In our previous video, we discussed how NAMPT
is controlled by the circadian clock. In this video, we will discuss the complex relationship
between metabolism, the circadian clock, and NAMPT.
Nampt encodes Nicotinamide Phosphoribosyltransferase, an enzyme that is critical for the production
of NAD+. NAD+ is both an electron carrier essential for cells to transfer chemical energy
and also a cofactor for enzymes that modify gene expression or repair damaged DNA. NAD
acts as an electron shuttle for various reactions including fatty acid oxidation, the Krebs
cycle, and glycolysis. These processes use interlinked reactions that ultimately use
NAD+ to carry chemical energy used to make ATP.
NAMPT converts one of the building blocks of NAD+, called NAM, into NMN. NMN is then
converted into NAD+. As NAD+ is used in reactions, it is converted back into NAM and NAMPT is
needed to generate more NAD+ NAD+ activates a protein called SIRT1. SIRT1
binds to the CLOCK/BMAL1 dimer and regulates the molecular clock. Thus, NAMPT activity
affects the level of NAD+, which affects the circadian clock, and the clock controls the
production of NAMPT. This feedback connects the circadian clock to metabolism.
As we discussed in our previous video, when the circadian clock is disrupted, NAMPT expression
decreases and becomes arrhythmic. What are the consequences of low NAMPT expression?
Well, mice with mutations that remove Nampt entirely are embryonic lethal - this means
that Nampt is so important to survival, that the Nampt knockout animals don’t live long
enough to be born. Instead, researchers can examine the role
of Nampt by making conditional knockouts, meaning that Nampt is not missing in all cells
all the time. Rather, loss of function can be specifically targeted to a tissue or developmental
stage. In many cases, animals can survive an embryonic lethal gene being knocked out
in certain tissues or later in development. To engineer mice for conditional loss of a
gene, the cre-lox system is used. This approach uses cre, a site-specific recombinase, and
lox p sites, sequences of DNA recognized by cre. Scientists engineer Cre to be expressed
only in a particular tissue or under specific conditions. Everywhere else in the body, Cre
is not made. For our example, let’s link the cre gene to the promoter of the gene that
codes for albumin, which is mostly made in the liver. Like all our genes, the genetic
code for albumin is stored in the DNA of every cell. But albumin is typically only expressed
in the liver. If we insert the cre gene under the control of the albumin promoter, then
although the cre gene will be in the DNA of every cell, the Cre protein will be made only
in the tissues where albumin is made, mostly in the liver.click] When a cell transcribes
the albumin gene, it will also turn on the Cre gene, and cells that do not synthesize
albumin, like in the kidney, will not make the recombinase. Lox P sites are placed around
an important part, such as a critical exon, of another gene - let’s say Nampt. When
Cre recombinase is expressed in cells that make albumin, Cre recognizes the lox p sites
and excises the Nampt gene sequence. Thus, these specific cells will not be able to express
functional NAMPT, while other cells are still able to.
Using Cre-Lox technology, scientists found that when NAMPT production is knocked out
in skeletal muscle, it causes muscle weakness. This is believed to be caused by an observed
decline in ATP content and NAD+ in the mitochondria. Loss of NAMPT in projection neurons, which
link the brain to the muscles, leads to neurodegeneration, causing motor abnormalities, muscle atrophy,
and eventually death. Deletion of NAMPT in adipose tissue causes severe insulin resistance.
Finally, loss of NAMPT in pancreatic beta cells leads to a decrease in the ATP/ADP ratio
and disrupts the insulin excretion signal. Having no NAMPT is problematic, but what about
the low, non-rhythmic NAMPT seen in circadian mutant mice? Let’s look at the role of NAMPT
in mitochondria: When an animal fasts, it oxidizes fat to form
ketones, and produces ATP in a process called fatty acid oxidation. In mitochondria from
the liver of mice that lack the core clock gene Bmal1, scientists found a decrease in
both NAD+ and activity of the oxidative pathways that carry out fatty acid oxidation. Bmal1
knockout mice die after relatively short periods of food restriction compared to wild type
animals. The sensitivity of the Bmal1 knockouts is believed to be due to a decrease in the
ability to oxidize their fat for energy. The scientists hypothesized that this decrease
in fatty acid oxidation and NAD+ is due to a decrease in NAMPT activity, so they gave
the circadian mutant animals NMN to bypass NAMPT. Once they did this, the liver mitochondria
in the circadian mutants had normal NAD+ levels, and recovered fatty acid oxidation activity
in these mice. These experiments show that the circadian clock controls mitochondrial
oxidative metabolism by regulating the Nampt-dependent production of NAD+.
So let’s review what we’ve learned. The circadian clock controls NAMPT production.
NAMPT production feeds back to the clock through NAD+ and SIRT1. NAMPT production is critical
for cellular metabolism, and some of the negative metabolic effects of disrupted circadian rhythms
occur because of decreased levels of NAMPT. These feedback processes are critical for
survival and reveal the interdependence of circadian timing and metabolic pathways.