Download Premigratory fat metabolism in hummingbirds: A Rumsfeldian

Current Zoology
59 (3): 371–380, 2013
Premigratory fat metabolism in hummingbirds:
A Rumsfeldian approach
Raul K. SUAREZ*
Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, California 93106, USA
Abstract Hummingbird migration is a remarkable feat, given the small body sizes of migratory species, their high metabolic
rates during flight and the long distances traveled using fat to fuel the effort. Equally remarkable is the ability of premigratory
hummingbirds in the wild to accumulate fat, synthesized from sugar, at rates as high as 10% of body mass per day. This paper
summarizes, using Rumsfeldian terminology, “known knowns” concerning the energetics of hummingbird migration and premigratory fattening. Energy metabolism during hover-feeding on floral nectar is fueled directly by dietary sugar through the pathway recently named the “sugar oxidation cascade”. However, flight without feeding for more than a few minutes requires shifting
to fat as a fuel. It is proposed that behavior and metabolic fuel choice are coadapted to maximize the rate of fat deposition during
premigratory fattening. The hummingbird liver appears to possess extraordinarily high capacities for fatty acid synthesis. The
analysis of “known knowns” leads to identification of “known unknowns”, e.g., the fates of dietary glucose and fructose, the
regulation of fat metabolism and metabolic interactions between liver and adipose tissue. The history of science behooves recognition of “unknown unknowns” that, when discovered serendipitously, might shed new light on fundamental mechanisms as well
as human pathological conditions [Current Zoology 59 (3): 371–380, 2013].
Keywords
1
Lipid synthesis, Metabolic rate, Metabolic regulation, Muscle energy metabolism, Sugar oxidation
Introduction
The remarkable ability of hummingbirds to hover, an
evolutionary adaptation that allows them to obtain most
of their dietary calories from floral nectar (Powers and
Nagy, 1988), has inspired numerous investigations concerning the biomechanics and energetics of flight. Some
species have become especially well known for their
long distance migrations. Rufous hummingbirds Selasphorus rufus, for example, are known to spend the
summer as far north as Alaska and overwinter as far
south as Mexico (Calder, 1987). Ruby-throated hummingbirds Archilochus colubris are known to fly across
the Gulf of Mexico, traveling nonstop for several hundred kilometers (Lasiewski, 1962). Migratory hummingbird species are typically small and accumulate
body fat synthesized mainly from dietary sugar. Like
other species of birds (Blem, 1976), they oxidize fat as
the main source of energy for migratory flight. Because
of their small size and body morphology, stationary and
directional flight both necessarily require high wingbeat
frequencies and, therefore, high rates of energy expenditure (Suarez, 1992). Small body size also results in
high rates of heat loss; this results in a high energetic
cost of thermoregulation (Bicudo et al., 2002). StationaReceived Feb. 20, 2013; accepted May 7, 2013.
∗ Corresponding author. E-mail: [email protected]
© 2013 Current Zoology
ry hovering also requires a higher rate of energy expenditure than forward flight (Berger, 1985; Clark and
Dudley, 2010). The ingestion of cold nectar on cold
days imposes further thermoregulatory costs, further
elevating the energetic cost of feeding (Lotz et al., 2003).
Despite the high energetic cost of being a hummingbird,
migratory species display the remarkable ability to
make a daily energetic profit by taking in more dietary
energy than they expend. Rates of fat deposition, representing the biochemical conversion of sugar to fat, have
been observed to occur in the wild at rates as high as
10% of body mass per day (Carpenter et al., 1993). This
invited essay focuses primarily on metabolism during
the period of premigratory fattening. A “Rumsfeldian”
approach is taken that begins with known knowns, proceeds with known unknowns and ends with unknown
unknowns. This logical structure is based on former U.S.
Defense Secretary Donald Rumsfeld’s words, uttered in
a news briefing on Feb. 12, 2002. Rumsfeld’s statements, arranged as poetry (Seely, 2003), read:
As we know,
There are known knowns.
There are things we know we know.
We also know
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There are known unknowns.
That is to say
We know there are some things
We do not know.
But there are also unknown unknowns,
The ones we don’t know we don’t know.
2
Known Knowns
2.1 Energetic requirements of hummingbird flight
It is well established that fat serves as the primary
fuel for migratory flight in birds (Blem, 1976). The fatty
acids made available from lipolysis are more highly
reduced than glycogen and yield more energy per unit
mass when oxidized to CO2 + H2O. Unlike glycogen,
which is stored in hydrated form in muscle and liver as
glycogen particles, fats are stored as lipid droplets in
these organs as well as in adipose tissue. The combination of these factors endows a unit mass of fat with
close to 8 times more energy than a unit mass of hydrated glycogen (Hochachka and Somero, 1973).
Many studies over several decades have involved
measuring the energetic cost of hovering in hummingbirds (Welch, 2011). Respiration rates obtained using
modern techniques generally agree with those reported
by Robert Lasiewski in the early 1960s of about 42 ml
O2 per gram bird per hour (Lasiewski ,1962, 1963).
Forward flight costs less energy than hovering (Berger,
1985; Clark and Dudley, 2010), so Lasiewski’s estimate
of migratory flight range in ruby throated hummingbirds
(Lasiewski, 1962) using hovering metabolic rate can be
considered conservative. Given fat content amounting to
40% of body mass (Odum et al., 1961) and assuming
that 1/6 of it is not depleted, Lasiewski calculated a
non-stop, migratory range of about 800 km. Certainly,
ruby-throated hummingbirds accumulate enough fat to
allow them to cross the Gulf of Mexico (Robinson et al.,
1996). Rufous hummingbirds, on the other hand, stop to
refuel along their migratory route (Carpenter et al., 1983;
Calder, 1987; Carpenter et al., 1993). Since they accumulate about as much fat as ruby-throated hummingbirds and the mass-specific metabolic rates of these species during flight are similar, Lasiewski’s estimate likely
applies to rufous hummingbirds as well. During migration, ruby-throated hummingbirds deplete fat stores at a
rate of 2% of body mass per meter per hour (Hedenstrom, 2010). This represents the highest energetic cost
of long-distance locomotion (i.e., lowest “gas mileage”)
known among migratory birds.
Since the energetic requirements of steady-state,
forward flight are more difficult to measure and less
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than that of hovering (Berger, 1985; Clark and Dudley,
2010), metabolic rates during hovering have served as
the foundation for the integration of much information
concerning the biomechanics of flight at the level of the
whole animal with structure and function at the level of
flight muscles, ATP turnover, mitochondrial respiration
rate and flux through pathways of substrate catabolism
(Suarez, 1992; Suarez et al., 2011). Hummingbirds possess high respiratory and cardiovascular capacities for
O2 delivery to flight muscles (Fig. 1). During steadystate, aerobic exercise, rates of O2 flux through the respiratory and cardiovascular systems are regulated to
match the demand for O2 by muscle mitochondria. In
hummingbirds, these capacities for O2 flux are exceptionally high and so it is useful to briefly summarize
some information, obtained mainly from rufous hummingbirds. In the flight muscles, mitochondria occupy
about 35% of fiber volume; this mitochondrial content
exceeds those in pigeons (Mathieu-Costello et al., 1994)
as well as geese (Scott et al., 2009) and approaches
those found in insects (Suarez 1996). Cristae surface
area per unit mitochondrial volume is about 58 m2/cm3
Fig. 1 Diagram of hummingbird in flight, drawn to emphasize extraordinarily high muscle power output, supported by high mitochondrial, respiratory and cardiovascular capacities for gas transport through the O2 transport
cascade, as well as high capacities for carbon flux from the
digestive system to muscle mitochondria through the sugar
oxidation cascade
The diagram illustrates how ATP hydrolysis drives mitochondrial
respiration rates within muscles. During aerobic exercise, e.g.,
hover-feeding and migratory flight, the rates of O2 flux through the
respiratory and cardiovascular systems equal the rate of O2 consumption by muscles. During repeated bouts of hover-feeding, the
time-averaged flux of dietary sugar through the intestinal epithelium
equals or exceeds the rate of sugar oxidation by the flight muscles.
From (Suarez, 1998) with permission.
SUAREZ RK: Premigratory metabolism in hummingbirds
(Suarez et al. 1991); this is similar to those in geese
(Scott et al., 2009) and also within the range found in
insects (Suarez, 1996; Suarez et al., 2000). These high
cristae surface densities are close to a theoretical upper
limit imposed, at least partly, by the need to accommodate Krebs cycle enzymes in the mitochondrial matrix
(Srere, 1985). The maximal capacities for flux, i.e., Vmax
values, at key enzymatic steps in pathways of carbohydrate and fatty acid oxidation are extremely high, reflecting high levels of expression of enzyme protein
(Suarez et al., 1986; Suarez et al., 1990; Fernandez et al.,
2011). During routine hovering flight, a 3-4 g hummingbird, displays a mass-specific, aerobic metabolic
rate about 10-fold higher than the maximum rates
achieved by human athletes. Under these conditions,
hummingbird flight muscles sustain ATP turnover rates
close to 500 µmol g-1 min-1 (Suarez et al. 1990; Welch et
al. 2007). In the fed state, as the birds go from flower to
flower, such ATP turnover rates are supported by glucose oxidation rates close to the maximal biochemical
capacities for glucose phosphorylation, i.e., the Vmax
values of muscle hexokinase (Suarez et al., 1990). During hovering in the fasted state, when fat stores are depleted and fatty acids are oxidized by the flight muscles,
flux through a key enzyme in the pathway, i.e., carnitine
palmitoyl transferase 2 (CPT2), is estimated to be 50%
of Vmax of this enzyme. This is much higher than fractional velocities at this step during exercise in other
species (Suarez et al., 1990). Thus, high metabolic rates
are achieved as a consequence of high mechanical
power outputs, directly supported by high rates of ATP
hydrolysis. High steady-state rates of O2 transport from
the external environment to the muscle mitochondria are
made possible by structural and functional enhancement
of capacities for flux through the cardio-respiratory system (Dubach, 1981; Johansen et al., 1987; MathieuCostello et al., 1992; Suarez, 1992). High rates of aerobic ATP synthesis are made possible by high mitochondrial oxidative capacities (Suarez et al., 1991), high levels of catabolic enzymes in pathways of carbohydrate
and fatty acid oxidation and the operation of key enzymes in these pathways at high fractional velocities
(Suarez et al., 1986; Suarez et al., 1990; Fernandez et al.,
2011). Data currently available indicate that the organization (evolutionary design) of pathways of substrate
oxidation is highly conserved across hummingbird species; interspecific variation in mass-specific hovering
metabolic rates is driven mainly by variation in muscle
power output (Fernandez et al., 2011).
373
2.2 Breakfast in cold meadows
The challenges faced by hummingbirds during migration are dramatically illustrated by considering a
rufous hummingbird on its way south in the late summer as it wakes up in a subalpine meadow in Oregon.
Hummingbirds forage in such habitats at this time of the
year, when morning temperatures can be close to freezing (C.L. Gass, personal communication). During the
overnight fast, fat stores are depleted and fatty acid
oxidation serves as a major source of energy (Powers,
1991). Low ambient temperature activates thermogenic
mechanisms, possibly involving non-shivering thermogenesis (Bicudo et al., 2002), that greatly elevate
resting metabolic rate (Lopez-Calleja and Bozinovic,
1995) and increase the energetic cost of overnight survival. The bird wakes up from sleep or arouses from
torpor (Carpenter and Hixon, 1988; Bucher and Chappell, 1997) and flies to forage, still relying on fat as its
main metabolic fuel. In the transition from rest to flight,
the bird undergoes a physiological transition from a
situation wherein various organs contribute (to various
degrees) to whole-body resting metabolic rate to one in
which flight muscles account for >90% of whole body
metabolic rate (Suarez, 1992). Low ambient temperature
also elevates the energetic cost of foraging flight
(Schuchmann, 1979; Chai et al., 1998; Welch and
Suarez, 2008) as well as the cost of warming up cold
nectar (Lotz et al., 2003). Despite these combined energetic costs, hummingbirds have been known to accumulate fat in preparation for migration or along their
migratory route (during refueling stopovers) up to about
40% of total body mass (Odum et al., 1961; Carpenter et
al., 1983).
Laboratory experiments were conducted in the morning during the late summer for 4 hour durations at 5oC to
simulate conditions in the wild during refueling stopovers. Results reveal that, even under these harsh conditions, rufous hummingbirds are able to gain mass by
accumulating fat when dietary sucrose is provided at a
concentration of 30% (Gass et al., 1999) (Fig. 2). However, lower concentrations (15 and 20%) result in mass
loss. When larger volumes of 15% sucrose are provided
at each visit to the feeder, hummingbirds decrease foraging frequency and increase the amount of time spent
sitting. 15% sucrose is within the range of concentrations found in hummingbird-visited flowers (Nicholson
and Fleming, 2003) and rufous hummingbirds are able
to stay in energy balance or gain mass when feeding on
floral nectar containing less than 30% sucrose at higher
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Fig. 2 Summary of results obtained in experiments
wherein premigratory rufous hummingbirds were kept
individually for 4 h on alternate mornings at 5oC in an
environment chamber
Within the chamber, one perch was provided on top of a digital balance interfaced with a computer; these allowed monitoring of mass
and activity (perched or flying). One feeder was provided in the
chamber that dispensed sugar solutions of various, randomly predetermined volumes of 15, 20 or 30% sucrose solution. During each
experiment, a bird had to sit on the perch after a feeding bout to electronically trigger the release of another bolus of sugar solution. In this
way, birds engaged in repeated bouts of sitting and hover-feeding.
Feeding frequencies were high at low ambient temperature but further
elevated when the amounts (volume and/or concentration) of sucrose
declined. The graph shows that energy balance is achieved, i.e., energy intake = energy expenditure, when mass change = zero. This occurs
at a time-averaged metabolic rate of 1 watt. Since birds weighed 4 g
on average, this corresponds to a time-averaged metabolic rate of 250
watts/kg, the highest known for vertebrate animals under conditions
wherein rates of energy intake = energy expenditure. Mass gain,
which represents fat deposition, is possible at 5oC when birds are
provided 30% sucrose for breakfast. This means that, under these
conditions, energy intake rate > rate energy expenditure. Mass loss
tends to result when birds are provided 15 and 20% sucrose, indicating
energy expenditure > energy intake even when large volumes of dilute
sugar are provided. From (Suarez et al., 2011) with permission.
ambient temperatures (Sutherland et al., 1982). Therefore, it is possible that, at low ambient temperature,
disposal of ingested water may limit the intake rate of
dilute floral nectars. These results are supported by a
theoretical model (Calder, 1979) that predicts hummingbirds should preferentially feed on floral nectars
with higher sucrose concentrations at lower ambient
temperatures. However, hummingbird-visited plant species found in higher, colder habitats do not tend to produce floral nectars with higher sugar concentrations
(Stiles and Freeman, 1993). The patterns of relationships in the compositions of nectars and the nectarivores
that consume them are more complex than physiology
alone would predict (see review by Nicolson and Fleming, 2003).
As fasted hummingbirds begin to feed on floral nec-
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tar, e.g., in the early morning after the overnight fast or
after 1–2 h interruption of feeding, the ratio of CO2
produced to O2 consumed (RQ, the respiratory quotient)
is close to 0.7, indicating that fatty acid oxidation fuels
flight. As feeding on sucrose-rich nectar continues, the
RQ rapidly increases to about 1.0 indicating a shift from
fatty acid to carbohydrate oxidation (Suarez et al., 1990;
Welch et al., 2006). The RQ of 1.0 is maintained during
repeated hover-feeding. Stable carbon isotope experiments, combined with measurement of RQ values, reveal that, under these conditions, recently ingested sugar
directly fuels close to 100% of the energy metabolism
occurring in the flight muscles (Welch et al., 2006;
Welch et al., 2007b). The pathway taken by sugar
molecules from flowers, through the gut, the circulatory
system and into oxidative pathways in muscles is called
the “sugar oxidation cascade” (Suarez et al., 2011) (Fig.
3). It operates in parallel and converges with the pathway for O2 from the external environment to the muscle
mitochondria. Thus, hummingbirds feeding on floral
nectar rapidly switch to sugar oxidation as their main
source of energy. Sugar in excess of that required to
satisfy energy requirements is converted to fat (Fig. 4).
Among small hummingbirds, the energetic cost of flight
is so high that, in the absence of nectar-feeding, it is
estimated that flight could be sustained for only several
minutes using stored glycogen (Suarez et al., 1990).
This means that flight without feeding for more than a
several minutes necessarily requires switching to fatty
acid oxidation (Fig. 4). Since the synthesis of fat costs
energy, it has been proposed that the direct oxidation of
dietary sugar enhances net energy gain because it avoids
the inefficiency of investing energy to make fat, only to
burn it shortly after to fuel foraging (Suarez et al., 1990).
Hummingbird foraging behavior which, in the case of
territorial individuals, involves brief foraging bouts
(Diamond et al., 1986), ensures that metabolism is fueled mainly by dietary sugar. This coadaptation between
behavior and metabolic regulation is hypothesized to
enhance the rate of fat deposition (Suarez and Gass,
2002). In the wild, hummingbirds are able to gain mass
at rates of up to about 10% per day (Carpenter et al.,
1983). The energetic profit or net energy gain, stored as
fat, represents the difference between daily energy intake rate and daily energy expenditure. Energy expenditure can be minimized, for example, by going into
torpor at night or during inactive periods during the day
(Carpenter and Hixon, 1988; Hiebert, 1990; 1993) while
energy intake rate can be enhanced behaviorally, for
example, by increasing feeding frequency (Gass et al.,
SUAREZ RK: Premigratory metabolism in hummingbirds
375
Fig. 3 The sugar oxidation cascade, as it operates in nectar-feeding hummingbirds during refueling stops
Fig. 4
birds
During repeated bouts of foraging, flight muscle energy metabolism is
directly fueled by ingested sugar, resulting in VCO2/VO2 = 1.0. The
diagram shows how the sugar oxidation cascade operates in parallel
and intersects with the oxygen transport cascade. Sugar (white diamonds) in excess of that required to directly support energy needs is
converted to fatty acids and esterified to form fat (triacylglycerol) in
the liver. Triacylglycerol is combined with cholesterol, phospholipids
and apolipoprotein B to form very low density lipoprotein (VLDL),
then released by the liver into the blood. For simplicity, the diagram
makes use of circles to represent VLDL, triacyglycerol and fatty acids.
Fat deposition in adipose tissue involves triglyceride hydrolysis to
fatty acids and glycerol by lipoprotein lipase, transport across cell
membranes, reesterification and storage as lipid droplets. From
(Suarez et al., 2011) with permission.
Lipid substrate is transported mainly as VLDL in the blood. Again,
white diamonds represent sugar and, for simplicity, circles represent
VLDL, triacyglycerol and fatty acids. Sugar oxidation is inhibited in
the flight muscles while oxidation of exogenous and endogenous lipid
results in VCO2/VO2 = 0.7. This occurs during flight in the fasted state
when birds wake up in the morning before feeding or when feeding is
interrupted for 1-2 h and during long-distance migratory flight.
1999) and adjustment of territory size (Carpenter et al.,
1983).
2.3 Fat-fueled hovering vs. forward flight
Capacities for carbohydrate and fatty acid oxidation
have been evaluated in light of the observation that
hummingbirds are able to support the energetic requirements of hovering using either fuel. Pyruvate (derived mainly from glycolysis in vivo) and palmitoylCoA
(an intermediate in long chain fatty acid oxidation) are
oxidized at similar rates to support ADP phosphorylation to ATP by hummingbird flight muscle mitochondria
in vitro (Suarez et al., 1986). This indicates that, at least
at the level of mitochondria, capacities for ATP synthe-
Fat-fueled flight in fasted or migrating humming-
sis are similar, regardless of whether sugar or fats are
metabolized to provide energy. The pathways for both
these processes are highly conserved in cardiac, fast and
slow oxidative muscles among vertebrate animals. As in
other vertebrate species, fatty acid oxidation is carnitine-dependent and fatty acid oxidation inhibits pyruvate oxidation (Suarez et al., 1986). The latter serves as
just one of several control mechanisms by which carbohydrate is “spared” when, for example, in the fasted
state, fatty acids serve as the main fuel for oxidative
metabolism. On the other hand, fatty acid oxidation is
inhibited, partly through inhibition of carnitine palmitoyl transferase 1 (CPT1), by malonylCoA (McGarry,
1995; but see Kim et al., 2002). Such a scenario can be
envisioned as hummingbirds forage while displaying an
RQ of 1.0 as they oxidize dietary sugar to fuel foraging
flight (Suarez et al.,1990; Welch et al., 2006). The oxidation of fatty acid requires about 15% more O2 per ATP
synthesized than the oxidation of carbohydrate (Brand,
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Current Zoology
2005). However, the mechanical power requirement and,
therefore, the ATP hydrolysis rate required for hovering
is independent of which fuel is oxidized. Thus, a fasted
hummingbird, as it oxidizes fatty acid, consumes O2 at a
rate about 15% higher than in the fed state when oxidizing dietary sugar (Welch and Suarez, 2007). An
added benefit of sugar oxidation during foraging is the
requirement for less O2, as many hummingbird species
forage in hypodense, hypoxic air at high elevation (Altshuler et al., 2004).
3
Known Unknowns
The Nobel prize winning biochemist, Albert Szent
Gyorgyi (Szent-Gyorgyi, 1963), is widely quoted as
having once said that “Discovery is seeing what everybody has seen and thinking what nobody has thought”.
Certainly, the assessment of what is known can lead to
new insights as well as the formulation of new questions.
This is true in general and true of metabolism during
premigratory fattening in hummingbirds.
3.1 Fatty acid oxidation in muscle
That fatty acid oxidation can fully support the power
requirements of stationary hovering (Suarez et al., 1990;
Welch et al., 2007) implies that the lower energy requirements of forward flight are supported by enzymatic
flux capacities in the pathway of fatty acid oxidation
well in excess of physiological requirements. It is not
known whether maximum rates of O2 consumption
(VO2max) can be supported equally well by both carbohydrate and fatty acid oxidation. Hovering VO2 values
have been reported to increase by significantly during
hovering in mixtures of helium and O2 (Chai and Dudley, 1996); it would be interesting to determine whether
the same VO2max values are displayed at RQ values of
1.0 and 0.7. It is also interesting that enhancement of
enzymatic capacities for fatty acid oxidation is observed
in various bird species as they prepare for or engage in
migratory flight (Marsh, 1981; Lundgren and Kiessling, 1986; McFarlan et al., 2009). Whether similar upregulation of capacities for flux through pathways of
fatty acid oxidation occurs in migratory hummingbirds
has not been examined. Small hummingbird species can
increase muscle power output, but only for brief durations, to lift loads of up to 80% of body mass (Chai et
al., 1997). In migratory hummingbirds, it would be reasonable to hypothesize that the increased mechanical
power requirements required to lift large stores of fat
might require higher levels of enzyme and transporter
protein expression in pathways of fatty acid oxidation in
muscles. Therefore, seasonal changes in hormone levels
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and their biochemical consequences (Zajac et al., 2011),
in general, might occur as preparation (physiological
adaptation) for increased load-lifting using fat as a fuel,
rather than preparation for long-distance flight per se.
3.2 Fat synthesis in liver and adipose tissue
Given the observation that hummingbirds accumulate
fat at high rates, two rather obvious questions follow.
First, what are the biochemical pathways involved and,
second, how are they regulated (e.g., “turned on”) to
achieve these high rates? It is well known that most of
the triacylglycerol synthesis in birds occurs in the liver
(Hermier, 1997). After synthesis in hepatocytes, most of
the triacylglycerol is combined with cholesterol, phospholipids and apolipoprotein B to form very low density
lipoprotein (VLDL), which is then released into the
blood. Most extrahepatic fat deposition occurs in adipose tissue and involves triglyceride hydrolysis to fatty
acids and glycerol by lipoprotein lipase, transport across
adipocyte cell membranes, reesterification and storage
as lipid droplets (Hermier, 1997). Information specifically concerning triacyglycerol synthesis in hummingbirds is virtually nonexistent. However, rufous hummingbirds possesses extremely high acetylCoA carboxylase activities (Vmax values) in their livers (Reaction 16 in Fig. 5) (Suarez et al., 1988). Since this is a
key regulatory step in fatty acid synthesis (catalyzing
the ATP-dependent synthesis of malonylCoA from acetylCoA and bicarbonate), it is reasonable to hypothesize
that hummingbird livers possesses high capacities for
fatty acid synthesis (Fig. 5). A likely scenario is that
during nectar-feeding, high blood sugar levels lead to
elevated insulin levels, resulting in activation of hepatic
acetylCoA carboxylase, at least partly by a change in its
phosphorylation state (Brownsey et al., 2006). The resulting high rate of fatty acid synthesis is combined with
elevated rates of triacyglycerol synthesis (Reaction 18
in Figure 5), VLDL formation and export into the blood
(Hermier, 1997). Again, drawing from data acquired
from chickens, it is known that the transition from the
fasted to the (high-carbohydrate) fed state results in increased expression of hepatic acetylCoA carboxylase by
transcriptional activation (Hillgartner et al., 1996). Isolated chicken hepatocytes provided high glucose concentration increase the expression of enzymes catalyzing other reactions essential to fatty acid synthesis, i.e.,
malic enzyme (which provides almost all of the
NADPH required for fatty acid synthesis in chicken
livers) and fatty acid synthase (which catalyzes the synthesis of palmitate from 1 acetylCoA, 7 malonylCoA,
and 14 NADPH) (Reactions 15 and 17 in Fig. 5),
SUAREZ RK: Premigratory metabolism in hummingbirds
Fig. 5
377
Pathways of hepatic triacyglyerol synthesis from glucose and fructose
Fatty acid synthesis occurs primarily in the liver in birds. Subsequent formation of VLDL, which makes use of the triacylglycerol synthesized from
sugar, is not shown. Numbers in circles denote the following enzyme-catalyzed reactions: (1) glucokinase, (2) phosphoglucoisomerase, (3) phosphofructokinase, (4) fructokinase, (5) aldolase, (6) glycerol 3-phosphate dehydrogenase, (7) triose kinase, (8) triosephosphate isomerase, (9) pyruvate kinase, (10) pyruvate dehydrogenase, (11) pyruvate carboxylase, (12) citrate synthase, (13) ATP-citrate lyase, (14) malate dehydrogenase, (15)
malic enzyme, (16) acetylCoA carboxylase, (17) fatty acid synthase, (18) esterification. Redrawn and modified from (Martin, 1987).
through transcriptional activation (Hillgartner et al.,
1998). Due to the highly conserved nature of metabolic
pathways and their regulation in birds (Suarez et al.,
1988; Fernandez et al., 2011), these regulatory mechanisms are likely to operate in hummingbirds as well.
Thus, it is worth asking whether it would be worthwhile
to perform research simply to determine whether hummingbird fat metabolism occurs as it does in chickens.
Perhaps it might be more interesting to ask whether
elevated rates of fat deposition in premigratory birds
result from changes in hepatocyte or adipocyte enzyme
expression, i.e., “hierarchical regulation”, or whether
“metabolic regulation” occurs, involving the control of
the activities of preexisting enzymes by allosteric
mechanisms and/or covalent modification (ter Kuile and
Westerhoff , 2001; Suarez et al., 2005). By quantifying
the relative roles played by hierarchical and metabolic
regulation, it should be possible to determine the
mechanisms by which high rates of fat synthesis are
achieved and to gain insights into their activation in
premigratory birds.
A question waiting to be answered concerns the rela-
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tive contributions made by dietary glucose and fructose
to the synthesis of triacyglycerol in nectarivorous animals. In humans, a large fraction of ingested fructose is
metabolized by the liver to glucose (Delarue et al.,
1993). The higher concentration of glucose in the blood
and the greater abundance of membrane glucose transporters than fructose transporters, in combination, result
in the preferential uptake and extrahepatic metabolism
of glucose (Zierath et al., 1995; Kristiansen et al., 1997).
As most of the energy contained in nectars of hummingbird-visited flowers is in the form of sucrose, glucose and fructose (Baker, 1975; Baker et al., 1998), the
possibility that hummingbirds maintain high concentrations of fructose in their blood and that their tissues
possess high capacities for fructose metabolism is worthy of investigation.
4
Unknown Unknowns
Learning to ask good questions is one of the challenges faced by those training to become scientists because, of course, not all questions are worthy of study.
As Peter Hochachka (see biography by Somero and
Suarez, 2005) once put it, some hypotheses are the
equivalent of “predicting that the sun will rise tomorrow
morning”. Nevertheless, there are many “known unknowns” worth pursuing that form the bases for thesis
proposals and grant applications worldwide. In addition
to these, the history of science is full of examples of
serendipitous discoveries, many of which occurred in
the course of performing curiosity-driven research.
Many such accidents occur as investigators delve deeply
into the “how and why” of phenomena such as those
described in this article. While a list of “unknown unknowns” is, by definition, impossible to construct, a
fitting conclusion would be to call attention to the fact
that one cannot predict what might be discovered or
what new insights might come from studying the
mechanisms underlying the phenomenal rates of fat
deposition observed in migratory hummingbirds. The
increasing ingestion of fructose by humans (largely due
to the marketing of manufactured foods containing
high-fructose corn syrup) over the past few decades has
been proposed to contribute to the development of obesity (Elliott et al., 2002; Bremer et al., 2012). The detrimental effects of fructose ingestion are said to be
partly due to the pathway for hepatic fructose metabolism that bypasses some of the reactions in glycolysis
(Fig. 5). Thus, research concerning the pathways of
sugar metabolism to triacylglycerol and their regulation
in hummingbirds may yield unanticipated insights into
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the causes of human obesity and ailments referred to as
the “metabolic syndrome” (Bremer et al., 2012). One
would also hope that better understanding of the challenges faced by migratory, nectarivorous species such as
hummingbirds would ultimately contribute to efforts to
mitigate the effects of climate change and other negative
human impacts on the diversity of life (Cooke et al.
2013).
Acknowledgements I am deeply grateful to P.W. Hochachka,
C.L. Gass, K.C. Welch, Jr., J.R.B. Lighton, C. Martinez del
Rio and O. Mathieu-Costello for their contributions to the
research that led to the ideas presented and questions raised in
this paper. Research on which this article is based was funded
by the U.S. National Science Foundation (IOB 0517694).
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