rathkolb and rozman / phenotyping glucose metabolism

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Glucose metabolism and phenotyping tests for disturbed glucose metabolism

Clinical Chemistry & Hematology

Birgit Rathkolb

Head Clinical Chemistry and Hematology Screen GERMAN MOUSE CLINIC

Jan Rozman

Head Energy Metabolism Screen GERMAN MOUSE CLINIC

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Known/Expected Phenotypes


Modified from: Keith N. Frain, Metabolic Regulation, 3rd edition, Wiley-Blackwell 2010

Incretins (GLP-1, GIP)

Overview Glucose Metabolism

Blood cells

Glycerol

Lactate + Pyrovate

Alanine

FFA

Kidneys

Gluconeo-genesis

Blue arrows: Pattern of glucose metabolism after carbohydrate uptake Red arrows: Energy substrate mobilisation under fasting condition Green arrows: Incretin effects

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Glucose Tolerance Test (GTT): General workflow


Start

Removal of food

T0 T15 T30 T60 T120 fasting period

Basal glucose level

Glucose challenge

i.p. or oral

Workflow GTT

Device used:

Hand-held Glucometer Parameters analyzed:

Basal fasting glucose level,

Area under the curve

Body

weight

Glucose level

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IpGTT: Protocol variations (fasting period)

1. Fasting period - Short food withdrawal (3-5 hours) >> emptying of

stomach and proximal gut >> low/no glucose uptake, low insulin level.

- Medium food withdrawal (6-8 hours) >> switch from predominant glycogenolysis to lipolysis for energy substrate allocation.

- Overnight food withdrawal (16-18h) >> challenge of lipolysis / gluconeogenesis capacities and energy saving mechanisms. Body weight loss as additional parameter.

Weight difference

Group

Over night fed_morning 5 hour fast fed_ afternoon

g

-5

-4

-3

-2

-1

0

1

2

3

Glucose

Group


mm

ol/l

2

4

6

8

10

12

14

16

18

20

22

NEFA

Group


mm

ol/l

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Glycerol

Group


mm

ol/l

0,05

0,10

0,15

0,20

0,25

0,30

0,35

Body weight difference

ON f fed 5-6h f fed morning 8 am afternoon 2 pm

Glucose level Glycerol level

NEFA level




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IpGTT: Protocol variations (fasting period)

Andrikopoulos et al. 2008, Am J Physiol Endocrinol Metab

IpGTT results (plasma glucose and insulin curves) comparing mice fed high fat diet (white) to mice fed control diet (black) using protocols with different fasting durations before testing.

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IpGTT: Protocol variations


2. Glucose dosage - Dosage according to body mass (1-2 g/kg) >> possible effects of body composition >> higher dose > stronger effect - Dosage according to lean mass (1-2 g/kg) >> requires previous analysis of body composition - Fixed dose (e.g. 50 mg/mouse) >> possible

effect of body weight/especially lean mass

Andrikopoulos et al. 2008, Am J Physiol Endocrinol Metab

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IpGTT: Protocol variations (glucose application route)

3. Glucose application route Commonly used in mice: - Intraperitoneal injection - Oral gavage

Rather uncommon: -intravenous application



Blood cells

Glycerol

Lactate + Pyrovate

Alanine

FFA

Kidneys

Gluconeo-genesis

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IpGTT: Protocol variations (glucose application route)

Clinical Chemistry & Hematology Andrikopoulos et al. 2008, Am J Physiol Endocrinol Metab

Pacini et al. 2013, J of Diabetes Research

Oral application: Incretin effects stimulate insulin secretion – lower peak glucose levels i.v. – application: Maximum glucose level reached at moment of injection >> immediate maximum insulin response >> short intervals required to follow glucose clearance

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Ip Glucose Tolerance Test (IpGTT): Standard procedure IMPC


17:00

Removal of food

T0 T15 T30 T60 T120 16-18 hours

Basal glucose level

Glucose i.p.

2 g/kg BW

Workflow IpGTT

Device used:

Handheld Glucometer

Parameters analyzed:

Body Weight loss by fasting,

basal fasted glucose level,

Area under the curve (T0-T30,

T30-T120)

Body

weight

Glucose level

Rozman et al. 2015, Glucose Tolerance Tests for Systematic Screening of Glucose Homeostasis Curr. Protoc. Mouse Biol. 5: 65-84

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IpGTT: Examples from IMPC Screen


Body mass loss vs. Initial body mass Glucose levels during IpGTT

Example for severely impaired glucose tolerance from the IMPC screen.

Cpetm1b: Knockout of Carboxypeptidase E

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IpGTT: Examples from IMPC Screen


Same data as displayed on the IMPC webpage. Line Cpetm1b: Knockout of carboxypeptidase E >> cleaves pro-insulin to release biologically active insulin

Insulin level Levels of mutants above limit of quantification (1500 pg/mL). High insulin levels in mutants, because test kit also detects pro-insulin.

Cpetm1b: Knockout of Carboxypeptidase E

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IpGTT: Examples from IMPC Screen)


Body mass loss vs. Initial body mass Glucose levels during IpGTT

Gfpt2tm1b : Knockout of Glutamine fructose-6-phosphate transaminase 2 Controls glucose flux to the hexoamine biosynthetic pathway >> one of two genes coding for the rate limiting enzyme of this pathway; variants associated with T2DM and diabetic nephropathy in humans.

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IpGTT: Approaches to data analysis


Glucose T0 AUC 0-30 AUC 30-120

Basal glucose level and Total AUC – above zero level Total AUC – above basal level Partial AUC values (above basal level or above zero) Glucose level increase (Maximum – basal level) Steepness of increasing and/or decreasing curves between single time points Glucose levels at single time points

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Clinical Chemistry & Hematology Clinical Chemistry & Hematology



Blood cells

Glycerol

Lactate + Pyrovate

Alanine

FFA

Kidneys

Gluconeo-genesis

Glucosuria

Impaired glucose tolerance: >> impaired insulin secretion? or >> biologically ineffective insulin? or >> impaired insulin signalling/insulin resistance? Improved glucose tolerance: >> impaired glucose resorption ? or >> increased glucose clearance ? - Increased insulin secretion - Increased insulin sensitivity - Improved insulin signalling - glucose loss by glucosuria?

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Insulin Tolerance Test (ITT) : Test for insulin sensitivity


Start

Removal of food

T0 T15 T30 T60 T120 3-4 hour fasting

i.p. Insulin injection

0,2-4,0 U/kg

Workflow ITT

Device used:

Hand-held Glucometer




Body

weight

Glucose level

Insulin dose has to be adapted according to insulin sensitivity of the genetic background of mice tested.

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Insulin Tolerance Test: Example from literature


Bowman et al. 2016, Mol. Metab. Example of ITT-results in ACSL5-KO mice

The degree of blood glucose level decrease in response to insulin injection reflects whole body insulin sensitivity. KO-mice (KO) show improved insulin sensitivity compared to floxed control mice (FL).

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Pyruvate or Lactate Tolerance Test (PTT / LTT)


Start

Removal of food

T0 T15 T30 T60 T120 6h – overnight

fasting

i.p. pyruvate or lactate

injection (1- 2 g/kg)

Workflow PTT/LTT

Device used:

Hand-held Glucometer




Body

weight

Glucose level

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Pyruvate Tolerance Test: Example from Literature


Wilfred et al. 2013, AJP – Endocrinol. Metab.

GLP-1 (28-36)a nonapeptide treatment improves glucose tolerance and pyruvate tolerance. The PTT or LTT is a test for gluconeogenesis response to pyruvate or lactate injection.

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Glucose INFusion Rate (GINF) - correlating with insulin sensitivity Endogenous Glucose Production - estimating hepatic insulin action

Hyperinsulinemic-euglycemic Clamp in conscious mouse A gold-standard method to assess insulin action and glucose utilization in vivo.

Basal period Clamp period

Steady state

Target glycemia

Glucose

Insulin

Incorporation of radioactive-labeled glucose measures glucose metablism in individual organs.

Blood glucose

Time [min]

mg/d

l

0 50 100 1500

50

100

150

200

250

Control

Mutant

Hyperinsulinemic-euglycemic Clamp GINF

mm

ol/k

g/m

in

Control Mutant0.0

0.5

1.0

1.5

2.0

****

Supression of EGP

%

Control Mutant

0

50

100

150

**

Gastrocnemius muscle Epididymal white adipose tissue

impaired insulin sensitivity of mutants (decreased insulin stimulated glucose turnover)

impaired insulin sensitive suppression of endogenous hepatic glucose production (EGP)

Glucose uptake rates in skeletal muscle or white adipose tissue were not altered.

Result Example

rathkolb and rozman / phenotyping glucose metabolism

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