pigs as models for metabolic research handout · 2020-05-26 · blood volume pig total blood...
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Pigs as models formetabolic research
Simone Renner
Chair for Molecular Animal Breeding andBiotechnology
LMU Munich, Germany
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Outline
• Animal model pig – comparative aspects• In vivo assessment of glucose homeostasis in the pig
• Porcine models for obesity, metabolicsyndrome, diabetes mellitus – different approaches
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The translational pathway
Structural biology Modeling Biobanking -OMICS
Genomics Transcriptomics Proteomics Metabolomics
Theranostics Imaging technologies Preclinical models
• Fly, Fish, Mouse• Pig, Sheep• Dog, NHP
Basicresearch
Industry
Knowledge Market value
Publications,Impact factors Patents,
Businessdevelopment
Publicfunding
Venture capital,Industry
G. Wess, 2008
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The productivity crisis in pharmaceutical R&D
Fabio Pammolli, Laura Magazzini and Massimo Riccaboni
Trends in attrition rates* of drug development projects started between 1990 and 2004 in the United States, Europe and Japan. *rate of loss of drug candidates during development
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Non-rodent mammalian models may bridge the gapbetween proof-of-concept studies and clinical trials
G A T C A A T G TC T A G T T A C A
G A T C A T T G TC T A G T A A C A
G A T C T A T G TC T A G A T A C A
Human patients
GWA studies
Pathophysiology
Proof-of-concept Translational model
Efficacy BiomarkersSafety
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Animal model pig –comparative aspects
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The perfect animal model
Desirable criterium Rodent Pig NHP
Best possible similarity to humans Limited Good Good
Ethically accepted Yes Yes No
High hygienic status Yes Yes withlimitations
Yes withlimitations
Low maintenance costs Yes No No
Good animal compliance Limited Yes Yes
Sufficient sample material Limited Good Limited
Good repoduction cirteria Yes Yes No
Genetic modification established Yes Yes Yes
http://whichboxmedia.comRenner et al. Theriogenology 2016
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Relevant advantages of porcine modelsfor diabetes research
• Similarities with humans in structure andfunction of the GI tract (monogastric omnivore, symbiotic micro-organisms play minor role, similar transit time and digestive effectiveness)
• Similar structure of the pancreas (size, shape, position, endocrine cell distribution, beta-cell content)
• Similar insulin structure
• Similar blood glucose levels
• Similar pharmacokinetics after subcutaneous administration ofcompounds
• No brown adipose tissue
-10 0 10 20 30 40 50 60 70 80 900
10
20
30
40
50 wt (n=6) tg (n=5)
Time (minutes)
Insu
lin (µ
U/m
l)
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Favorable reproduction criteriain the pig
Sexual maturity: 3 – 5 months (Minipig) 6 – 7 months (Domestic pig)
Gestation length: 114 days
Litter size: 6 – 18
~ 2.5 litters per year
http://www.supercoloring.com
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Historyof GMof pigs
Dmochewitz & Wolf, Anim Front 2015
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Geneticmodificationof pigs via somatic cellnuclear transfer
Kurome et al., Methods Mol Biol 1222, 37-59 (2015)
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In vivo assessment of glucosehomeostasis in the pig
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Accessible blood vessels in the pig
Blood vessel Application Permanent catheter
Auricular vein Single or frequentsampling Yes
Jugular vein Single or frequentsampling Yes
Carotic artery Frequent sampling Yes
Femoral vein Frequent sampling Limited
Femoral artery Frequent sampling Limited
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Auricular vein
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Jugular vein (external/internal)
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Placement of permanent catheters –auricular vein
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Placement of permanent catheters –jugular vein/carotid artery
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Catheter placement
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Blood volume pig
Total blood volume: 6.5% of the body weight*: Single blood collection$: over a max. period of 2 weeks
PigAbsolute bloodvolume
Max. bloodsampling *
Daily bloodsampling $
Adult Minipig(35 kg)
2300 ml 230 ml 23 ml
Young adult domestic pig (200 kg)
13000 ml 1300 ml 130 ml
Neonate Minipig(450 g)
29 ml 2.9 ml ‐
Neonate Domesticpig (1500 g)
97 ml 9.7 ml ‐
Society for Laboratory Animal Science, 2009
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Methods for the evaluation of glucose homeostatis in the pig
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Variables affecting glucose homeostasis
Zoological variablesBreedSexAgePhysiological statusBody composition
Experimental variablesFasting durationTime of the dayStress
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Available pig breeds
Minipig breeds Domestic pig breeds
GöttingenOssabaw
Yucatan
German Landrace
Iberian
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Glucose tolerance test (GTT)
Fasting (12‐18h)
Glucose bolus
Fasting glucose& Insulin levels
15 30 45 60 90 120 150 180
Oral 2 g Glc./kg BW
Mixed‐meal2 g Glc./kg BW + food
Intravenous0.3/0.5 g Glc/kg BW
Blood collection at defined intervals
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Glucose tolerance test (GTT)
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Hyperinsulinemic‐euglycemic clamp
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Hyperinsulinemic‐euglycemic clamp
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Porcine models for obesity, metabolic syndrome, diabetesmellitus – different approaches
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Porcine model for themetabolic syndrome/diabetes
Surgical Chemical Geneticmodification
Dietaryintervention(Pancreatectomy) (Streptozotocin)
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Example 1 Permanent diabetes in transgenic pigs expressing
the mutant insulin C94Y
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Stoy et al., Proc Natl Acad Sci U S A. 2007 Sep 18;104(38):15040
Insulin gene mutations as a cause of permanent neonatal diabetes mellitus
> 50 mutations within the insulin gene in humans
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INSC94Y („Akita mutation“)
Cysteine Tyrosine
wt Akita
DIABETES 2013
hu-INS MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCNpo-INS MALWTRLLPLLALLALWAPAPAQAFVNQHLCGSHLVEALYLVCGERGFFYTPKARREAENPQAGAVELGGGLG--GLQALALEGPPQKRGIVEQCCTSICSLYQLENYCNmu-INS1 MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPKSRREVEDPQVEQLELGGSPG--DLQTLALEVARQKRGIVDQCCTSICSLYQLENYCNmu-INS2 MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQVAQLELGGGPGAGDLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN
Signal peptide B-chain Connecting peptide A-chain
hu-INS MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCNpo-INS MALWTRLLPLLALLALWAPAPAQAFVNQHLCGSHLVEALYLVCGERGFFYTPKARREAENPQAGAVELGGGLG--GLQALALEGPPQKRGIVEQCCTSICSLYQLENYCNmu-INS1 MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPKSRREVEDPQVEQLELGGSPG--DLQTLALEVARQKRGIVDQCCTSICSLYQLENYCNmu-INS2 MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQVAQLELGGGPGAGDLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN
Signal peptide B-chain Connecting peptide A-chain
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Mutant insulin molecules – mode of action
ER
Unfoldedproteins
TranslationalAttenuation
Induction of ER Chaperones
(Bip etc)
Nucleus
Apoptosis
Cell death
ProteasomeDegradation(ERAD)
Araki et al., Exp Biol Med. 2003 Nov;228(10):1213‐7
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Elevated fasting blood glucose levels inan INSC94Y transgenic founder boar
40 60 80 100 120 140 160 180 200 2200
50
100
150
200
250
300
350
400
450
5009725972697279728974597469747
Age (days)
Glu
cose
(mg/
dl)
Renner et al., Diabetes. 2013 May;62(5):1505‐11
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Early onset diabetes depends on theINSC94Y : INS transcript ratio
0.0 0.3 0.6 0.9
F19747972597269727972897459746
F0
INSC94Y : INS transcript ratio
Renner et al., Diabetes. 2013 May;62(5):1505‐11
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Early on hyperglycemia in INSC94Y transgenic pigs
0 2 4 6 80
100
200
300
400
500 wt (n=4)tg (n=6)
non fasting
*
*** ***
Age (days)
Glu
cose
(mg/
dl)
A
4.5 mon
ths
wt tg0
2
4
6
8 wt (n=6)tg (n=6)
**
ddddddd
Insu
lin (µ
U/m
l)
8 da
ys
B
wt tg0
5
10
15
20 wt (n=4)tg (n=6)p=0.38
dddddd
Insu
lin (µ
U/m
l)
0 25 50 75 100 125 1500
100
200
300
400
500 wt (n=7)tg (n=9)
non fasting fasting
******
** ****** ***
*****
***
***
Age (days)
Glu
cose
(mg/
dl)
Renner et al., Diabetes. 2013 May;62(5):1505‐11
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Ede looking forward to his insulin shot
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Progressive β‐cell loss in INSC94Y transgenic pigs
wt tg0
50
100
150
200 wt (n=4)tg (n=4)
p=0.682
V bcell,Pan
V (
-cel
l,Pan
) (m
m3 )
wt tg0
500
1000
1500
2000
***
wt (n=7)tg (n=7)
dddddV
( -c
ell,P
an) (
mm
3 ) -74%
Renner et al., Diabetes. 2013 May;62(5):1505‐11
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Altered ultrastructural morphology of pancreaticβ‐cells in INSC94Y transgenic pigs
Renner et al., Diabetes. 2013 May;62(5):1505‐11
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Thank you for your attention!