Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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
2
Chair of
Animal Nutrition
3
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%
Chair of
Animal Nutrition
4
Zinc feeding significantly affects the zinc fluxes within animal
production chains
Zn2+
UBA 2004, Wuana and Okieimen 2011, Romeo et al. 2014
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
5
Chair of
Animal Nutrition
6
Most essential trace elements are transition metals
Weller et al. 2014
Chair of
Animal Nutrition
7
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
Chair of
Animal Nutrition
8
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
9
Highly diverse genetic architecture of zinc transport
Lichten and Cousins 2009; Schweigel-Röntgen 2014, NCBI 2015, ENSEMBL 2015
Chair of
Animal Nutrition
10
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)
Chair of
Animal Nutrition
11
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)
Chair of
Animal Nutrition
12
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
Chair of
Animal Nutrition
13
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
14
„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
15
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
16
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)
Chair of
Animal Nutrition
17
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
Chair of
Animal Nutrition
18
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?
Context: Why does monogastric zinc metabolism matter?
Mechanisms of molecular zinc homeostasis
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
19
Chair of
Animal Nutrition
20
Trace elements can be grouped by their major homeostatic switch
absorption
Zn
Windisch 2002
body inside
Chair of
Animal Nutrition
21
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
22
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
23
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
Chair of
Animal Nutrition
24
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
25
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
Chair of
Animal Nutrition
26
Urinary zinc exhibits no response over dietary zinc doses
High degree of regulation of renal Zn fluxes!
Windisch and Kirchgessner 1994
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
27
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
28
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
]
Feed zinc content [mg/kg]
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]
Feed zinc content [mg/kg]
Trypsin Chymotrypsin Lipase α-Amylase
Higher zinc accumulation was also accompanied by increased oxidative stress!
Chair of
Animal Nutrition
29
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
30
Oversupplemented feed Zn amounts are directly transferred to
faeces through homeostatic counterregulation at the gut barrier
Windisch and Kirchgessner 1995
Study in 65Zn labeled adult rats
Chair of
Animal Nutrition
31
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
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
32
„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
34
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
Chair of
Animal Nutrition
35
Potential differences in feed zinc bioavailability between severely and
latent zinc deficient animals
Reduced slope in response
Chair of
Animal Nutrition
36
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
edom
-muse.c
om
/2015/0
6/2
2/w
hic
h-c
om
es-f
irst-
plo
t-vs
-chara
cte
rs/
Chair of
Animal Nutrition
37
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
38
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
39
>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
43
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
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
44
Chair of
Animal Nutrition
45
Outlook: Future challenges for zinc metabolism research
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
46
Thank you!