In parallel to time-resolved integration of the multiple afferent signals
conveying at central circuits regulating energy balance, these neuroendocrine
networks have to simultaneously orchestrate an appropriate efferent response.
While it is clear that these efferent signals, such as hormonal, behavioral,
parasympathetic and sympathetic output from the brain to the periphery, are
inducing changes in energy expenditure, it is still unclear how appetite induction
is triggered by hypothalamic circuits involving the arcuate nucleus and
other hypothalamic centers of feeding control.
The autonomic and enteric nervous systems, and the hypothalamic-pituitaryendocrine
axes significantly modulate gastrointestinal handling and metabolic
processing of food as well as the partitioning and oxidation of metabolites, and
thus codetermine the efferent side of the regulatory loop. Behavioral changes
expressed through the skeletal motor system (e.g. decreased spontaneous
locomotor activity, fidgeting) are one way to maintain or restore energy homeostasis.
In addition, temporary deficits can be rapidly restored by increased
absorption from the gastrointestinal tract, mobilization from stores, or changes
in utilization rates. An important efferent determinant of energy balance is the
vagal parasympathetic outflow. Neurons involved in parasympathetic outflow
of central and peripheral circuits regulating energy balance are connected with
a large number of nuclei and neurons in forebrain, midbrain, caudal medulla,
hypothalamus, and dorsal vagal complex, and receive viscero-sensory input via
the solitary nucleus as well as endocrine input via the are postrema. Vagalparasympathetic
outflow directly modulates several physiological parameters
such as pancreatic secretion, hepatic glycogenesis, salivation, gastric emptying,
intestinal absorption and gastrointestinal hormone secretion, to just name a few
mechanisms involved in energy balance regulation. Sympathetic outflow regulating
energy balance receives neuronal input from the hypothalamus, frontal
cortex, amygdala, midbrain, caudal medulla and directly influences energy
homeostasis via targeting brown as well as white adipose tissue, adrenal
medulla, skeletal muscle, hepatocyte, pancreatic alpha- as well as beta-cells or
skin vasomotor tone.
The classical endocrine axes consisting of hypothalamic releasing
hormones, pituitary hormones and peripheral endocrine signals are heavily
involved in maintaining the balance of metabolism and energy homeostasis.
Hypothalamic releasing hormone (TRH, CRH, GHRH, LHRH, Ghrelin)
expression is influenced by activity levels of hypothalamic circuitry known to
be crucially involved in central energy balance regulation (e.g. NPY/AGRPneurons,
POMC/CART neurons). Central activation of the hypothalamicpituitary-
adrenal (HPA) axis induces a positive energy balance, while
stimulation of the hypothalamic-pituitary-thyroid (HPT) axis produces energy
deficits via an increased metabolic rate. The growth hormone (GH) – insulinlike-
growth factor I (IGF-1) axis (also called somatotropic axis) promotes
lipolysis and muscle growth. Stimulation of the hypothalamic-pituitary-sex hormone
axis (gonadotropic axis) causes a negative energy balance via increased fat
oxidation and decreased food intake in women (estrogens) and promotes an
increased ratio of muscle tissue vs. fat tissue in men (androgens). All these
efferent systems described above are usually modulated in concert to achieve
the appropriate adaptational metabolic changes as based on information
received by a panel of afferent signals and processed by specific neuronal
circuitry in the brain. The central networks that are regulating energy homeostasis
by balancing endocrine and neuronal efferences with afferent signals
indicating energy requirements, also have to adjust to unusual environmental
challenges and disease states which might require drastic changes in orexigenic
drive or metabolic efficiency. While the respective adjustments of the neuroendocrine
networks governing energy balance will mostly reflect an appropriate
attempt to counterbalance pathophysiological situation, in many cases these
reactive changes will not suffice to re-establish energy homeostasis (i.e. cancer
cachexia, diet-induced obesity). To understand how the human central nervous
system functions to control and adjust appetite, energy expenditure and body
composition in health and disease, researchers are facing multiple difficulties.
Firstly, in vivo imaging techniques such as positron emission tomography
(PET scan) or functional magnetic resonance (FMRI) imaging of the human
brain can not yet provide information that is detailed enough to detect, e.g.
relevant changes of blood flow in specific hypothalamic nuclei. Secondly,
rodent brains are not identical with human brains, although pathology studies
reveal to a surprisingly large extent similar neuronal modalities when comparing
mouse, rat and human brains. NPY neurons for example exhibit a different
distribution pattern in mice as compared to humans. However, for most neuronal
structures involved, primate studies confirm findings from rodents and
monogenetic causes of obesity first described in mice or rats (such as ob/ob
mutations causing leptin deficiency and therefore morbid obesity) have been
confirmed in humans [14–22].
Rodent Models
The analysis of targets and pathways leading to or preventing from obesity
is a complicated task, since almost all known naturally occurring models of
obesity in rodents may affect several pathways and tissues, hence are not
particularly useful to dissect peripheral targets from brain-driven phenomena.
Therefore, researchers have been creating genetically modified rodent models
to circumvent these problems. Genetic modifications have the unambiguous
advantage that they can be restricted to defined cell types by the use of tissue
specific promoters. For example, the promoter of the UCP1 gene has been used
to direct expression restricted to brown fat cells, the aP2-promoter for white
adipocytes, the albumin promoter for liver-specific expression, the muscle
creatine kinase promoter for expression restricted to skeletal muscle and heart,
etc. Employing this approach researcher were enabled to introduce novel genes
into tissue where they normally to not occur, or the expression levels of genes
were elevated (so-called overexpression) within any tissue of interest to obesity
research. Shortly afterwards, techniques were invented to disrupt the expression
of a given gene in mice. This so-called knock-out technique is a versatile tool
to inactivate candidate genes which might contribute to obesity in both mice
and men. While the first-generation knock-outs were equally affecting any
cell within the rodent body, subsequently developed techniques gave rise to
the option to disrupt gene expression in defined tissues (as for overexpression,
see above). To obtain such a tissue specificity parts of the gene of interest
were genomically flanked with so-called loxP sites, essentially recognition
sequences for a highly specific restriction enzyme called cre recombinase.
While mice carrying the loxP sites only are indistinguishable from their genetically
unaltered littermates, and show unaffected expression levels of the
flanked gene, they lose the expression of the targeted gene when intercrossed
with mice transgenically overexpressing cre recombinase in a tissue specific
manner. Using this approach, cre recombinase can be expressed under the control
of the promoter listed above (and many others) and directs a tissue specific
knock-out of any loxP flanked gene. Lastly, recent techniques additionally
allow the disruption of genes at a defined time point: While the non-conditional
as well as the tissue specific knock-out is activated as soon as the promoter
becomes switched on during embryonic development, certain hypothesis
require the inactivation of genes in a later stage of development. To obtain this
goal, the transgenically overexpressed cre recombinase was fused to mutants of
ligand-binding domains (LBD) of steroid receptors. While this fusion protein is
readily expressed in any cell activating the preceding promoter, in stays in the
cytosol, i.e. does not exert its action as a restriction enzyme since chromosomal
DNA is located within the nucleus and cannot be reached by the recombinase.
To obtain a knock-out event in such animals these are injected with a artificial
steroid hormone derivative which specifically enables cytosolic cre recombinase
to move into the nucleus where it finally causes chromosomal recombination,
i.e. a knock-out event. Employing this technique disruption of genes can
be obtained in both, spatially and temporally restricted, fashion. Taken together,
these techniques have been of tremendous impact for the dissection of pathways
leading to or preventing from obesity [23–25].
The analysis of central neuroendocrine pathways controlling energy homeostasis,
mainly based on neuroanatomy and molecular genetic approaches, aims
to elucidate how communication networks involving humoral factors, neuropeptides
and neuronal circuitry including synapses and neurotransmitters, regulate
acute and chronic supply, metabolism and storage of energy (fig. 1, 2).
Functional relevance of the putatively involved pathways and factors, however,
can only be proven, if equally sophisticated methods are used to detect and analyze
in vivo changes in caloric intake, energy expenditure or body composition.
To achieve that goal, animal models (specifically rodent models) relevant for
the (patho-)physiology of (disease)states defined by (impaired)energy balance,
have to be employed.
The best animal model for a disease is one that closely represents most
or all of its pathophysiological characteristics. Animal models available for
obesity research differ widely in food intake, metabolism, presence of diabetes
or insulin resistance, and the extent of obesity presented. Energy balance is
the match of energy intake to energy expenditure. Energy intake (EI) can be
measured by calculating the weight of food consumed over a period of time and
converting mass to kilocalories (kcal). More sophisticated methods have been
used to measure meal size, meal frequency and feeding state. However, for most
studies of obesity and energy homeostasis, calculation of daily caloric intake is
sufficient. Measurement of energy expenditure (EE) requires a calorimeter.
Some instruments directly measure the heat dissipated from an experimental
animal or human while more modern equipment indirectly measures heat
liberated by assaying carbon dioxide expired and oxygen consumed within a
sealed chamber. Energy is consumed and used for work, or it is stored in the
form of glycogen (liver and muscle), fat (adipose) or protein (muscle). Energy
balance is achieved when energy intake is equal to energy expenditure. A positive
energy balance occurs when calories ingested, digested and resorbed are
greater than calories expended. A negative energy balance can be produced by
decreasing energy intake, increasing energy expenditure, or both. There are
many aspects of energy intake that must be considered for the careful study of
energy homeostasis. Not all food eaten is digested, and not all digested foods
are absorbed. Such variables as the composition of food, feeding pattern, rate
of gastric emptying and intestinal transit time may all alter energy intake.
Careful study of these variables requires not only measurement of food intake,
but also measurement of fecal composition. Study of energy expenditure permits
assignment of calories to basal life-sustaining functions such as the energy
required for respiration, cardiac function, and all the specialized functions of
each organ. In addition, energy is also spent for muscular activity as well as
support of the digestive process itself.
Energy is required for generation (growth) and maintenance (repair) of
body mass. Thus, the age of animals studied is a very important variable for
consideration when studying energy balance. For example, pubertal rats
rapidly gain lean mass and show completely different metabolic features than
aged rats that may be losing lean mass and accruing fat mass. Environmental
conditions must also be considered when studying energy homeostasis.
A small, sustained increase in the environmental temperature can induce a positive
energy balance in rodents because they expend less energy to maintain
body temperature. Although creating an artificial situation, it is necessary to
study animals that are single-housed to accurately measure food intake. While
this prevents competition for feeding, it also decreases the environmental
temperature within a cage and increases the area for locomotor activity or
exercise. All manipulations to the animal and its environment must be considered
with each experimental design, because just weighing an animal or its
remaining food will alter both energy intake and energy expenditure. Like
humans, animals have taste preferences and tend to overeat when presented
with certain diets or may eat less when presented less palatable diets that are
of low caloric density. Moreover, manipulations of food presentation and its
timing can drastically alter caloric intake. Rodents tend to overeat when food
is presented on the cage floor. Changing the feeding system from cage top to
hanging feeders or other containers may cause very significant reductions in
food intake. Rodents are nocturnal animals and eat during the dark photoperiod.
Longer dark periods may favor hyperphagia, while presentation of food
only during the light period may reduce feeding. Finally, infections may not
necessarily present with obvious signs but can still severely influence feeding
patterns, energy expenditure and ultimately change body weight. The obesity
epidemic documented in modern societies and in populations that have moved
from impoverished communities to those with abundant food and modern
conveniences is used as evidence that obesity results from an interaction of
inherited genes with the environment. In particular: (a) decreased energy
expenditure (lack of physical exercise), and (b) increased caloric intake (abundant
high caloric foods), or both, are blamed for the etiology of obesity in
modern societies. Thus, many investigators use wild-type rodents maintained
on a high fat diet as a model to study obesity. For example, Long-Evans rats
are susceptible to gaining fat when placed on a high fat diet. When they are
housed in groups, a dominant social hierarchy is established for feeding and
the group conserves heat by assembling together. The rats can be housed singly
to prevent competition for food and the ambient temperature of the animal
facilities (usually around 728 F) can be increased (for example to 76 808 F)
to prevent them from expending energy in purpose to maintain body temperature
(‘thermoneutral zone’ ambient temperature where a specific strain
burns the least amount of calories to generate or reduce heat for the maintenance
of body temperature). The rats can be weaned onto a palatable diet that
is comprised of 40% fat, 40% carbohydrate and 20% protein (based on calories).
Alternatively, a ‘cafeteria diet’ can be used. Here researchers try to match
eating habits that lead to human obesity by offering the studied rodents large
variety of cafeteria food including chocolate, cookies, bread, biscuits, ham,
cakes, peanuts, cheese, etc. However, with this diet, nutrient intake is difficult
to quantify or analyze. 100 days is the recommended age to start an obesity
study, because at this age rats cease to gain lean mass, but readily accrue fat
mass. Long-Evans rats are an out-bred strain and body weight is characterized
by a normal distribution. About 4% become morbidly obese ( 50% fat mass),
4% remain lean ( 10% fat mass) and the remainder is represented by a continuum
between the 2 extreme phenotypes. The same principle can be applied
to other rat strains (i.e. male Wistar) or mice.
Several strains of rodents have been identified and propagated because
of spontaneous mutations that resulted in an obese phenotype. The animals are
not only useful when studying biology underlying a specific mutation, but can
be used to study obesity itself and are particularly helpful to dissect individual
pathways triggered by pharmacological studies of energy balance
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