zinc metabolism in monogastric...

Hans-Eisenmann-Zentrum

for Agricultural Science

TUM School of Life Sciences Weihenstephan

Technische Universität München

Chair of Animal Nutrition

Daniel Brugger

Chair of

Animal Nutrition

Zinc metabolism in monogastric animals

Context: Why does monogastric zinc metabolism matter?

Mechanisms of molecular zinc homeostasis

Present knowledge on zinc homeostatic adaption to varying supply

Short term vs. long term shortage in zinc supply and

methodological considerations arising thereof

Outlook: Future challenges for zinc metabolism research

Chair of

Animal Nutrition

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Chair of

Animal Nutrition

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We have to supplement zinc to the diets in order to ensure

sufficient supply

ZnFe

Cu Mn

pH 6 - 7

Phytate-complex

reorganizes within the

small intestine and

binds multivalent ions

Windisch and Kirchgessner 1999

Range of

phytate in

cereals

Study in 65Zn labeled adult

rats

R² = 0.98

28%

9%

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Animal Nutrition

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Zinc feeding significantly affects the zinc fluxes within animal

production chains

Zn2+

UBA 2004, Wuana and Okieimen 2011, Romeo et al. 2014







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Animal Nutrition

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Chair of

Animal Nutrition

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Most essential trace elements are transition metals

Weller et al. 2014

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Animal Nutrition

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Homeostasis maintains a steady state of free and total (bound)

metal ion load behind the gut barrier

body insideEquillibrium of metal load by regulating

uptake and efflux under varying conditions:

changes in metal supply levels

changes in metal demand

(physiological state)

M

M

M

M

M

M

M

M

M

M

M M

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Animal Nutrition

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Zinc homeostasis is achieved by fine-tuning of transmembrane fluxes

and intracellular zinc buffering and muffling Secretory granules,

vesicles…

Cell organelles

Plasma membrane

Gut lumen/ Extracellular space

Nucleus

Molecular Zn

Molecular Zn

Molecular Zn

Zn2+ Zn2+

Zn2+ Zn2+

Molecular Zn:

Zn binding enzymes

Zn sensors

Metallothionein….

Cousins 2009, Lichten and Cousins 2009, Colvin et al. 2010, Wang and Chou 2010,

Holt et al. 2012, Schweigel-Röntgen 2014, Blindauer 2015

Zn2+Zn2+

Zn2+

Zn2+

Zn2+

Zn2+

Zn2+

Zn2+

Zn2+ Zn2+

Molecular Zn

Zn2+Orchestrated regulation

of ionic Zn transport is

a key function

of homeostatic regulation

… and birds?

no gene sequence available for

ZIP1, ZIP2, ZIP4 and ZIP7

within published Gallus gallus

chromosome allignments…

Not existing? Incomplete alignment?

Competition with other trace elements

(e.g. iron, copper) for transport mechanisms

(e.g. DMT1)

non-regulated, non-saturable Zn2+

fluxes

ZnT (SLC30) family: ZIP (SLC39) family:

10 members: 14 members:

ZnT1 to ZnT10 ZIP1 to ZIP14

High specificity for Varying data regarding

Zn2+ ions ion specificity:

e.g.:

ZIP4 highly specific for Zn2+

ZIP14 Active transport

of Zn2+ and non-transferrin Fe2+

some ZIP´s transport Mn2+ in the

brain

At least 24 distinct genes are responsible for

regulated, saturable Zn2+ transport in mammals

Chair of

Animal Nutrition

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Highly diverse genetic architecture of zinc transport

Lichten and Cousins 2009; Schweigel-Röntgen 2014, NCBI 2015, ENSEMBL 2015

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Animal Nutrition

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Zinc transporters exhibit differences in tissue and subcellular distribution

Romeo et al. 2014

Pig: mRNA expression evident for all transporters exept for

ZnT3, ZIP10, 12, in Jejunum and Colon, ZnT1, 8, ZIP4 in

pancreas, ZIP10 in kidney

(Kaler and Prasad 2007, Sieber et al. 2013, Schweiger et al. 2013,

Pieper et al. 2015, Brugger et al. 2015, yet unpublished data)

Chicken: mRNA expression of ZnT1, 2, 5, 7, ZIP9,

13 in Duodenum, Jejunum and Ileum, ZnT5, 7,

ZIP9, 13 in Caecum, ZIP9 in B-Cells, ZnT1, 2, 5 in

pancreas, ZIP11 in muscle

(Yu et al. 2008, Jorg et al. 2010 Liao et al. 2012, Paris

and Wong 2013, Tamiguchi et al. 2014, Yin et al.

2015, Su et al. 2015, Lietal 2015, Troche et al. 2015)

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Animal Nutrition

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Not all zinc transporters respond to variations in zinc feeding/status

Modified according to Lichten and Cousins 2009

Abbreviations: ZnD, dietary Zn restriction; ZnE,

Dietary Zn excess; +, up-regulated; -, down-regulated

Zn status/feeding dependent

gene expression in

Pig:

jejunal ZnT 1, 2, 4, 9

jejunal ZIP 1, 3, 4, 5, 7, 8, 11, 13

colonic ZnT 1, 2, 8

colonic ZIP 2, 3, 4, 5, 7, 11, 14

(Brugger et al. 2015, yet unpublished data)

Chicken:

ZnT1, 2 and 5 in duodenum, jejunum, ileum

(Yu et al. 2008)

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Animal Nutrition

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ZnT and ZIP peptides transport Zn2+ ions in opposite directions

ZIP peptides transport

Zn ions to the cytosol

ZnT peptides transport

Zn ions away from the cytosol

Lichten and Cousins 2009; Schweigel-Röntgen 2014

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Animal Nutrition

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What is known on the regulation of „relevant“ intestinal Zn transporters (in

mammals)

Wang and Zhou 2010, Cousins 2010

ZIP4 : Main transporter from GIT lumen into the cell

Basline expression under sufficient and oversupply conditions

Increased expression under deficient conditions (Krüppel-like-factor 4)

ZnT1: Main transporter from GIT cells to the circulation

Expression correlates to intracellular free Zn2+ contents (MTF 1)

ZnT5: seems to transport zinc from the cell into the GIT lumen

potentially bidirectional

may serve as a detoxification mechanism

ZIP5: located at the basolateral membran of GIT cells

not expressed during deficiency but acute administration

(Pig: colonic ZIP5 upregulated in deficiency (Brugger et al. 2015, yet unpublished data)

may import oversupplied zinc from the blood into the cell

may serve as sensor of body zinc status

ZnT7: Imports zinc into the Golgi complex

Directly involved in Alkaline Phosphatase maturation

Potentially involved in regulation of cytosolic Zn2+ contents

Chair of

Animal Nutrition

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„Relevant“ zinc transporters within the gastrointestinal tract (of mammals)

Intestinal Lumen Blood

ZIP4 ZnT1

ZnT5

Golgi complex

ZnT7

ZIP5

Wang and Zhou 2010, Cousins 2010

??? ???

Other

compartments

???

???

Many more zinc transporters are present in the gastro-intestinal tract.

Their precise function and regulation is yet unknown.

Chair of

Animal Nutrition

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Regulation of ZIP4 and ZnT1 synthesis within entherocytes (of

mammals)

Cousins 2009, Lichten and Cousins 2009, Colvin et al. 2010, Wang and Chou 2010,

Holt et al. 2012, Schweigel-Röntgen 2014, Blindauer 2015

ZnT1ZIP4

Transcription factor Krüppel-like-factor 4

senses zinc status through a

yet unknown mechanism

and increaes ZIP4 transcription

at zinc deficiency

Furthermore, ZIP4 mRNA degradation

is decreased in zinc deficiency

Activation of MTF-1 by intracellular

free Zn2+ ions.

Chair of

Animal Nutrition

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Species differences in main zinc absorption sites in the GIT?

Holt et al. 2012, Romeo et al. 2014

ZIP4 and ZnT1 have been found in all parts

of the digestive tract of monogastric mammals!

Stomach Small Intestine Large Intestine

Little absorption has

been reported in:

Humans

Considered as main

absorption site:

Humans:

distal Duodenum or

proximal Jejunum

Chicken:

Ileum

Pigs:

Duodenum or Jejunum

Significant absorption has

been reportet in:

Rats

Pigs

(Ruminants)

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Animal Nutrition

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1st Short interim conclusion

What do we really know about molecular Zn homeostasis?

High diversity of Zn transport peptides in vertebrates

Zn transporters seem to be highly conserved over (mammal) species

Transporters can be grouped in regard to their transport direction

into cytosol

away from cytosol

Transporters differ in regard to tissue and subcellular distribution

Molecular zinc homeostasis is an orchestrated interplay between Zn transporters

and Zn acceptors/donators

Intestinal zinc aquisition is mainly due to luminal Zn2+ uptake by ZIP 4 and

serosal Zn2+ efflux by ZnT1

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Animal Nutrition

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1st Short interim conclusion

What are we still not knowing about molecular Zn homeostasis?

Precise role of all described Zn transporters in Zn homeostasis

Are there differences in transporter abundance, distribution and regulation

between mammal/vertebrate species (livestock species)?

What are the precise electrophysiological mechanisms of Zn transport?

What are the important Zn donators/acceptors (apart from Metallothionein)

within cells and how are their interactions with each other?

How does the organism orchestrates zinc homeostasis on level of

the whole system (within and between tissues)?

Are there status dependent changes in the intestinal main absorption site?



Zinc homeostatic adaption to varying supply




Chair of

Animal Nutrition

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Chair of

Animal Nutrition

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Trace elements can be grouped by their major homeostatic switch

absorption

Zn

Windisch 2002

body inside

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Animal Nutrition

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Absorption efficiency is a major switch of zinc homeostatic adaption

intestinal lumen

ZnZn Zn ZnZn

ZnZn

Zn

Zn Zn

Zn

Zn

Zn

Zn

Zn

Zn Zn Zn

Basal condition:

Basal activity of active transport mechanisms to compensate

basal Zn losses (secretion, excretion, surface)

Zn

Zn

Zn

urinary excrection

pancre

atic

/bili

ary

secre

tion

body inside

Chair of

Animal Nutrition

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Homeostatic adaption to dietary zinc deficiency

intestinal lumen

ZnZn Zn

Zn

Zn

Zn

Zn

Zn

Zn

Increase in active Zn (re)absorption efficiency at the gut barrier

(and renal reabsorption ???)

Limitation of endogenous faecal Zn losses (and renal secretion???)

Zn

Znurinary excrection

pancre

atic

/bili

ary

secre

tion

Zn

Zn

Zn

Zn

Zn Zn

Zn

Zn

Zn

???

Holt et al. 2012

??????

Chair of

Animal Nutrition

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Systemic Zn status dependent behaviour of ZIP4 and ZnT1 in

piglets

Brugger et al. 2014, yet unpublished data

0

0,5

1

1,5

2

2,5

3

27 37 47 57 67 77 87

Rela

tive i

nte

sti

nal

gen

e e

xp

ressio

n

[xfo

ldre

gu

lati

on

]

Feed zinc content [mg/kg]

Intestinal ZIP4 gene expression Intestinal ZnT1 gene expression

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Animal Nutrition

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Parameters of total Zn absorption reveal a breakpoint in response

behaviour above the requirement threshold

Weigand and Kirchgessner 1980, Windisch and Kirchgessner 1994

Growing rats

Adult rats

Chair of

Animal Nutrition

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The organism reduces endogenous zinc losses in response to a

decrease in true daily Zn absorption

Modified according to Weigand and Kirchgessner 1980, Windisch and Kirchgessner 1994

5.6 10.618.2

39

70

ppm diet. Zn

141

Growing ratsAdult rats

R² = 0.99

19

23

2937

46

58

73

92

ppm diet. Zn

114

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Animal Nutrition

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Urinary zinc exhibits no response over dietary zinc doses

High degree of regulation of renal Zn fluxes!


R² = 0,0002

4

4,5

5

5,5

6

0 20 40 60 80 100 120Uri

na

ry z

inc

ex

cre

tio

n [

µg

/d]

Dietary zinc content [mg/kg]

Adult rats

Growing rats: „…urinary zinc excretion remained rather constant regardless of the large differences

in zinc intake between groups.“ (Weigand and Kirchgessner 1980)

body inside

Chair of

Animal Nutrition

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Homeostatic adaption to non-toxic dietary Zn oversupply

intestinal lumen

ZnZn Zn

Zn

Zn

Zn

Zn Zn

Down-regulation of active transport from the GIT lumen

(and reabsorption in the kidney, respectively???)

Only reduced, non-regulated Zn influx occurs

Increased pancreatic Zn secretion (and renal excretion???)

Zn

urinary excrection

pancre

atic

/bili

ary

secre

tion

Zn

Zn

???

ZnZn

Zn

ZnZn

Zn

Zn Zn

ZnZn

Zn

ZnZn

Zn

ZnZn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn

Zn ZnZn

Zn Zn

???

???

Holt et al. 2012

Chair of

Animal Nutrition

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Non-toxic oversupply associated passive zinc influx

promotes jejunal zinc detoxification mechanisms in piglets

Martin et al. 2013

0

20

40

60

80

100

120

0

0,2

0,4

0,6

0,8

1

1,2

1,4

57 164 2425

Je

juna

lzin

cconte

nt[m

g/k

g]

Rela

tive g

ene

expre

ssio

n[x

fold

]


Jejunal ZnT1 (xfold) Jejunal ZIP4 (xfold) Jejunal Zn (mg/kg)

0

200

400

600

800

1000

1200

1400

1600

57 164 2425

Pan

cre

ati

c e

nzym

e a

cti

vit

y

[U/m

g p

rote

in]


Trypsin Chymotrypsin Lipase α-Amylase

Higher zinc accumulation was also accompanied by increased oxidative stress!

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Animal Nutrition

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The exogenous pancreas increases its secretory activity

under the condition of non-toxic zinc oversupply in piglets

Pieper et al. 2015

Chair of

Animal Nutrition

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Oversupplemented feed Zn amounts are directly transferred to

faeces through homeostatic counterregulation at the gut barrier


Study in 65Zn labeled adult rats

Chair of

Animal Nutrition

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2nd Short interim conclusion

Absorption efficiency reacts status dependent with an upregulation of active Zn

transport under zinc deficiency or, respectively, down-regulation at sufficient zinc

supply/oversupply

Endogenous faecal zinc losses decrease to an inevitable amount under the terms

of zinc deficiency whereas under oversupply conditions the exogenous pancreas

increases its secretory activity







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Animal Nutrition

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„State-of-the-art“ experimental design of

zinc deficiency trials:

Phase 1: Long term Zn depletion

(2 - 4 weeks)

Phase 2: Repletion with varying supply

Monitoring the physiological response

during repletion

Induction of zinc deficiency disease by „state-of-the-art“ experimental

approaches

Chair of

Animal Nutrition

33Windisch and Kirchgessner 1994, Windisch et al. 2003

Chair of

Animal Nutrition

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The physiological status may determine the magnitude of homeostatic

response

18.2

39

70

ppm diet. Zn

Growing rats Adult rats

19

37

73 ppm diet. Zn

87 124 151

70

107

132

25

106

191

183 350 425

Modified according to Weigand and Kirchgessner 1980, Windisch and Kirchgessner 1994

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Animal Nutrition

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Potential differences in feed zinc bioavailability between severely and

latent zinc deficient animals

Reduced slope in response

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Animal Nutrition

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Secondary metabolic imbalance is a result of zinc deficiency disease

Increased oxidative stress

Impaired immune functionImpaired digestive function

Reduced protein turnover

Cummings and Kovacic 2009, Eide 2011, Romeo et al. 2014, Maywald and Rink 2014

http://fre

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om

/2015/0

6/2

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Animal Nutrition

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3rd short interim conclusion

• The degree in Zn store depletion determines the magnitude

in homeostatic response

possible overestimation of feed Zn bioavailability

in severely deficient animals?

• Long-term models compare sick with healthy individuals

Bias in assessment of feed efficiency?

Bias in monitoring Zn assoicated metabolic function?

Chair of

Animal Nutrition

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Modelling latent zinc deficiency in weaned piglets – no visible signs

of zinc deficiency but dose/status dependent adaption of homeostasis

8 days

varying Zn supply

Brugger et al. 2014

Short-term vs. long-term shortage in supply – differences in response?

Chair of

Animal Nutrition

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>10d shortage in dietary Zn supply

Weigand and Kirchgessner 1980, Wedekind et al. 1992, Brugger et al. 2014

<10d shortage in dietary Zn supply

Bone Zn

AP activity

Short-term vs. long-term shortage in supply – differences in response?

Chair of

Animal Nutrition

40Windisch and Kirchgessner 1994, Brugger et al. 2014

Muscle Zn

Heart Zn

>10d shortage in dietary Zn supply <10d shortage in dietary Zn supply

Feed zinc content (mg/kg)rats

rats

piglets

piglets

Significant metabolic shifts in latent zinc deficient piglets Digestion

Chair of

Animal Nutrition

41Brugger et al. 2014

Zn-status-dependent decrease in digestive capacity!

Significant metabolic shifts in latent zinc deficient piglets

Antioxidative capacity

Chair of

Animal Nutrition

42Brugger et al. 2014

Cardiac α-tocopherol content (ng/mg)Cardiac H2O2-detoxification

efficiency (mU/mg)

Reduction in parameters of anti-oxidative defense!

High correlation to Metallothionein

Gene expression (r > 0.90)

Chair of

Animal Nutrition

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4th short interim conclusion

Short-term experimental approaches allow an early view on

zinc associated metabolism

Many response patterns currently lack appropriate explanation

and foster the need for follow-up investigations

Even short-term insufficiencies in alimentary zinc supply can

cause significant metabolic shifts







Chair of

Animal Nutrition

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Chair of

Animal Nutrition

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Are species dependent differences in regard to zinc

homeostatic adaption evident?

How does tissues sense and communicate zinc status in order to

maintain whole body homeostasis?

What are the early events in the zinc homeostatic response?

How can we measure true zinc absorption in large animals under

basal conditions?

Chair of

Animal Nutrition

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Thank you!

zinc metabolism in monogastric...

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