1) Type 2 diabetes is associated with disrupted intracellular metabolite homeostasis in addition to disrupted blood glucose homeostasis. Many current diabetes therapies do not address intracellular homeostasis. 2) Glucokinase activators were predicted to lower blood glucose by increasing liver glucokinase activity but lost efficacy after 4 weeks, possibly due to increased hepatic G6P repressing the glucokinase gene. 3) Metformin may lower hepatic G6P through inhibition of mitochondrial complex 1, thereby attenuating high glucose-induced changes in gene expression and potentially restoring metabolite homeostasis. Understanding metabolite control of gene expression provides insights into drug mechanisms of action.

1. Control metabólico de la expresión de genes hepáticos
por antiguos y nuevos fármacos antihiperglicémicos
Metabolite control of hepatic gene expression
by old and new antihyperglycaemic drugs
Loranne Agius,
Newcastle University, Institute of Cellular Medicine

2. Overview:
1. Metabolite control of liver gene expression in Type 2 diabetes (T2D)
2. Glucokinase Activators-drugs for T2D:
Do they fail because of metabolite control gene expression?
3. Metformin- Old drug for T2D:
Does it work because of metabolite control  gene expression?
4. How does metformin affect liver cell metabolites?
5. Selected papers of Alberto Sols  Relevance for T2D and liver gene expression

3. Conclusions:
1. Type 2 diabetes: a disease of compromised intracellular metabolite homeostasis.
2. Glucokinase Activators- ↑ G6P and repress the GCK gene.
3. Metformin- ↓ G6P and induces the GCK gene.
4. How does metformin lower G6P?
5. Relevance of several papers of Alberto Sols for understanding metabolite changes.

4. Type 2 diabetes: defined by defect in blood glucose homeostasis
Treated by ↓ Blood glucose: by ↑ Insulin or ↑Glucose uptake
Triglyceride
G
G
GLUCOSE > 7mM
G G6PTP Pyruvate
GK
G6PC
Insulin
Sulphonylureas
GLP1, DPP4I
TZDs,
Glitazones
Metformin

5. Triglyceride
G
G
GLUCOSE > 7mM
G G6PTP Pyruvate
GK
G6PC
Insulin
Sulphonylureas
GLP1, DPP4I
TZDs,
Glitazones
Metformin
Type 2 diabetes: also characterised by high lipids in tissues and blood
In liver: due to combined effects of ChREBP and SREBP
ChREBP SREBP-1c
De novo lipogenesis TAG =TAG
G Insulin

6. What is the function of ChREBP?
Paradigm-1 Carbohydrate  Fat (efficient energy storage & survival)
Paradigm-2 Maintaining intracellular phosphate ester and ATP homeostasis
Agius L (2016) Proc Nutr Soc. 75:10-18.
Glucose G6P <> F6P <> FDP <> TP <> PEP
Pyruvate
Oxaloacetate
GK
GKRP
G6PC
PKLR
PCK1

7. Evidence supporting a role of ChREBP in cell Phosphate ester homeostasis
1. Major target genes of ChREBP are Pklr, G6pc & GCKR which when induced ↓ [cell G6P]
2. Deficiency of G6PC, PKLR and GCKR causes compromised homeostasis (raised uric acid)
3. ChREBP is activated by ↑ [phosphate ester metabolites] not by glucose itself.
4. ChREBP-KO is associated ↑ G6P and ↓ ATP
5. Fructose in lethal in ChREBP-KO models
 High ChREBP levels in Insulin resistance and T2D indicate compromised intracellular homeostasis.
 High G6PC may be due to ChREBP activation not insulin deficiency.
Is compromised intracellular homeostasis the primary event which then leads to failure of blood
glucose homeostasis?

8. Rationale for the development of Glucokinase Activators for Type 2 diabetes (2000)
1. GK is expressed in the pancreatic β-cells where it is the glucose sensor of insulin secretion
 Activating β-cell GK stimulates Insulin secretion (Potential risk hypoglycaemia)
2. GK is expressed in the liver where it is the major control enzyme for glucose metabolism
 Activating liver GK stimulates glucose disposal (Potential risk raised trriglycerides)
3. GK activity is low in Type 2 diabetes
 Activating liver GK should restore it to normal
Transcriptional regulation of the liver GCK gene is by insulin and glucagon
Iynedjian PB (2009) Cell Mol Life Sci. 66:27-42.

9. Selection of papers on Glucokinase
1: VINUELA E, SALAS M, SOLS A. Glucokinase and hexokinase in liver in relation to glycogen synthesis.
J Biol Chem. 1963;238:1175-7.
2: SALAS M, VINUELA E, SOLS A. Insulin-dependent synthesis of glucokinase in the rat. J Biol Chem.
1963;238:3535-8.
3: SALAS J, SALAS M, VINUELA E, SOLS A. Glucokinase of rabbit liver. J Biol Chem. 1965;240:1014-8.
4: Sols A, Sillero A, Salas J. Insulin-dependent synthesis of glucokinase. J Cell Physiol. 1965;66:23-38.
5: Sapag-Hagar M, Marco R, Sols A. Distribution of hexokinase and glucokinas between parenchymal
and non-parenchymal cells of rat liver. FEBS Lett. 1969;3:68
6. Regulation of the level of key enzymes of glycolysis and gluconeogenesis in liver
Sillero A, Sillero MA, Sols A. Eur J Biochem. 1969;10:351-4.

10. Regulation of the level of key enzymes of glycolysis and gluconeogenesis in liver
Sillero A, Sillero MA, Sols A. Eur J Biochem. 1969 Sep;10(2):351-4.
Control + Fructose diet Diabetic are Fructose diet
Glucokinase 0.6  0.3 0.05 > 0.05
PFK-1 0.4  0.3 1 > 1
Pyruvate Kinase 30  60 22  40
G6Pase 3.5  7.5 15 == 15
F-1,6-P2 3.6  9 9 == 10
PEPCK 15  6
Conclusion: non co-ordinated regulation of glucokinase and pyruvate kinase

11. Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward
glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048.
van Dijk TH et al J Biol Chem. 2001;276:25727-35.
Theo H. van Dijk et al. J. Biol. Chem. 2001;276:25727-25735
Glucose G6P
GK
G6PC
S4048
S4048 S4048
Insulin Glucagon

12. In isolated hepatocytes the liver GCK gene is repressed by cell metabolites but not by
metabolic flux.
G6pc
inhibitor
S4048
1,2
↓
3
↓
4
↓
Arden et al (2011) Diabetes 60;3110
Elevated glucose represses liver glucokinase and induces its regulatory protein to safeguard hepatic phosphate homeostasis.

13. Summary
 Type 2 diabetes is defined by disruption of blood glucose homeostasis, but it is also associated with raised
lipids and de novo lipogenesis and increased ChREBP expression.
 ChREBP target genes include G6PC and other genes involved in control of metabolite homeostasis.
 Increased ChREBP expression is a marker of compromised intracellular homeostasis
 Many therapies for Type 2 diabetes focus on lowering blood glucose and do not take into account
intracellular homeostasis.

14. GKAs: arguments supporting validity as a therapeutic strategy for Type 2 diabetes
 Glucokinase is expressed in islet- β-cells where it functions as the sensor for insulin secretion
 Glucokinase is the major control enzyme for liver glucose metabolism
 Inactivating mutations in the GCK gene in man cause diabetes (MODY-GCK diabetes)
 Activating mutations in the GCK gene in man (PHHI): ↑ insulin & ↓glucose
 Liver Glucokinase activity is low in Type 2 diabetes
Prediction: Increasing glucokinase activity with drugs (GKAs)
should lower blood glucose in Type 2 diabetes
Concerns / Risks: Hypoglycaemia & Hypertriglyceridaemia
Matschinsky FM (2013) Trends Pharmacol Sci 34:90

15. Glucokinase Activators (GKAs) as new drugs for Type 2 diabetes:
Predicted result: remarkably effective at ↓ blood glucose after single dose treatment
GKA
GKA
Improves GTT
GKA
GK Kinetics (enzyme assay)
Blood glucose
Bristol Myers
Squibb
Roche,
AstraZeneca
Banyu
Lilli
Novo Nordisk,
Merck GmbH
Novartis
Takeda
Pfizer
Amgen
Tanabe
Advinus
Array
Open Low-affinity
Conformation
GKA

16. Glucokinase Activators (GKAs) as new drugs for Type 2 diabetes:
Unpredicted result: Efficacy in Phase 2 trials was lost after 4 weeks
Merck GKA M-0941
Meininger (2011) Diabetes Care 34:2560
GKA
Astrazeneca GKA AZD1656
Wilding (2013) Diab Obesity Metab 15:750
Kiyosu A (2013) Diabetes Obes Metab. 2013;15:923
Efficacy during first 4 wks
Loss of efficacy after 4 wks
Why do they lose their efficacy?
Merck GKA: showed a 11.5% increase in blood TAGs at 4 mo
AstraZeneca GKA: showed 7-5% increase (Wilding study) or no increase (Kiyosue study)

17. In hepatocyte studies:
A GKA causes a left-shift in the glucose effect on G6P and on gene regulation
Al-Aonzi ZH et al. Diabetes Obes Metab. 2017

18. 2-Deoxyglucose ↑ hexose 6-P and represses GCK but does not induce ChREBP genes
Al-Aonzi ZH, Diabetes Obes Metab. 2017

19. Summary GKAs In vivo (in man):
 Very effective in ↓ blood glucose but only during short-term therapy.
 Small to moderate ↑ in blood TAGs (7-12%) in some but not all studies.
GKAs in hepatocytes
 ↑ cell G6P by more than occurs at 25mM glucose
 Repress the GCK gene and induce G6PC
 2-Deoxyglucose mimics the GCK repression but not ChREBP activation
Hypothesis  Raised G6P by the GKA represses the liver GCK gene. This may contribute to the loss of
GKA efficacy chronically

20. 3. Does G6P have a role in the metformin mechanism?

21. Previous evidence for ↓G6P at high metformin concentration?
 Owen MR, Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the
mitochondrial respiratory chain. Biochem J. 2000;348:607
 Guigas B, et al. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and metformin inhibit hepatic glucose
phosphorylation. Diabetes 2006;55:865
 Mukhtar MH, Inhibition of glucokinase translocation by AMP-activated protein kinase is .. Am J Physiol 2008;294:R766
Owen & Halestrap (2000) Biochem J. 348:607

22. Does metformin lower G6P in hepatocytes at “therapeutic doses”?
Christensen (2013) Eur J Clin Pharmacol 71:691
 Nakamichi (2013) J Pharm Sci. 102:3407
Wilcock & Bailey (1994) Xenobiotica 24:49
Liver content of metformin at blood peak
180-280 nmol/ g wet wt liver
1-2 nmol / mg protein
17-23 µM

23. Therapeutic doses of metformin ↓ G6P and attenuate gene regulation by high glucose
G6P
Al-Aonzi ZH, Diabetes Obes Metab. 2017

24. Metformin mechanism: current hypotheses
Inhibition of Complex I : Owen & Halestrap (2000) Biochem J 348:607
Inhibition of Complex I (direct): Bridges et al (2014) Biochem J 462:475
Activation of AMPK: Zhou et al (2001) J Clin Invest. 108:1167
AMPK-independent by ATP depletion: Foretz et al. (2010) J Clin Invest. 120:2355
AMPK-independent by inhibiting glucagon signalling: Miller et al (2013) Nature, 494:256
Inhibition of mitochondrial mG3PD & glycerophosphate shuttle: Madiraju et al (2014) Nature 510:542

25. 1. Inhibitors of mG3PD and AMPK activators do not ↓ G6P
Inhibition of mG3PD Activators of AMPK
Al-Aonzi ZH, Diabetes Obes Metab. 2017

26. 2. Mitochondrial inhibitors e.g. rotenone, uncouplers, Rhein ↓ G6P (without changing ATP)
2 5 m M g lu c o s e S 4 0 4 8
R o te n o n e ( M )
G6P(%Control)
0
.0
0
.2
5
0
.5
1
.0
0
50
100
150
**
**
**
2 5 m M g lu c o s e S 4 0 4 8
D N P ( M )
G6P(%Control)
0
5
20
40
0
50
100
150
**
**
**
2 5 m M g lu c o s e S 4 0 4 8
A m m o n iu m ( M )
G6P(%Control)
0
.0
0
.5
5
.0
0
5 0
1 0 0
1 5 0
**
2 5 m M g lu c o s e S 4 0 4 8
R h e in ( M )
G6P(%Control)
0
1
0
2
0
4
0
0
5 0
1 0 0
1 5 0
**
*
Complex I (rotenone) Uncoupler (DNP)
Nnt inhibitor Ammonium ion

27. Glucose G6P F6P FDP TP Pyruvate
Glycogen
Glucokinase
G6Pase
H6PD
GLUCONEOGENESIS
GLYCOLYSIS
PENTOSE
PHOSPHATE
PATHWAY
GS GP
> 7 Possible mechanisms for ↓ G6P by metformin (> 15mM glucose, 5mM DHA, 2 mM Xylitol)
Inhibition of
1. Glucokinase
2. Gluconeogenesis
3. G phosphorylase
Stimulation of
4. G6Pase
5. GS
6. Glycolysis
7. PPP
↑
↑
↑
↑
↑
25mM Glucose DihydroxyacetoneXylitol

28. Inhibition of
1. Glucokinase
2. Gluconeogenesis
3. G phosphorylase
Stimulation of
4. G6Pase
5. GS
6. Glycolysis
7. PPP
Glucose G6P 
Glucokinase
G6Pase
H6PD
S4048
2 5 m M g lu c o s e
[
14
C]glycogen(%control)
-
S
4
0
4
8
+
S
4
0
4
8
0
2 0 0
4 0 0
6 0 0
**
[
14
C]glycogen(%control)
0
0
.1
m
M
m
e
tfo
rm
in
0
.2
m
M
m
e
tfo
rm
in
0
.5
m
M
m
e
tfo
rm
in
5

M
A
7
6
9
6
6
2
1
0

M
A
7
6
9
6
6
2
0
5 0
1 0 0
1 5 0
2 5 m M g lu c o s e
2 5 m M g lu c o s e + S 4 0 4 8
*
**
**
**
**
**
**
**
**
Glycogen synthesis parallels G6P
?
0 0.2 0.5 1 0 1 2 4
0
50
100
150
Metformin
GKRP
Metformin GKRP
*
*
* *
GlucosePhosphorylation
0 0.2 0.5 1 0 1 2 4
0
5
10
15
20
Metformin GKRP
*
* *
*
*
*
G6P(nmo/mg)

29. Inhibition of
1. Glucokinase
2. Gluconeogenesis
3. G phosphorylase
Stimulation of
4. G6Pase
5. GS
6. Glycolysis
7. PPP
 Unlikely
Biphasic effect of fructose 2,6-bisphosphate on the liver
fructose-1,6-bisphosphatase: mechanistic and physiological
implications
Corredor C, Boscá L, Sols A. FEBS Lett. 1984;167:199
 F2,6P2
 F2,6P2
INHIBITION and ACTIVATION of FBPase-1

30. Testing involvement of the pentose phosphate pathway with DHEA or G6PD-KD in the ↓ G6P
DHEA a G6PD inhibitor counteracts the effect of metformin but not ammonium ion on G6P
5 m M D H A S 4 0 4 8
G6P(%control)
0
0
.1
m
M
m
e
tfo
rm
in
0
.2
m
M
m
e
tfo
rm
in
0
.5
m
M
m
e
tfo
rm
in
2
m
M
N
H
4
0
5 0
1 0 0
1 5 0
C o n tro l
2 0  M D H E A
*
**
**
**
**
**
** **
2 5 m M g lu c o s e S 4 0 4 8
G6P(%Control)
0
0
.1
m
M
m
e
tfo
rm
in
0
.2
m
M
m
e
tfo
rm
in
0
.5
m
M
m
e
tfo
rm
in
2
m
M
N
H
4
0
5 0
1 0 0
1 5 0
C o ntrol
20 M D H E A
*
**
**
**
**
** **
2 m M X y lito l S 4 0 4 8
G6P(%control)
0
0
.1
m
M
m
e
tfo
rm
in
0
.2
m
M
m
e
tfo
rm
in
0
.5
m
M
m
e
tfo
rm
in
2
m
M
N
H
4
0
5 0
1 0 0
1 5 0
C o n tro l
2 0  M D H E A
**
****
**
**
**
**

31. Glycolysis vs PPP by 13C-metabolomics: Metformin favours glycolysis relative to PPP
An AMPK activator has the converse effect
Silvia Marin & Marta Cascante, Unpublished

32. Conclusions: mechanism by which metformin lowers G6P
 Metformin ↓ G6P in hepatocytes at therapeutic (1-2 nmol/mg protein) doses, irrespective of
substrate (high glucose, DHA or xylitol) and without changes in ATP.
 The metformin effect is not mimicked either by AMPK activators or mG3PD inhibitors.
 It is mimicked by mitochondrial inhibitors (rotenone, rhein, uncouplers and also ammonium ion).
 The ↓ G6P may be due to increased glycolytic flux.
 Metformin inhibits complex I at ≥ 0.5mM but ↓ G6P at ≥ 0.1mM.
 Mechanisms other than Complex I may account for the increased glycolysis

33. Selection of papers on Hexokinases
1: CRANE RK, SOLS A. The association of hexokinase with particulate fractions of brain and other tissue homogenates. J
Biol Chem. 1953;203:273-92.
2: SOLS A, CRANE RK. The inhibition of brain hexokinase by adenosinediphosphate and sulfhydryl reagents. J Biol Chem.
1954 Feb;206(2):925-36.
3: CRANE RK, SOLS A. The non-competitive inhibition of brain hexokinase by glucose-6-phosphate and related
compounds. J Biol Chem. 1954;210:597-606.
4: VILLAR-PALASI C, CARBALLIDO A, SOLS A, ARTETA JL. Sensitivity of pancreas hexokinase towards alloxan and its
modification by glucose. Nature. 1957;180:387-8.
5: Apparent unbalance between the activities of 6-phosphogluconate and glucose-6-phosphate dehydrogenases in rat
liver Sapag-Hagar M, Lagunas R, Sols A. Biochem Biophys Res Commun. 1973;50:179
6: Regulation of liver pyruvate kinase and the phosphoenolpyruvate crossroads
Llorente P, Marco R, Sols A. Eur J Biochem. 1970;13:45-54.
7. Studies on the mechanism of the antifungal action of benzoate
Krebs HA, Wiggins D, Stubbs M, Sols A, Bedoya F. Biochem J. 1983;214:657-63.

34. Studies on the mechanism of the antifungal action of benzoate
Krebs HA, Wiggins D, Stubbs M, Sols A, Bedoya F. Biochem J. 1983;214:657-63.
Benzoate (2-10mM):
1. ↓ cell pH by 0.1– 1.0 pH units (enhanced by low extracellular pH)
2. ↑ G6P & F6P
3. ↓ Fru-1,6-P2 & triose phosphates
4. ↓ ATP, ↑ AMP (no Δ ADP; no Δ adenine nucleotides)
5. Functional inhibition (indirect) of PFK1 by intracellular acidification.
Effects of benzoate (pKa 4.2) mimicked by salicylate, sorbate, acetate:
(i) Similar pKa
(ii) Undissociated acid is sufficiently lipophilic to cross membranes
(iii) Anion does not cross membranes
G6P F6P F16P2 TP Pyr
Glucose  G6P F6P  F1,6P2  Triose P
Hk Pfk1

35. Thank you
Colleagues
Ziad Al-Oanzi
Sue Tudhope
Sophia Fountana
Tabassum Moonira
Ahmed Alsawi
John Petrie
Gillian Patman
Catherine Arden
Medical
Research
Council
Collaborators
Benoit Viollet
Marta Cascante
Silvia Marin
Helen Reeves

37. Apparent unbalance between the activities of 6-phosphogluconate and glucose-6-
phosphate dehydrogenases in rat liver
Sapag-Hagar M, Lagunas R, Sols A. Biochem Biophys Res Commun. 1973;50:179

38. Regulation of liver pyruvate kinase and the phosphoenolpyruvate crossroads
Llorente P, Marco R, Sols A. Eur J Biochem. 1970;13:45-54.
Pyruvate kinase in liver and kidney:
(a) Co-operative substrate (PEP) kinetics
(b) Strong allosteric inhibition by ATP and alanine (↑ S0.5 for PEP)
(c) Strong activation by fructose 1,6-P2
Conclusion > In gluconeogenic tissues pyruvate kinase has regulatory
mechanisms to prevent diversion of PEP from gluconeogenesis.
The regulatory switch from Glycolysis to Gluconeogenesis comprises:
1. Feed-back inhibitors: Alanine and ATP
2. Forward activator: Fructose 1,6-bisphosphate (0.5-1.0 µM)
Fru-6-P
Fru-1,6-P2
PEP
OAA Pyruvate Alanine
ADP
ATP
Glucose