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Piperine’s mitigation of obesity and diabetes can be
explained by its up-regulation of the metabolic rate
of resting muscle
Leonardo Nogara
a
, Nariman Naber
b
, Edward Pate
c
, Marcella Canton
a
, Carlo Reggiani
a
, and Roger Cooke
b,1
a
Dipartimento di Scienze Biomediche, University of Padua, Padua, Italy 35122;
b
Department of Biochemistry, University of California, San Francisco,
CA 94158; and
c
Voiland School of Bioengineering, Washington State University, Pullman, WA 99163
Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved September 27, 2016 (received for review May 12, 2016)
We identify a target for treating obesity and type 2 diabetes, the
consumption of calories by an increase in the metabolic rate of resting
skeletal muscle. The metabolic rate of skeletal muscle can be increased
by shifting myosin heads from the super-relaxed state (SRX), with a
low ATPase activity, to a disordered relaxed state (DRX), with a higher
ATPase activity. The shift of myosin heads was detected by a change
in fluorescent intensity of a probe attached to the myosin regulatory
light chain in skinned skeletal fibers, allowing us to perform a high-
throughput screen of 2,128 compounds. The screen identified one
compound, which destabilized the super-relaxed state, piperine (the
main alkaloid component of black pepper). Destabilization of the SRX
by piperine was confirmed by single-nucleotide turnover measure-
ments. The effect was only observed in fast twitch skeletal fibers
and not in slow twitch fibers or cardiac tissues. Piperine increased
ATPase activity of skinned relaxed fibers by 66 ±15%. The K
d
was
∼2μM. Piperine had little effect on the mechanics of either fully active
or resting muscle fibers. Previous work has shown that piperine can
mitigate both obesity and type 2 diabetes in rodent models of these
conditions. We propose that the increase in resting muscle metabo-
lism contributes to these positive effects. The results described here
show that up-regulation of resting muscle metabolism could treat
obesity and type 2 diabetes and that piperine would provide a useful
lead compound for the development of these therapies.
myosin
|
fluorescence
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skeletal muscle
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super-relaxed state
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obesity
An epidemic of obesity and type 2 diabetes is currently affecting
a large fraction of the world population (1, 2). This is primarily
due to an overconsumption of food coupled with a reduction in
physical activity. A natural antidote to both obesity and type 2 di-
abetes is to choose a healthy lifestyle, including a low-calorie diet
and increased physical activity. However, many do not choose this
option, and for some advanced patients, increased physical activity
is not possible. The response to overfeeding in humans is diverse:
some store the excess calories almost entirely, whereas others
metabolize most of them (3). The variation is strongly dependent
on both lifestyle and genetics. As strenuous activity was limited in
some of these protocols, the diversity of weight gain was attributed
to light activities, such as fidgeting (3, 4). Alternatively, the diversity
may be due to variation in the metabolic rate of resting muscle and
to how this responds to activity (5).
A pharmacological approach to combating obesity has been of
limited value, of the order of 5–10% weight loss when combined
with lifestyle changes (for review, see refs. 6 and 7). Therapies for
type 2 diabetes produce little effect, with the exception of met-
formin (for review, see ref. 8). Lifestyle changes, including better
diets and increased activity levels, have a larger effect than phar-
maceuticals. Here we suggest a target for combating obesity and
type 2 diabetes, increasing the metabolic rate of resting skeletal
muscle. The metabolic rate of resting, living skeletal muscle is
variable, responding to a number of factors, including the hor-
mones leptin and epinephrine (for review, see ref. 5). Although the
resting metabolism of muscle is low compared with many other
tissues, due to its large mass, ∼40% of body weight, its contribution
to whole-body resting metabolic rate is appreciable, ∼25%. This
strategy will address the fundamental problem in these conditions:
fat and glucose intake exceed the amount consumed by metabo-
lism. A common problem following weight loss is regain of weight
due to a reduction in metabolic rate (9). A pharmaceutical that
increases metabolic rate may help achieve weight loss and maintain
it. Skeletal muscle is an ideal tissue for increasing thermogenesis
because of its large metabolic capacity. The resting metabolic rate
is ∼15W (for review, see ref. 5). Human subjects can raise the
metabolic rate to 600 W for 1 h (10, 11).
We have recently identified a new mechanism for thermogenesis
in resting muscle, variation in the ATPase activity of the motor
protein myosin (5, 12, 13). Measuring single-cycle ATP turnover
rates in skinned resting muscle, we showed that myosin has two
states, one with a turnover time of ∼20 s, similar to the rate for
purified myosin at 25 °C, and one with a turnover time of about
250 s. It had been shown many years earlier by a comparison be-
tween the in vivo rate of resting muscle and the activity of purified
myosin that the myosin ATPase activity in resting skeletal fibers
was highly inhibited (14). A correlation with structural data sug-
gested that the myosin heads with the slow turnover time were
bound to the core of the thick filament in a complex known as the
interacting-heads motif (12, 15–17). The myosin heads with the
faster ATPase activity are not bound to the thick filament and are
disordered and capable of binding weakly to actin in resting fibers.
We have called the myosin state with the slower ATPase activity
the super-relaxed state (SRX) and the one with the faster ATPase
activity the disordered relaxed state (DRX) (5, 12, 13). The low
metabolic rate of resting skeletal muscle requires that myosin heads
Significance
We have developed a method for finding pharmaceuticals that
would treat obesity and type 2 diabetes by increasing the met-
abolic rate of resting skeletal muscle. The metabolic rate is in-
creased by shifting the motor protein myosin from a low activity
state to a higher activity state. We devised an assay, screened for
compounds, and found one molecule, piperine. Piperine increased
the metabolic rate of resting muscle fibers. Piperine does not
have the properties required to be a pharmaceutical in humans,
but it would make a good lead compound for finding compounds
that do. Our results provide proof of concept that these metabolic
diseases can be treated by future pharmaceuticals that target
myosin to increase the metabolism of excess calories.
Author contributions: L.N. and R.C. designed research; L.N. and N.N. performed research;
L.N., E.P., and R.C. analyzed data; and L.N., M.C., C.R., and R.C. wrote the paper.
Conflict of interest statement: R.C. is a member of the scientific advisory board of
MyoKardia.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. Email: cooke@cgl.ucsf.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1607536113/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1607536113 PNAS Early Edition
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BIOCHEMISTRY
spend most of their time in the SRX in both animals and in humans.
A simple calculation shows that if all myosin heads were transferred
from the SRX to the DRX, human whole-body metabolic rate
would increase by ∼2–4MJ·d
‒1
(5). Thus, a pharmaceutical that
destabilizedalargefractionofmyosinintheSRXwouldbean
effective therapy for obesity and type 2 diabetes.
A major hurdle to discovering small molecules that destabilize
the SRX has been the limitations imposed by the single-nucleotide
turnover assay previously used and the requirement of observing it
in fibers. We recently overcame these obstacles by finding a fluo-
rescent probe on a subunit of myosin, the regulatory light chain
(RLC), which showed an increase in emission intensity and shift to
shorter wavelengths upon the transition from the SRX to the DRX
(Figs. S1 and S2) (18). We used this signal to carry out a high-
throughput screen of 2,128 compounds approved for human con-
sumption by the FDA (see Supporting Information for description).
This screen identified one compound, piperine, the main alkaloid
component of black pepper.
Results
The Signal Used in the Screen. Our previous work had identified the
ratio of fluorescent intensities as a reporter of the population of
the SRX (18). We used an RLC mutant labeled with a coumarin
maleimide (MDCC) on cysteine 5. The emission of the probe
shifted to lower wavelengths upon the transition from rigor or the
DRX to the SRX. The intensity obtained at shorter wavelengths,
440 nm, was divided by that obtained at longer wavelengths,
520 nm, to produce a ratio that was intrinsic to the state of myosin
and independent of the number of fibers being visualized, intensity
of excitation, and photobleaching.
To strengthen the hypothesis that this signal reports the
population of the SRX, we measured the population of the SRX
by the ratio signal at different temperatures. The population of
the SRX measured by the mant–chase protocol and the binding
of myosin heads to the core of the thick filament, measured
by X-ray diffraction, have both shown a strong temperature de-
pendence (12, 19). As shown in Fig. S3, the population of the
SRX measured by the ratio signal also has a strong temperature
dependence, increasing by a factor of 2.2 ±0.2 (SEM, n=6) on
raising the temperature from 15 °C to 30 °C. This change in
population is similar to that observed previously using the chase
protocol, which found that the population increased by a factor
of 1.8 over the same temperature range. These observations
provide additional support for the hypothesis that the fluores-
cence ratio reports the population of the SRX.
High-Throughput Screen. The screens were carried out by observing
the change in fluorescence intensity of probes attached to a sub-
unit of myosin, RLC, in skinned rabbit fast skeletal muscle fibers.
The wells were loaded using the labeled and chopped fiber
preparation as described in Materials and Methods and Supporting
Information (Fig. S1). Because of the low homogeneity of the fiber
preparation, fiber number varied among wells. Even if fibers were
loosely attached to the bottom of the well, it was not possible to
exclude movements during the assay. To further complicate mat-
ters the fluorescent excitation intensity was not uniform across the
well. These problems have been addressed by the discovery of a
fluorescence ratiometric reporter of the myosin state, described
above (18). The images were analyzed at two wavelengths by the
Fiji macro, and outliers were reexamined by manual analysis. See
Supporting Information for a more detailed description of the fiber
preparation and the high-throughput screen.
In only one well did the intensity ratio indicate that the SRX of
the fibers had been destabilized by the compound present in that
well. The compound was piperine, which is the main alkaloid
component of black pepper (Fig. 1). Piperine is a well-known
compound in ayurvedic medicine and has been shown to interact
with a number of targets in biological systems.
Flow Cells. To confirm that piperine was a true hit, we observed its
effect on a labeled fiber mounted in a flow cell on an inverted
fluorescence microscope. This setup allowed the quick exchange of
solutions with continuous measurement of the fluorescence emis-
sion at both wavelengths. In a typical experiment the fiber started
in rigor buffer, and a relaxing solution was added to populate the
SRX state (Fig. 1 and Fig. S2). In the presence of ATP, the ratio of
the fluorescence intensity increased by about 15%. The solution
was replaced by a relaxing solution containing 100 μM piperine,
producing about a 10% drop in the intensity ratio and indicating
that piperine had destabilized ∼60% of the previously existing
myosin in the SRX. The solution was again exchanged with a
relaxing solution without piperine, and the ratio returned to its
previous level, showing that the effect of piperine was reversible.
Data from a number of similar experiments showed that 100 μM
piperine destabilized the population of the SRX by 46 ±3%
(SEM, n=22).
The data from the screen used the fluorescent signal of a la-
beled subunit of myosin to monitor the population of the SRX. A
more definitive experiment is to directly measure the rate of
nucleotide turnover in the fibers, using the single-nucleotide
turnover experiments that were first used to identify the SRX
(12, 13). In these experiments the fiber is first incubated in
mantATP, a fluorescent analog of ATP. This is followed by the
chase phase, using unlabeled ATP to displace the fluorescent
nucleotides as soon as they are released (Fig. S4C). In this ex-
periment, fluorescence decays in two phases: a fast phase in-
volving the release of mant nucleotides from ATPases with rapid
turnover rates, followed by a slow phase, which arises from the
release of mant nucleotides from myosin in the SRX.
Typical data for fast skeletal fibers are shown in Fig. 2A, where
it is clear that both the magnitude and lifetime of the component
of the fluorescence that decays slowly are diminished in the
presence of 100 μM piperine. Data obtained in a number of
similar experiments showed that piperine destabilized the pop-
ulation of myosin heads that were in the slow component by 34 ±
4% (SEM, n=10). The lifetime of the heads remaining in the
slow component is decreased by 29 ±11% (SEM, n=9). In fi-
bers exchanged with RLC-MDCC, there is a small inhibition of
Fig. 1. The effect of 100 μM piperine on the fluorescence of fast skeletal
RLC-MDCC fibers. A single labeled fiber was mounted in a simple flow cell,
and fluorescence was measured at two wavelengths. The ratio of the two
intensities, which measures the population of the myosin heads in the SRX
(18), is shown as a function of time. The fiber starts in rigor where the
population of the SRX is zero. The ratio increases upon addition of ATP
showing an increased population of the SRX. Subsequent addition of 100 μM
piperine to the relaxing solution decreases the ratio showing that ∼60% of
the myosin heads have transferred out of the SRX into the DRX. A sub-
sequent wash with ATP shows that the effect of piperine is reversible. The
structure of piperine is shown in the lower right.
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the population of the slow component, 27%, but little change in
the inhibition caused by piperine (Fig. S4 Avs. B).
The screen and all of the fiber experiments discussed above were
performed using rabbit psoas, a fast twitch muscle predominantly
composed of myosin type 2B fibers (20–22). To assess a possible
fiber-type specificity, the experiment was repeated using fibers
isolated from the soleus muscle, which uses a slow twitch type 1
myosin isoform. In contrast to fast twitch fibers, piperine showed
little effect on the SRX in the slow twitch fibers (Fig. 2B). The
addition of piperine also showed little effect on the SRX in strips
of rabbit cardiac tissue (Fig. 2C). Slow twitch skeletal fibers share
the same myosin heavy-chain and regulatory light-chain isoforms as
cardiac ventricle (20–22), so it is expected that if piperine targets
one of these chains, the results would be similar in the two muscles.
These observations have important consequences for the devel-
opment of piperine as a therapeutic compound. An effect of pip-
erine in cardiac tissue would have been very undesirable in a
therapeutic agent targeting skeletal muscle. In addition, if the
compound only affects fast twitch fibers in humans, as it does in the
rabbit fibers, this would limit its power to elevate thermogenesis.
ATPase Activity. The data described above show that piperine de-
stabilizes the SRX, shifting a large fraction of its population into
the DRX state, where its ATPase activity is about tenfold greater.
This destabilization would be expected to increase fiber ATPase
activity. To explore this possibility, the ATPase activities of bundles
of skinned fibers were measured by the amount of inorganic
phosphate produced over time (Fig. 3). In the absence of piperine
the fiber ATPase activity is 0.06 s
−1
(expressed per head of myosin).
This result is within the range of values obtained previously, 0.05–
0.1 s
−1
(23–25), which has been noted to be too high to be com-
patible with the low resting metabolic rate of living muscle (24).
The addition of 100 μM piperine to the solution bathing the fibers
produced a 66 ±10% increase in the ATPase activity of the fibers.
The skinned fiber sample observed here is a complex system,
containing a number of other ATPases in addition to myosin. To
determine whether piperine affects these activities, we used a spe-
cific myosin inhibitor, blebbistatin, which decreases myosin ATPase
activity by about 90% (26, 27). The ATPase activity of nonmyosin
proteins accounts for about one third of the total, and the small
increase seen with piperine may be due to blebbistatin-free myosin
heads (Fig. 3, columns 3 and 4). Piperine had little effect on the
ATPase activity of purified myosin (Fig. 3, columns 5 and 6). This
result suggests that the effect of piperine is specific for the myosin
heads in the SRX complex and not to myosin itself. Exchange of
fibers with RLC-MDCC did not alter the effect of 100 μM piperine
on fiber ATPase activity (Fig. S5). Together, the data show that
piperine increases the ATPase activity of skinned fibers by desta-
bilizingtheSRX.Thefractionofmyosinheadsinvolvedcanbe
Fig. 2. The single-nucleotide turnover experiment used to measure the SRX. Single skinned skeletal muscle fibers or strips of cardiac tissue were mounted in a
flow cell, incubated in a fluorescent analog of ATP, mantATP, and chased with ATP. The intensity of the fiber fluorescence is plotted as a function of time during
the chase phase. Each plot shows samples in the absence (open squares) and presence (solid circles) of 100 μM piperine. Fiber fluorescence decreases in two phases:
a fast phase that is largely over in about 20 s, followed by a slow phase with a lifetime of minutes. The slow phase arises from the slow release of nucleotides by
myosin in the SRX. (A) Fast twitch skeletal fibers. Data averaged from a number of such experiments showed that populations of the slow fluorescent component
were control, 32 ±3% (SEM, n=10), piperine, 21 ±2%, (SEM, n=18), and the lifetimes were control, 191 ±13 s (SEM, n=9), piperine, 136 ±8 s (SEM, n=8). Both
the populat ion and the l ifetime show that the S RX has been partially destabilize d. (B) Slow twitch fibers, showing little effect of piperine. Averaged populations
of the slow fluorescent component were control, 33 ±4% (SEM, n=4), piperine, 33 ±3% (SEM, n=8), and the lifetimes were control, 107 ±10 s (SEM, n=4),
piperine, 98 ±8 s (SEM, n=8). (C) Cardiac tissues showing little effect of piperine. Averaged populations of the slow fluorescent component were control, 21 ±
2% (SEM, n=11), piperine, 21 ±3% (SEM, n=13), and the lifetimes were control, 146 ±24 s (SEM, n=11), piperine, 178 ±28 s (SEM, n=13).
Fig. 3. The effect of piperine on the ATPase activity of skinned skeletal muscle
fibers and purified myosin. Multiple single fibers were incubated in a small
aliquot of relaxing solution, without (column “fiber”)orwith100μM piperine
(column “fiber P”), and their ATPase activity was expressed as the turnover rate
per myosin head per second (Fig. S5). Piperine increases the ATPase activity of
control fibers by 66 ±10%. Samples were also run in the presence of 40 μM
blebbistatin (columns “fiber B”and “fibers B P”with piperine), which inhibits
myosin ATPase activity but not the activity of other enzymes in the fiber, and
piperine has little effect on these enzymes. Piperine has a small and not sig-
nificant effect on the ATPase activity of purified myosin (columns “myosin”
and “myosin P”). Together these data show that piperine increases fiber
ATPase activity by destabilizing the SRX. Errors are SEM (n=8–12).
Nogara et al. PNAS Early Edition
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BIOCHEMISTRY
estimated by comparing the increase in fiber activity, ∼0.04 s
−1
,with
that of pure myosin, ∼0.06 s
−1
. This shows that a large fraction of
the myosin heads, >50%, would have to be shifted from the SRX
to the DRX to produce the observed increase.
Measuring Affinity. Estimating the affinity for the binding of pip-
erine to its target is complicated, becausepiperineisveryhydro-
phobic and binds to the many hydrophobic surfaces available in the
interior of the fiber. Nonspecific binding acts in two ways: it de-
creases the concentration of free piperine inside the fiber and it
provides a larger pool of available binding sites that need to be
filled. The full effect of piperine requires about 80 s at a concen-
tration of 100 μM(Fig.1).Thetimeincreasedwithdecreasing
piperineandreachedmorethan1hat10μM. We observed
chopped fibers in plates, where they were stable for long periods,
and we measured ATPase activities after preincubation in relaxing
solutions plus piperine, as described in Materials and Methods.In
each experiment, data were obtained at a particular concentration
and also at a high concentration, 80–100 μM. The value obtained
at the lower concentration was normalized by that obtained at
the higher concentration, providing a more accurate concentration
dependence. In the case of the plate-reader, different concentra-
tions were observed in different wells (Fig. S6). In the case of the
ATPase activities each set of fibers was first incubated in a lower
concentration of piperine in a relaxing solution, followed by mea-
surement of ATPase activities at that concentration and finally by
measurement in 100 μM piperine (Fig. S7).
As the concentration of piperine was lowered, its effect on the
ratio of fluorescent intensities and on the fiber ATPase activities
decreased. Measurement of the fluorescent intensities in plates
produced a K
d
of 1.7 ±0.4 μM (Fig. 4A). Observation of ATPase
activities showed a similar K
d
of 3 ±0.8 μM (Fig. 4B). The affinity
of piperine for its target is not sufficiently high to be used as a
pharmaceutical in humans. However, it would be a good candi-
date as a lead compound for molecule optimization.
Mechanics. To be an effective therapeutic for metabolic diseases,
a compound must increase the metabolic rate of resting fibers
without having an effect on the mechanics of active muscle. To
explore this possibility, single skinned muscle fibers were
mounted on a tensiometer, which could measure tension and
shortening velocities (28). Each fiber was observed in the pres-
ence or absence of 100 μM piperine, at 25 °C, using a temper-
ature jump protocol to provide better sarcomere stability at
25 °C (Fig. S8). In fully relaxed fibers, piperine had no effect.
Addition of 100 μM piperine to fully activated fibers had no
significant effect on isometric tension, ratio of piperine to con-
trol =1.05 ±0.05 (SEM, n=8) (Fig. S8). Isotonic velocities at
25% of isometric tension were measured (Fig. S9). Piperine had
no significant effect on the shortening velocity, ratio of piperine
to control =0.97 ±0.08 (SEM, n=10).
Components of Piperine. Once a compound with the desired
properties has been identified in a high-throughput screen, a
customary next step is to examine similar compounds for further
drug development. Piperine consists of two ring systems con-
nected by a short conjugated leash (Fig. 1). The compound is
easily broken down into two components: piperidine, the six-
membered ring on the left, and piperic acid. The effects of both
of these compounds were examined using the single-nucleotide
turnover protocols. Neither compound at concentrations up to
200 μM had any effect on either the population or the lifetime of
the SRX. Thus, the action of piperine requires that the two ring
systems be connected together to produce an effect.
Discussion
After identifying piperine in the high-throughput screen as a
destabilizer of the SRX, we were pleased to find in the literature
that piperine had already been associated with attenuation of
weight gain in rodents. Piperine is efficiently absorbed by the gut
and is widely distributed in the various tissues (29, 30). However,
most of the piperine has been conjugated with glucuronic acid,
which may influence its activity. In a typical experiment, rats or
mice were fed a high-fat chow for an extended time. All animals
gained fat mass, but those also receiving piperine gained less than
the controls. The difference in fat mass gain varied between 20 and
70% in different studies (31–35). Changes in lean mass were small,
∼10%. One study showed that administration of piperine to mice
during caloric restriction had no effect on a series of parameters
(36). Thus, the main effect of piperine appears to be to mitigate fat
gain during caloric overload. In the studies above there was no
change in the amount of food consumed; therefore piperine must
affect the amount of fat stored by increasing the amount metab-
olized. Piperine is approved by the FDA for human consumption
but at doses, 20 mg/d, much lower than used in the experiments in
rodents cited above, 20–50 mg ·kg
‒1
·d
‒1
. It is approved, not for
weight control, but for increasing the bioavailability of other drugs,
which it does by inhibiting liver enzymes that metabolize them (37).
Piperine also has a beneficial effect in rodent models of type
2 diabetes (31, 32). Rodents fed high-fat and glucose diets for
extended periods have higher blood glucose levels and show in-
sulin resistance. Addition of piperine to their diets lowers levels
of blood glucose and insulin. Piperine also improves the rate of
glucose removal in a glucose tolerance test (32). All of the above
Fig. 4. The concentration dependence of the effects of piperine on the SRX
in fast skeletal fibers. (A) The inhibition of the SRX by piperine was measured
using the ratio of fluorescent intensities of fibers in wells in a 384-well plate
with different concentrations of piperine. Values are calculated by taking
the rigor state as 0% SRX and the ATP state as 100% SRX. The fit to the data
defines a K
d
of 1.7 ±0.4μM. (B) The effect of different concentrations of
piperine on the increase of fiber ATPases, obtained as shown in Fig. 3. Values
from different experiments were compared as described in Fig. S7. The fit to
the data defines a K
d
of 3 ±0.8 μM. Errors on the data are SEM (n=4–12).
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observations could be explained by an increase in glucose con-
sumption by resting muscle resulting from the increased metabolic
rate of myosin produced by piperine.
The mechanisms by which piperine promotes weight loss or
improves diabetes were unclear. A number of targets have been
identified, but none have been shown to be causative (31, 33, 38–40).
Our data show that piperine enhances thermogenesis of resting
muscle via a perturbation of the SRX/DRX ratio, thus providing
a mechanism. As originally recognized by Ferenczi and coworkers
(14) over 30 y ago, in vivo ATP turnover requires almost all
myosin in relaxed skeletal frog muscle to be in what we now term
the SRX. A transfer of only 20% of the myosin in relaxed muscle
from the SRX to the DRX would cause thermogenesis of resting
muscle to double (5).
Could the activation of resting muscle metabolism by piperine
found here explain the difference in fat gain produced by piperine in
rodents fed high-fat chow for an extended period? In the experi-
ments by BrahmaNaidu et al. (31), rats were fed high-fat chow for
42 d, producing one of the largest observed gains in fat mass. All rats
gained fat during this time, but those who were also fed piperine
gained ∼80 g less fat than controls. The oxidation of this amount of
fat will yield 40 mol of ATP (41). We first compare this value to the
amount of ATP that would be consumedifallofthemyosinheads
were in the DRX. The amount of myosin is calculated by taking the
lean mass of the rats consuming the high-fat chow, 375 g, assuming
50% is muscle and multiplying by the concentration of myosin heads
in mammalian muscle, 360 μM (42), to give 72 μmol of myosin. The
basal ATPase activity of rat myosin has, to our knowledge, not been
measured. To estimate this rate we extrapolated from the values for
other myosin using the known variation of basal metabolic rate with
body mass (43), producing an estimate of ∼0.5 s
−1
(see Supporting
Information for a more detailed description). Multiplying the total
amount of myosin heads times the activity and the duration of the
experiment we find that 129 mol of ATP would be consumed if all
myosin heads were in the DRX. To estimate the fraction of the
myosin heads that would be transferred from the SRX to the DRX,
we assume that 50% of the heads are affected and only in fast twitch
fibers, taken to be 50% of total fibers, giving an ATP consumption of
∼32 mol. This energy is similar to the energy involved in the dif-
ference in fat gain observed by BrahmaNaidu et al. (31). Thus, if the
piperine concentration used was far above its K
d
,>5μM, it would
explain much if not all of the attenuation in fat gain. BrahmaNaidu
et al. measured the attenuation of fat gain at different concentra-
tions of piperine, finding only a modest decrease in effect when the
piperine dose was lowered from 40 mg ·kg
‒1
to 20 mg ·kg
‒1
,sug-
gesting that the doses used were far above the K
d
(31). Although it
requires a number of assumptions, this calculation suggests that the
metabolic changes found here for piperine are of the magnitude to
explain its effect on weight gain. The changes amount to an increase
in the basic metabolic rate of about 25%. We propose that this is a
major factor in the effect of piperine in mitigating weight gain
and diabetes.
At saturating levels, piperine destabilizes ∼50% of the myosin
heads that are in the SRX. This may be because the binding of
piperine only provides enough energy to destabilize 50% of the
heads in the array. Alternatively, the two myosin heads in the
interacting-heads motif are in different configurations, and one
of them, the free head, is less stable (15). It could be that pip-
erine acts on that head only.
Although the affinity of piperine for its target is reasonably
high, sufficient to produce beneficial effects at the high doses
used in rodents, it is probably not high enough to be an effective
therapy in humans. Piperine has been identified as binding to a
number of other molecular targets with a similar affinity, dis-
cussed above, which would probably lead to unfavorable side
effects if taken at the quantities that would be necessary for ef-
fective thermogenesis. Although piperine is probably not an ef-
fective therapeutic, it is an excellent lead compound, which could
be used to find similar compounds whose properties could be
optimized using our in vitro muscle assay systems.
Piperine has many of the qualities that would be required for
any compound to be useful as a therapeutic treatment for met-
abolic diseases in humans: (i) It destabilizes the SRX and can
lead to substantial thermogenesis. (ii) It functions only in fast
twitch muscle fibers with a marginal effect in cardiac muscle fi-
bers. (iii) It has little effect on fully active muscle fibers. (iv)Itis
well tolerated at high doses in rodents, with no obvious side ef-
fects. If a piperine-like pharmaceutical were developed that
destabilized 50% of the myosin heads in the fast muscles of a
70-kg human [assuming 50% fast fibers and a myosin activity in
the DRX of 0.09 s
−1
(44)], it would increase metabolic rate by
1.2 MJ ·d
‒1
. This represents 15% of total daily energy expendi-
ture (TEE) and would consume 32 g fat ·d
‒1
or 12 kg fat ·y
‒1
.
How well will an increase of 15% of TEE be tolerated? Over-
expression of uncoupling protein 3 or ectopic expression of
uncoupling protein 1 in mice raised TEE by 15–25% (45–47). It
was also well tolerated and led to decreased adiposity and im-
proved insulin resistance, suggesting that the approach proposed
here could be successful.
In summary, our results provide the proof of concept that
pharmaceuticals targeting resting muscle thermogenesis can be
found and that they will effectively treat the metabolic diseases,
obesity and type 2 diabetes, in humans. Muscle is an ideal tissue
to target for increasing thermogenesis, as it has a large-reserve
metabolic capacity, and the modest increase suggested here can
be accommodated. These pharmaceuticals will directly address
the fundamental problem in these conditions: the consumption
of more fuels than are metabolized. Here we show that high-
throughput screens can be performed and that the protocols we
used can find molecules that do increase the metabolic rate of
resting muscle. Given the immensity of the problem and the need
for effective therapies, this approach should be attempted. We
suggest that this will open up an area in the field of muscle re-
search and a race to be the first to market with a new class
of pharmaceuticals.
Materials and Methods
Fibers and Solutions. White adult New Zealand rabbits were killed according
to protocols approvedby the Universityof California, San Francisco Institutional
Animal Care and Use C ommittee #AN108976-02. Psoas and soleus muscle fibers
were harvested and stored at ‒20 °C in a solution of rigor buffer and glycerol
mixed 50/50. The Rigor buffer contained 50 mM 3-(N-morpholino)pro-
panesulfonic acid, 120 mM potassium acetate, 5 mM magnesium chloride,
5 mM EGTA, 4 mM DTT, 5 mM potassium phosphate, pH =6.8. Relaxing
buffers were obtained by addition of 4 mM ATP or 250 μM mantATP to the
Rigor buffer. Activating solutions were obtained by addition of 3 mM CaCl
2
to the relaxing buffer. Strips of rabbit cardiac tissues were obtained from the
left ventricle, mounted with aluminum clips, and measured as described in
ref. 48.
Proteins. The RLC used was from mouse skeletal muscle (MLC2F, National
Center for Biotechnology Information identification NP_058034.1) and was
expressed in bacteria, labeled, and exchanged into fibers as described in ref.
18. Myosin was made as described by ref. 49. Labeling with MDCC was 60%,
and ∼50% of the endogenous RLC was replaced by mutant RLC during the
exchange (18).
Characterization of Fiber Properties. Single-nucleotide turnovers were mea-
sured in flow cells as described previously (12). The ATPase activities of a
group of single fibers, 8–12, were measured by direct determination of
phosphate using malachite green (50), For a more detailed description of the
assay, see Figs. S5 and S7. At concentrations of piperine of 25 μM and below
the fibers were preincubated in piperine in relaxing solution for 60–120 min.
Single-fiber mechanics were measured using methods and apparatus de-
scribed previously (28) and in Figs. S8 and S9. All experiments were per-
formed at room temperature, ∼22 °C. For more detailed descriptions, see
Supporting Information.
Nogara et al. PNAS Early Edition
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BIOCHEMISTRY
High-Throughput Screening. Fibers were chopped, producing a preparation of
predominantly single fibers 50–200 μm in length (Fig. S1). The fibers were
exchanged with the labeled light chains as described in Supporting In-
formation and in ref. 18. The labeled fibers were loaded into the wells of
384-well plates. Fibers in relaxing solution without compounds served as
negative controls, fibers in rigor buffer served as positive controls, and fibers
in relaxing solution with 10-μM compounds were the experimental samples.
Images. The images were collected using epifluorescence on a Nikon 6D High-
Throughput fluorescent microscope. The excitation filter was 382–393 nm,
and emission filters were set at 420–460 nm and 500–550 nm. Two images
obtained at different emission wavelengths for each well were first analyzed
by a macro written in Fiji (51). The macro used a series of cutoff filters to
determine the intensities of the fibers; the background; and bright objects,
such as dust, clumps of fibers, and so forth. After correction for background
and bright spots, fiber fluorescence was determined and used to calculate
the ratio of fiber intensity in the two images, providing a signal that was
sensitive to the population of the SRX. Possible hits were checked by ana-
lyzing individual wells, via picking fibers and their adjacent background
manually. The macro was able to analyze most wells, leaving only a few
dozen wells with an outlying ratio in each plate to be manually checked.
ACKNOWLEDGMENTS. We thank Dr. Kurt Thorn and Ms. Delaine Larson for
their generous help in using the microscopes. Troponin C used in the
preparation of labeled fibers was a gift from Dr. Wen-ji Dong (Washington
State University). The screen was run by Kenny Ang at the Small Molecule
Discovery Center at University of California, San Francisco (UCSF), Michelle
Arkin, director. We thank Fondazione Cassa di Risparmio di Padova
e Rovigo for its support. This research was supported by a grant from the
NIH (AR062279) (to E.P. and R.C.). Data for this study were acquired at
the Nikon Imaging Center at UCSF/California Institute for Quantitative
Biosciences (QB3).
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