Bato Korać, Univerzitet u Beogradu, SrbijaTreći kongres o dijetetskim suplementima

25 - 26. novembar 2011.Beograd

Lupinus (lupins, lupines)

Scientific classification

Xiao Shi Xiong Huang San

Kingdom: Plantae(unranked): Angiosperms(unranked): Eudicots(unranked): Rosids(unranked): RosidsOrder: FabalesFamily: FabaceaeSubfamily:  FaboideaeTribe:  GenisteaeSubtribe:  LupininaeGenus: LupinusSpecies: Lupinus albusSpecies: Lupinus albus

Lupinus albus

Protein39.70 %

Campos and El-Dash, Cereal Chem, 1978, 55:619-627.

(2-amino-5-guanidinovaleric acid)

1886: Schuize i Steiger; izolovan L-arginin iz sadnica (semena) lupina

1895: Hedin; L-arginin je komponentaproteina životinja.

1897: Schuize i Winterstein; pokazanastruktura L-arginina.

1924: Kossel i Gross; L-arginin je glavna amino kiselina baznih proteina spermatozoida riba.1930: Scull i Rose; sisari sintetišu L-arginin.1932: Krebs i Henseleit; uloga L-arginina u fiziologiji i metabolizmu.

• “Slao sam 240 pacijenata godišnje kardiologu. Od kada sam počeo sa korišćenjem L-arginina ja sam poslao jednog pacijenta u poslednje dve

di ” (J P d t)

; g g f g j

godine” (Joe Prendergast).

• 1988: Hibbs i sar.; NO je identifikovan kao biološki aktivni intermedijer L-arginin → nitrit+ nitrat puta u makrofaga.

Metabolic fates of arginine in mammalian cells

Wu and Morris JR, Biochem J, 1998, 336:1-17.

Nitric oxide timeline... 1 billion years ago78% N2 and 21% O2...

1847 Nitroglycerin A. Sobrero

1863 Dynamite A. Nobel

8 Nit l i l t W M ll

1979 NO cGMP, F. Murad

1975 nitroglycerin cGMP, F. Murad

1870 Nitroglycerin as coronary relaxant,W. Murrell

1987 EDRF NO, J.L. Ignarro

1980 EDRF, R.F. Furchott and J. Zawadski

1988 L‐arginine ‐NO, S. Moncada NO toxic gas

SENSATIONSENSATIONcommon air pollutant could be produced in cells and exert

SURPRISESURPRISE

… common air pollutant could be produced in cells and exert biological effects in higher animals such as mammals

… totally different from any other known signaling moleculeand very unstable

Nitric Oxide Synthase (NOS)

Homodimer, 130-150 KDa

• products of 3different genes

• 50-60% homology

NOS NOS M M

Nitric oxide synthase isoenzymes:

eNOS , nNOS, nM- µMiNOS, µM - mM

Nitric oxide synthase isoenzymes:

- Neuronal NOS (nNOS, NOS-I)- Inducible NOS (iNOS, NOS-II)

eNOS and nNOS - calcium sensitive

iNOS - calcium insensitive - Endothelial NOS (eNOS, NOS-III)- Mitochondrial NOS (mtNOS) mt NOS - organ specific different

splice variants

The potential role of the nitrate–nitrite–NO pathway in therapeutics

L-arginine

NO

Everything should be madeas simple as possible,

b t t i lbut not simpler.A. Ajnštajn

OPTION A 911 Call Free Ambulance Trip ($500-1000)* $1,000.00

OPTION B

•1 DPA Test $30 00p ( ) , ER Specialist Care/Treatment$350.00 EKG Test $366.00 Cardiac Catheterization $5,609.00 Open Heart or Brain Surgery $50,000.00

1 DPA Test $30.00•1 Month of Arginine     $75.00•Peace of Mind Priceless

p g y $ ,(Angioplasty, Stent or Bypass)

Anesthesia Costs for 7 Hours $7,078.00 12 Hours in Recovery Room $2,895.00 2 Days in Intensive Care $5,292.00

Argininey $ ,

Hospital Stay ($1880 x 5) $9,400.00

Cost that vary which are not included) Salary/Leave Loss (while off work)Salary/Leave Loss (while off work) Medications Home Health/Hospice Care

Complete Funeral Arrangements $7500.00Complete Funeral Arrangements $7500.00(Programs, Casket, Plot, Services, etc)

Approximate Total $93,490.00 Actual Total    $105

Interscapular Brown Adipose

Termogenicinactive tissue

Termogenicactive tissueAdipose

TissueIBAT

inactive tissue active tissue

4 ± 1 ºC22 ± 1 ºC

hyperplasticbrown

adipocyte

dormantbrown

adipocyteNorA βAR cAMP

C O L D

HEAT

UCP1

Is •NO involved in regulation of key thermogenic molecule expressionthermogenic molecule expression

45 days at 22±1 ºC Control

Mill Hillrat males

Untreated

1 3 7 12 21 d 45 L-arginine (2.25%)

L NAME (0 01%)

1, 3, 7, 12, 21 and 45 days at 4±1 ºC

L-NAME (0.01%)

NO-increased noradrenalin immunostaining and sympathetic innervation that could be one of possible ways of NO-acting in improvement of IBAT thermogeneis

Noradrenaline InnervationC 22°C 22°

day 1

day 3day 3

day 7

day 12

day 21

d 45day 45

Untreated L-arginine L-NAME Untreated L-arginine L-NAMEon cold

C 22°

L-arginine accelerated and prolonged cold-induced activation of PCNA as well asVEGF immunopositivity. L-NAME acted in the opposite way.

PCNA VEGF C 22°

day 1

day 3y

day 7

day 12

day 21

day 45

Untreated L-arginine L-NAME Untreated L-arginine L-NAMEon cold

L-arginine intensified cold-induced IBAT hyperplasia, blood flow and capillary remodeling. L-NAME acted in the opposite way.

cont

rol

untr

eate

dL-

arginine

E

day 1 day 3 day 7 day 12 day 21 day 45

L-NAM

E

Ekspresioni profili iNOS i eNOS proteina

Bez tretmana L-arginin L-NAME

iNOS

eNOS

L-arginine augmented, accelerated and prolonged cold-induced increase of thermogenic genes expression. L-NAME had an opposite effects.

Untreated L-arginine L-NAME

UCP1

PPARγPPARγ

PGC-1α

BAT

Fenotipske adaptacije u BAT koje omogućavaju metaboličke adaptacije (intenziviranje metabolizma)

BAT,4 ºC

BAT,

COLD

22 ºC

COLD

povećanje sadržaja UCP1, dekuplovanje

proliferacija prekursorskih ćelija

...UCP1.......PCNA

hipertrofija “zrelih” adipocita

povećanje protoka krvi, angiogeneza

sinteza DNA i proteina..... VEGF

......... PPARγ

PCNAsinteza DNA i proteina

Mitohondriogeneza ................. PGC-1α................PCNA

Cold-induced mitochondrial molecular and structural remodeling is intensified by L-arginine. L-NAME had an opposite effects.

Control Untreated L-arginine L-NAME

Control Untreated L-arginine L-NAME

room temperature 4 5 d a y – c o l d a c c l i m a t i o n

Group22 1 C 4 1 C 4 1 C 4 1 C

Mitochondrialnumber per

cell 66 ± 5 76 ± 4*

97 ± 5*** ###

61 ± 11###

Cristae number per

mitochondrion14 ± 1 18 ± 1

**22 ± 1

* #12 ± 1

###

Untreated L-arginine L-NAME

Complex I

Cyt cy

COX

Pathways of arginine synthesis

1, phosphate-dependent glutaminase;2 i 3, pyrroline-5-carboxylate synthetase;Step 4 is a spontaneous;p p ;5, ornithine aminotransferase;6, ornithine carbamoyltransferase;7, argininosuccinate synthase;8, argininosuccinate lyase;9 N acetylglutamate synthase;9, N-acetylglutamate synthase;10, carbamoyl-phosphate synthetase I;11, proline oxidase;12, aspartate aminotransferase;

Reactions 1-6 and 9-11 occur in mitochondria, reactions 7 and 8 take place in the cytosol, and reaction 12 can occur in both mitochondria and the cytosol.OAA, oxaloacetate;CP, carbamoyl phosphate.

Wu and Morris JR, Biochem J, 1998, 336:1-17.

Beneficial effects of dietary L-arginine supplementation on diet-induced obese rats

Size distribution of adipocytes from retroperitoneal adipose tissue of rats

McKnight et al., Amino Acids, 2010, 39:349-357.

Effects of dietary supplementation with arginineor N-acetylglutamate on concentrations of aminoacids in the plasma of weanling pigs

“Dietary supplementation with 0.6% L-arginine or 0.08% N-carbamylglutamate enhances intestinal growth and integrity, improvenutrition health and growth performance and the availability of dietary

Wu et al., Amino Acids, 2010, 39:831-839.

nutrition, health, and growth performance, and the availability of dietarynutrients for whole-body weight gain in postweaning pigs”.

6.2 μm 9.3 μm

Effects of dietary supplementation with arginine on intestinal morphology in rats

control 22 °C control 4 °C12.7 μm 9.3 μm

L-arginine 4 °CL-arginine 22 °C5 7 μm 4 4 μm5.7 μm 4.4 μm

L-NAME 4 °CL-NAME 22 °C

Experimental design

Non-diabetic

Control

L-arginine (2 25%)

Mill Hill

Non diabetic L arginine (2.25%)

L-NAME (0.01%)

Mill Hillrats

Control

Diabetic(alloxan, 120 mg/kg)

L-arginine (2.25%)

L-NAME (0.01%)

30,00

l / l)

Beneficial effects of L-arginine in pancreas of diabetic animals

20,00

entr

atio

n (m

mo

treatmentGlucose concentration

0,00

10,00

1 2 3Glu

cose

con

c

control level

200

diabetes diabetes + L-arginine diabetes + L-NAME

100

150

atio

n (m

mol

/ l)

control level

50

100

nsul

in c

once

ntra

Insulin concentration

01 2

In

diabetes diabetes + L-arginine diabetes + L-NAME

Vasilijević et al., J Physiol, 2007, 584:921933.

Non-diabetic Diabetic

Beneficial effects of L-arginine in pancreas of diabetic animals – Insulin immunopositivity

Control

L-arginineL arg n ne

L NAMEL-NAME

Non-diabetic Diabetic

Beneficial effects of L-arginine in pancreas of diabetic animals – PDX-1 immunopositivity

Control

L-arginineL-arginine

L-NAME

Non-diabetic Diabetic

Beneficial effects of L-arginine in pancreas of diabetic animals – Insulin/PDX-1 colocalization

Control

L-arginineL-arginine L-arginine-NO producing pathway

plays a role in t i f restoring of

insulin-producing cells acting as a

positive modulator L-NAME positive modulator of PDX-1 in

diabetic pancreas.

Non-diabetic Diabetic

Beneficial effects of L-arginine in pancreas of diabetic animals – nNOS immunopositivity

ControlControl

L-arginine

L-NAME

Beneficial effect of L-arginine in

di b i i L-NAME diabetic pancreas is mediated by nNOS.

Vasoregulation Nitrergic inervation

nNOS

LL--argininarginineNOS

··NONOnNOSnNOS

Exocrine secretion Endocrine secretion

.NO i O2.-, rapidno reaguju. Konstanta brzine ove reakcije je 3 puta

veća od konstante brzine reakcije dismutacije O2.-, katalizovane SOD.

Km 2x109 M-1s-1

Km 6.7x109 M-1s-1

Redoks status je specifično moduliran primenom sintetskogRedoks status je specifično moduliran primenom sintetskogmimetika superoksid dismutaze - [MnII(pyane)Cl2]

.

Heptakoordinovani Mn-makrociklični SOD mimetik [MnII(pyane)Cl2]

Experimental design

Untreated

[Mn(pyane)X2]

Non-diabetic

Mill Hillrats

[Mn(pyane)X2](5mg/kg)

Untreated

Diabetic

[Mn(pyane)X2](5mg/kg)

Diabetic(aloxan, 120 mg/kg)

(5mg/kg)

Beneficial effects of SOD mimics in pancreas of diabetic animals

100 Control100

tion

(mm

ol /

l)

Control

Diabetes

Diabetes + SOD mimic

50ul

in c

once

ntra

tControl level

01 2

Insu

Control Diabetes Diabetes + SOD mimic

Beneficial effects of SOD mimic in pancreas of diabetic animals – Vascularity

Control Diabetes Diabetes + SOD mimic

Beneficial effects of SOD mimics in pancreas of diabetic animals– NO metabolites -

Beneficial effects of SOD mimics in pancreas of diabetic animals– NO metabolites -

Nit it h lNitrite S-nitrosothiols

cGMP eNOS mRNA level

I

KontrolaTirodovmedijum

0h

Spermatozoidiič ih

j

normospermičnihpacijenata (30)

3h, 37°C %

Tirodov medijum Bez tretmana

6% CO2

Tirodov medijum 50 M SOD i tiksa 50 µM

[MnII(pyane)Cl2]SOD mimetik

[MnII(pyane)Cl2] povećava koncentraciju .NO u spermatozoidima

Kontrola Bez tretmana [MnII(pyane)Cl2]Kontrola Bez tretmana [Mn (pyane)Cl2]

Diamino-fluorescein-2-diacetat (Daf2-DA)

Mitochondrial functionality determines sperm quality

spermatogeneza

pokretljivost

spermatogeneza

hiperaktivacija

kapacitacijakapacitacija

vezivanje zazona pelucidu

Ca 2+

signaling

sintezaproteina

zona pelucidu

akrozomalnareakcija

fuzija saoocitom reakc jaoocitom

Sousa et al., PLos One, 2011.

Kontrola Bez tretmana [MnII(pyane)Cl2]

II Fragmentacija - najuočljiviji defekt ranih in vitro embriona,jasan i prihvaćen indikator lošeg kvaliteta embriona

K NF VF

sazrevanjep pt

< 30% > 50%

joociteapoptoza

ATPROS/RNSfragmentacija fertilizacija

razvoj ranogembriona

implantacija

Distribucija mitohondrija u blastomerama preimplantacionih embriona(MT-G)( )

Embrion bez fragmentacije Delimično fragmentisan embrionEm n z f gm n j D m n f gm n n m n

Blastomere delimično fragmentisanih preimplantacionih embrionakaraterišu se poremećenom, nejednakom distribucijom mitohondrija i .NO

Distribucija mitohondrija, MT-G

Distribucija .NO, Daf2-DA

TEM

eNOSiNOS

nNOS

TEM

Lokalizacija nNOS u mitohondrijama koje šse grupišu u klastere i povezuju sa

membranom vakuola obrazujućistrukture koje svedoče o njihovojeliminaciji fizičkom ekstruzijomj j

potvrđuje uključenost NO u fragmentaciji

Imunocitohemijska detekcija izoformi NOS u fragmentisanim embrionima

B e o g r a d s k a š k o l a f i z i o l o g i j e

1910.

Ivan Đaja1884 – 1957.

University of BelgradeInstitute for Biological Research

Dr Biljana BuzadžićDr Vesna OtaševićDr Ana StančićDr Aleksandra Janković

Faculty of BiologyCenter for Electron Microscopy

Milica Vučetić, PhD student

Center for Electron Microscopy

Dr Aleksandra KoraćKsenija Veličković, PhD studentMilica Markelić PhD studentMilica Markelić, PhD studentIgor Golić, PhD student

Anita Lazarević, BScMaja Bogdanović, BSc

University of Erlangen –Nurnberg GermanyNurnberg, Germany

Dr Ivana Ivanović-BurmazovićDr Miloš Filipović