Credit SeminarMOLECULAR ASPECTS OF

HYPOXIAL STRESS IN LIVESTOCK ANIMALS

By: Dr Wani Ahad M.V.Sc Scholar Animal

Biotechnology

The term “stress” was borrowed from the field of

physics by Hans Selye (Hans Selye, 1926) And has been widely used in biology to describe a

set of physiological and behavioural changes elicited by adverse stimuli

He proposed that “stress is a non-specific response of the body to any change”

Stress

In 1929, Cannon described stress as the

sympatho-adreno medullary (SAM) system attempt to regulate homeostasis when threatened by a variety of adverse stressors

(Walter Cannon, 1929)

Is the condition “where the environmental demand exceeds the natural regulatory capacity of an organism’’

(Bruce McEwen and Jaap Koolhaas, 2011)

Stress

HISTORY

Bernard recognized that the stability of the milieu interieur depended on ensembles of compensating mechanisms 

(Bernard, 1878)

Fifty years later, Walter Cannon (1929) introduced the term “homeostasis” to describe the dynamic, interactive nature of these mechanisms in maintaining the stability of the internal environment

Endocrine and neuroendocrine events proceed in an interdependent manner to

regulate multiple, variable stress responses, each unique, but influenced by previous events

Summers (2001)

Thermal stress on livestock particularly cattle and buffaloes decreases oestrus expression and conception rate

Upadhya et al., (2007)

HISTORY

ABIOTIC Environmental Changes in temperature (Heat or Cold) and relative humidity Ventilation High altitude(Hypoxia)

Managemental Fear

STRESS FACTORS

Social Isolation High population density Mixing Pathology Pain

Feeding Hunger Thirst

STRESS FACTORS

BIOTIC Diseases: Bacterial Viral Fungal Parasitic

STRESS FACTORS

Stressful events can activate the Hypothalamo-Pituitary-Adrenal

(HPA) axis or sympatho-adrenomedullary (SAM) system

The short term responses are produced by The Fight or Flight Response via SAM pathway and long term responses via HPA pathway

HPA axis increases the release of Corticotrophin Releasing Hormone (CRH) from the hypothalamic paraventricular nucleus

(Campagne, 2006)

General pathways involved in stress

Stress pathways

(HPA)Hypothalamus (PVN)

CRH

Ant.pituitary

ACTH

Cortisol

Release of Neurotransmitters

(i.e. NE, cholecystokinin,

serotonin)-

Adrenal Gland

Catecholamines

Stress

event

+

Energy

Metabolism

High Altitude Hypoxia

Is the reduction of oxygen supply to a tissue below

physiological levels despite adequate blood perfusion to the tissue

(Illingworth et al., 2014)

Is a condition in which the body or a region of the body is deprived of adequate oxygen supply

(Woorons & Xavier, 2014)

Hypoxia (Hypoxiation or Anoxemia)

Low partial pressure of oxygen in the blood

a) Low oxygen inspired air e.g high altitude

b) Inadequate ventilation due to lung disease or depression of breathing by drugs

c) Defective transfer of oxygen from lung alveoli to blood

Causes

Low content of oxygen in the blood due to inadequate or

abnormal haemoglobin e.g anemia

Failure of the heart and circulation to deliver an adequate oxygen supply to the tissues, even though the content in the blood may be normal

Poisoning of cells so that they cannot use the oxygen delivered to them

Causes

Generalized hypoxia affecting the whole body

Local hypoxia affecting a particular region of the body

Acute hypoxia a sudden or rapid depletion in the availability of oxygen at the tissue level

Chronic hypoxia a usually slow, insidious reduction in tissue oxygenation

Classification

Anemic hypoxia is due to reduction of the oxygen-

carrying capacity of the blood due to decreased total hemoglobin or altered hemoglobin constituents

Histotoxic hypoxia is due to impaired use of oxygen by tissues

Ischemic hypoxia is the local deficiency of arterial blood in an organ

Types

Hypoxic hypoxia is due to insufficient oxygen reaching

to the blood

Stagnant hypoxia due to the failure to transport sufficient oxygen because of inadequate blood flow e.g Hypo-volumic shock

Embolic hypoxia due to an emboli in the blood vessels e,g air,blood clot etc

Types

Hypoxia Inducible Factors (HIFs) are important

transcription factors in the cellular adaptation to hypoxia by regulating different sets of genes involved in angiogenesis, metabolism and cell homeostasis

(Semenza and Wang, 2011)

They are heterodimeric transcription factors consisting of two structurally related subunits, one is an oxygen sensitive HIFα subunit (HIF-1α , HIF-2α or EPAS1 and HIF3α)

Counter Mechanism

and the other is the stable subunit, HIF-1β/ARNT-subunit (Aryl

hydrocarbon Receptor Nuclear Translocator) (Wang et al., 1995)

HIF-1α is expressed in all cells

HIF2α and HIF3α are selectively expressed in certain tissues, including vascular endothelial cells, type II pneumocytes, renal interstitial cells & liver parenchymal cells

(Bertout et al., 2008)

Hypoxia Inducible Factor

HIF1α and HIF1β structures

774/789 aaHIF-1β/ARNT

bHLH A PAS B

826 aaHIF-1α bHLH A PAS B TAD

CIDTADN

NLS-N NLS-C

Protein Expression as a Function of [O2]

Oxygen ConcentrationRela

tiv e

HIF

-1 E

x pre

ssio

n

HIF-1 expression increases exponentially when O2 concentration decreases. The curve shows a point of inflection around 4-5% O2, which is the O2 concentration in normal human tissues (Semenza GL. 1997)

Conserved proline residue in HIF-1α are hydroxylated by

prolyl hydroxylase/PHD (oxygen dependent) (Ivan et al., 2001) In normal conditions hydroxylation of proline causes the

binding of von Hippel-Lindau tumor suppressor (VHL) protein by an E3 ubiquitin ligase

(Kaelin and Ratcliffe, 2008) The binding leads to the ubiquitination of HIF-1α &

degradation by proteasome (Giaccia A J et al., 2004)

Mechanism

Under hypoxic conditions prolyl hydroxylase is not activated HIF-1α accumulates and translocates into nucleus

In the nucleus, it binds to HIF-1β through their HLH and PAS domains forming HIF-1 and binds to HRE present in target genes (Mole et al., 2009; Xia et al., 2009)

Mechanism

HIF-1 is a heterodimer

HIF-1

hypoxia

HIF-1

HREHIF-1

Pol IIcomplexCBP/p300

Angiogenesis (VEGF)

Glucosemetabolism

Cellproliferation

Helps normal tissues as well as tumors to survive under hypoxic conditions

Stimulates lipid storage and inhibits lipid catabolism through β-oxidation (Bostrom et al., 2006)

HIF1α and HIF2α can modulate the expression of cytochrome c oxidase isoforms so as to maximize efficiency of the ETC (Gordan et al., 2007)

Role of HIF-1

Regulate angiogenic genes such as vascular endothelial growth factor (VEGF) (Manalo et al., 2005) HIF activation has been observed in the tissue from patients with inflammatory conditions such as arthritis, artherosclerosis, and autoimmune diseases (Nizet and Johnson, 2009) So far, more than 40 target genes have been found to be regulated by HIF-1

These genes can be classified into 3 main groups:

Role of HIF-1

HIF-1HYPOXIA

Vascular Permeabili

tyVEGF

VEGFR-1 Vasodilation

Nitric oxide synthases

Endothelial SproutingAngiopoietin-1

Proliferation &

migration of

endothelial cellsVEGF PGF

Inhibitory Factors

Angiopoietin-2

Extra Cellular Matrix

Matrix Metalloprotein

ases

Erythropoeitin (EPO) Nitric oxide synthase (NOS) Transferrin Transferrin receptor Vascular endothelial growth factor (VEGF) VEGF receptor -1 (Skuli et al., 2009)

HIF-1 Target Genes

Group 1: O2 Delivery

Hexokinase Phosphofructokinase Aldolase 3-Phospho Glyceraldehyde dehydrogenase Phosphoglycerate kinase Enolase (ENO) Pyruvate kinase Glucose transporter 1 Lactate dehydrogenase (Gordan et al.,

2007)

Group 2: Glucose/Energy Metabolism

HIF-1 Target Genes

Insulin-like growth factor 2 (IGF-2) IGF binding protein 1 IGF binding protein 3 P21/WAF1/CIP1 p35srj

Group 3: Cell Proliferation/Viability

HIF-1 Target Genes

Symptoms Signs

Dyspnea Restlessness Anorexia Confusion Agitation Headache Tremor Nausea Fatigue

Respiratory distress Cyanosis Tachypnea Tachycardia Cardiac arrhythmias Hypertension Hypotension Lethargy Coma

Impacts of Hypoxia

Brisket disease: Subcutaneous accumulation of fluid under the abdomen,brisket,neck and jowl

(John H Newman et al., 2011) Hypertrophy of myocardium Valvular incompetency Polycythemia Polypnoea

Hypoxia induced oxidative stress

for short or long time periods affects corpus luteum development and function, leading to the decreased sheep fertility at high altitude

(Parraguez VH et al., 2013)

Respiratory alkalosis

Impacts of Hypoxia

Cyanosis bluish tinge to the

skin, lips, and nails Acute mountain sickness

accompanied by loss of appetite, nausea, vomiting, fatigue, weakness, irritability or trouble sleeping

(Hall D.P et al., 2014)

Impacts of Hypoxia

High-altitude pulmonary

edema (HAPE) usually develops with in 24 to 96 hours 

(Kleinsasser et al., 2003)

High-altitude cerebral edema (HACE) is a rare but potentially fatal condition

(Patir H et al., 2012)

Impacts of Hypoxia

Pregnant animals are at high risk Premature labor Small birth weights The average survivability of these breed has been

reported to be 60% Inverse correlation between hypoxic zone & sperm concentration Decrease sperm motility (Sokol, 2006)

Impacts of Hypoxia

Early embryonic loss in livestock (Reynolds, 2005) Cortisol alters oxytocin receptor expression (Champagne, 2006) Reduced oocyte quality in cattle (Rocha, 2003) Heat stress reduces the developmental ability of embryos (Rutledge, 2001 )

Impacts of Stress

To limit the impact of extreme climatic events

Genetic selection for breeds resistant to the extreme climatic conditions

Optimisation of gradual shift of flock from one altitude to another

Careful management and well designed housing at the level of the farming system for different altitudes are important in achieving the optimum animal performance

Conclusion

THANK

YOU