundibulum (latin) means funnel. There are quite a few anatomical structures that carry the name infundibulum, such as the entry path of air chambers (alveoli) in the lungs, the outflow portion of the right ventricle of the heart or the mammalian oviduct (fallopian tube) when it approaches the ovary. Here it refers to the funnel shape of the stalk that connects the pituitary with the brain. The arcuate nucleus sits just above that funnel structure and therefore carries the alternative name of “infundibular nucleus”. In figures often shortened as “Inf”.
Figure 9 Anatomical localization of the arcuate nucleus and the surrounding hypothalamic nuclei in a coronal (left) and sagittal (right) section of the human brain.
The arcuate nucleus harbours two types of neurons (figure 10):
Figure 10 (a) Anatomical position-indicators. For whole organisms: dorsal, ventral, rostral, caudal, dexter and sinister are used. Dexter is on the right when looking from the back. For tissues, organs and cells, a second set of position-indicators is employed: lateral, medial, apical and basal. With respect to the “ventromedial” location of centrally-projecting cells in the arcuate nucleus; this means that you find them “on the belly side of the thalamus towards the middle”, which in this particular case equals ”basomedial” location.
Nomenclature of brain nuclei
For those who are not fully initiated in the field, like us, the naming and abbreviations employed for brain nuclei are hard to grasp. Not only do some use Latin and other English descriptions, the employment of different abbreviations makes it difficult to compare different studies or illustrations. Examples relevant for this webpage; the lateral hypothalamic area is shortened as LHA or LT and the paraventricular nucleus as PVN, PVH or PN. Some figures will shorten the arcuate nucleus as Arc, others as Inf (infundibular nucleus). We learned that the “anterior hypothalamic area” is also referred to as “the anterior nucleus” and that the “pre-optic areas” represent the same anatomical structures as the “medial and lateral pre-optic nuclei”. Or not? Moreover, despite many good brain atlases, navigation in the brain is not particular easy; sagittal or coronal projections give quite different images. And then, for some illustrations mice brains are used and for others humans. Finally, the understanding of neuronal networks is still in its infancy; what is connected to what and what exactly creates the sense of appetite or satiety still requires a lot of fine-tuning. If you find this webpage somewhat confusing with respect to neuronal connections and the neurotransmitters employed, than you now know why.
Centrally projecting neurons of the arcuate nucleus
Among the centrally-projecting neurons, two different sub-populations are distinguished, each characterized by the neurotransmitters they produce (figure 12):
Insulin, ghrelin and leptin interact with centrally projecting neurons and modulate their activity
Ghrelin (appetite, orexigenic), insulin and leptin (satiety, anorexigenic) enter the arcuate nucleus. They interact with specific receptors exposed at the surface of the neurons.
The above mentioned signals are combined with signals coming from the afferent branches of the vagus nerve (see paragraphs about CCK and PPY). Consult the web page of team 4 for information about the molecular mechanisms that bring about changes in neurotransmitter release.
Figure 13 Regulation of food intake at the level of the arcuate nucleus (ventro-medial hypothalamus).
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