pigs as models for metabolic research handout · 2020-05-26 · blood volume pig total blood...

Pigs as models formetabolic research 

Simone Renner

Chair for Molecular Animal Breeding andBiotechnology

LMU Munich, Germany

Outline

• Animal model pig – comparative aspects• In vivo assessment of glucose homeostasis in the pig

• Porcine models for obesity, metabolicsyndrome, diabetes mellitus – different approaches

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

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

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

Animal model pig –comparative aspects

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

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)

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

Historyof GMof pigs

Dmochewitz & Wolf, Anim Front 2015

Geneticmodificationof pigs via somatic cellnuclear transfer

Kurome et al., Methods Mol Biol 1222, 37-59 (2015)

In vivo assessment of glucosehomeostasis in the pig

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

Auricular vein

Jugular vein (external/internal)

Placement of permanent catheters –auricular vein

Placement of permanent catheters –jugular vein/carotid artery

Catheter placement

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

Methods for the evaluation of glucose homeostatis in the pig

Variables affecting glucose homeostasis

Zoological variablesBreedSexAgePhysiological statusBody composition

Experimental variablesFasting durationTime of the dayStress

Available pig breeds

Minipig breeds Domestic pig breeds

GöttingenOssabaw

Yucatan

German Landrace

Iberian

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

Glucose tolerance test (GTT)

Hyperinsulinemic‐euglycemic clamp

Hyperinsulinemic‐euglycemic clamp

Porcine models for obesity, metabolic syndrome, diabetesmellitus – different approaches

Porcine model for themetabolic syndrome/diabetes

Surgical Chemical Geneticmodification

Dietaryintervention(Pancreatectomy) (Streptozotocin)

Example 1 Permanent diabetes in transgenic pigs expressing

the mutant insulin C94Y

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

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

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

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

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

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

Ede looking forward to his insulin shot

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

Altered ultrastructural morphology of pancreaticβ‐cells in INSC94Y transgenic pigs

Renner et al., Diabetes. 2013 May;62(5):1505‐11

Thank you for your attention!