A seminar on Leptin and Nitric Oxide detailing the real world clinical uses and the way they have revolutionized the laboratory medicine.

1. Leptin and Nitric Oxide
Dr. Abhishek Roy
Junior Resident (2nd Year)
Dept. of Biochemistry,
Grant Govt. Medical College and
Sir J.J. Group of Hospitals, Mumbai
Email: mail@abhishek.ro

2. What is Leptin?
• It’s the “satiety hormone” in our body.
• Human leptin is a 16 kDa, 146 amino acid residue, non-glycosylated
polypeptide.
• Leptin is encoded by the ob gene.
• Its major source is adipose tissue, and its circulating concentrations indirectly
reflect body fat stores.
• Circulates in the body eventually activating leptin receptors in Arcuate
Nucleus of Hypothalamus.
• Plasma or serum concentrations of leptin are increased in obese humans and
strongly correlate with the degree of adiposity as expressed by percentage of
body fat or body mass index.

5. Leptin Signalling

7. Interactions of
Leptin

9. Genetically mutated obese (ob/ob)mouse Jackson lab, 1949

11. Nitric Oxide

12. What is Nitric Oxide?
 Nitric oxide (NO) is a reactive and toxic free radical gas.
 Thus, it came as a great surprise that this molecule functions as
paracrine signal in regulating blood vessel dilation and serves as a
neurotransmitter.
 It also functions in the immune response.
 It was proclaimed “Molecule of the Year” in 1992.
 Research into its function led to the 1998 Nobel Prize for discovering
the role of nitric oxide as a cardiovascular signaling molecule.

13.  The role of NO in vasodilation was discovered through the
observation that substances such as acetylcholine and
bradykinin which act through the phospho-inositide
signaling system to increase the flow through blood vessels
by eliciting smooth muscle relaxation, require an intact
endothelium overlying the smooth muscle.
 Evidently, endothelial cells respond to the presence of these
vasodilation agents by releasing a diffusible and highly labile
substance (half-life ~5 s) that induces the relaxation of
smooth muscle cells.
 This substance was identified as NO, in part, through parallel
studies identifying NO as the active metabolite that
mediates the well-known vasodilating effects of antianginal
organic nitrates such as nitroglycerin.

14. Nitric Oxide Synthase Requires Five
Redox-Active Cofactors
 NO is synthesized by NO synthase (NOS), which catalyzes the
NADPH-dependent 5-electron oxidation of L-arginine by O2 to yield
NO and the amino acid L-citrulline with the intermediate formation
of Nω-hydroxy-L-arginine.
 Three isozymes of NOS have been identified in mammals, neuronal
NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS),
which are also known as NOS-1, -2, and -3, respectively.

16.  An N-terminal, ~500-residue, oxygenase or heme
domain that catalyzes both reaction steps of
and contains the dimer interface. This domain
binds the substrates O2 and L-arginine and two
redox-active prosthetic groups, Fe(III)-heme and
5,6,7,8 tetrahydrobiopterin (H4B)
 A C-terminal, ~600-residue, reductase domain
that supplies the electrons for the NOS reaction.
It binds NADPH and two redox-active prosthetic
groups, an FAD and a flavin mononucleotide via
three nucleotide binding modules. This domain is
homologous to cytochrome P450 reductase, an
enzyme that participates in detoxification
processes.

17. a) Its N-terminal oxygenase domain
in complex with heme, L-arginine,
and H4B.
b) Its C-terminal reductase domain in complex with FMN, FAD,
and NADP+. In both structures the homodimeric protein is
viewed in semitransparent ribbon form with its 2-fold axis
vertical and with one subunit colored in rainbow order from
N-terminus (blue) to C-terminus (red) and the other subunit
blue-gray.

18.  NOS requires bound H4B to produce NO. In the absence of this prosthetic
group, NOS efficiently catalyzes the NADPH-mediated oxidation of O2 to
H2O2. Investigations by Steuhr have established that H4B functions as an
internal redox agent in that, during the reactions forming both NOHA and NO,
it is oxidized to its radical form (H4B+) and then re-reduced to its reduced form
(H4B). Thus, H4B does not undergo net oxidation in the NOS reaction.
 NO rapidly diffuses across cell membranes, although its high reactivity
prevents it from traveling >1 mm from its site of synthesis (in particular, it
efficiently reacts with both oxyhemoglobin and deoxyhemoglobin: NO +
HbO2 → NO- + metHb; and NO + Hb → HbNO).
3
 The physiological target of NO in smooth muscle cells is guanylate cyclase
(GC), which catalyzes the reaction of GTP to yield 3’,5’-cyclic GMP (cGMP)

19. cGMP causes smooth muscle relaxation
through its stimulation of protein phosphorylation by
cGMP-dependent protein kinase. NO reacts with GC’s
heme prosthetic group to yield nitroso-heme, whose
presence increases GC’s activity by up to 200-fold,
presumably via a conformation change resembling that in
hemoglobin on binding O2

20. eNOS and nNOS but Not iNOS Are
Regulated by [Ca2+]
 Ca2+–calmodulin activates eNOS and nNOS by binding to the ~30-
residue segments linking their oxygenase and reductase domains.
 NO functions to transduce hormonally induced increases in
intracellular [Ca2+] in endothelial cells to increased rates of
production of cGMP in neighboring smooth muscle cells.
 NO produced by nNOS mediates vasodilation through endothelium-independent
neural stimulation of smooth muscle. In this signal
transduction pathway, which is responsible for the dilation of
cerebral and other arteries as well as penile erection, nerve impulses
cause an increased [Ca2+] in nerve terminals, thereby stimulating
neuronal NOS.

21.  Inducible NOS (iNOS) is unresponsive to Ca2+ even though it has two tightly
bound calmodulin subunits. However, it is transcriptionally induced in
macrophages and neutrophils (white blood cells that function to ingest and kill
bacteria), as well as in endothelial and smooth muscle cells (in contrast, eNOS
and nNOS are expressed constitutively, that is, at a constant rate).
 Several hours after exposure to cytokines and/or endotoxins, these cells begin
to produce large quantities of NO and continue to do so for many hours.
Activated macrophages and neutrophils also produce superoxide ion (O2
- ),
which chemically combines with NO to form the even more toxic peroxynitrite
(OONO-, which rapidly reacts with H2O to yield the highly reactive hydroxide
radical, OH-, and NO2) that they use to kill ingested bacteria. Indeed, NOS
inhibitors block the cytotoxic actions of macrophages.

22. Clinical Scenario
 Heart: In atherosclerosis, the endothelium has a reduced capacity to
produce NO. However, NO can be furnished by treatment with
nitroglycerin. Large efforts in drug discovery are currently aimed at
generating more powerful and selective cardiac drugs based on the
new knowledge of NO as a signal molecule.
 Shock: Bacterial infections can lead to sepsis and circulatory shock. In
this situation, NO plays a harmful role. White blood cells react to
bacterial products by releasing enormous amounts of NO that dilate
the blood vessels. The blood pressure drops and the patient may
become unconscious. In this situation, inhibitors of NO synthesis may be
useful in intensive care treatment.
 Lungs: Intensive care patients can be treated by inhalation of NO gas.
This has provided good results and even saved lives. For instance, NO
gas has been used to reduce dangerously high blood pressure in the
lungs of infants. But the dosage is critical since the gas can be toxic at
high concentrations.

23.  Cancer: White blood cells use NO not only to kill infectious agents such as
bacteria, fungi and parasites, but also to defend the host against tumours.
Scientists are currently testing whether NO can be used to stop the growth
of tumours since this gas can induce programmed cell death, apoptosis.
 Impotence: NO can initiate erection of the penis by dilating the blood
vessels to the erectile bodies. This knowledge has already led to the
development of new drugs against impotence.
 Diagnostic analyses: Inflammatory diseases can be revealed by analysing
the production of NO from e.g. lungs and intestines. This is used for
diagnosing asthma, colitis, and other diseases.
 NO is important for the olfactory sense and our capacity to recognise
different scents. It may even be important for our memory.

24. Thank You