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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
2
Known/Expected Phenotypes
Clinical Chemistry & Hematology
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
3
Glucose Tolerance Test (GTT): General workflow
Clinical Chemistry & Hematology
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
4
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
Over night fed_morning 5 hour fast fed_ afternoon
mm
ol/l
2
4
6
8
10
12
14
16
18
20
22
NEFA
Group
Over night fed_morning 5 hour fast fed_ afternoon
mm
ol/l
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Glycerol
Group
Over night fed_morning 5 hour fast fed_ afternoon
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
ON f fed 5-6h f fed morning 8 am afternoon 2 pm
ON f fed 5-6h f fed morning 8 am afternoon 2 pm
ON f fed 5-6h f fed morning 8 am afternoon 2 pm
5
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.
6
IpGTT: Protocol variations
Clinical Chemistry & Hematology
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
7
IpGTT: Protocol variations (glucose application route)
3. Glucose application route Commonly used in mice: - Intraperitoneal injection - Oral gavage
Rather uncommon: -intravenous application
Modified from: Keith N. Frain, Metabolic Regulation, 3rd edition, Wiley-Blackwell 2010
Incretins (GLP-1, GIP)
Blood cells
Glycerol
Lactate + Pyrovate
Alanine
FFA
Kidneys
Gluconeo-genesis
8
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
9
Ip Glucose Tolerance Test (IpGTT): Standard procedure IMPC
Clinical Chemistry & Hematology
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
10
IpGTT: Examples from IMPC Screen
Clinical Chemistry & Hematology
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
11
IpGTT: Examples from IMPC Screen
Clinical Chemistry & Hematology
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
12
IpGTT: Examples from IMPC Screen)
Clinical Chemistry & Hematology
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.
13
IpGTT: Approaches to data analysis
Clinical Chemistry & Hematology
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
14
Clinical Chemistry & Hematology Clinical Chemistry & Hematology
Modified from: Keith N. Frain, Metabolic Regulation, 3rd edition, Wiley-Blackwell 2010
Incretins (GLP-1, GIP)
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?
15
Insulin Tolerance Test (ITT) : Test for insulin sensitivity
Clinical Chemistry & Hematology
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
Parameters analyzed:
Basal fasting glucose level,
Area under the curve
Body
weight
Glucose level
Insulin dose has to be adapted according to insulin sensitivity of the genetic background of mice tested.
16
Insulin Tolerance Test: Example from literature
Clinical Chemistry & Hematology
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).
17
Pyruvate or Lactate Tolerance Test (PTT / LTT)
Clinical Chemistry & Hematology
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
Parameters analyzed:
Basal fasting glucose level,
Area under the curve
Body
weight
Glucose level
18
Pyruvate Tolerance Test: Example from Literature
Clinical Chemistry & Hematology
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.
19
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