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Glucose levels are maintained within a narrow
range in mammals through the effects of
pancreatic hormones on liver gluconeogenesis
gt5 mmol/l
lt4 mmol/l
Normal blood glucose level 4.5 mmol/l
Diabetic hyperglycemia?
http//www.endocrineweb.com/insulin.html
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Insulin modulates gluconeogenesis by inhibition
of the coactivator TORC2

• Renaud Dentin,Yi Liu, Seung-Hoi Koo, Susan
  Hedrick, Thomas Vargas, Jose Heredia, John Yates,
  III Marc Montminy
• Peptide Biology Laboratories, Salk Institute For
  Biological Studies, La Jolla, California

Nature 449, 366-369 (20 September 2007)
Shiuh-Rong Ho Department of Cell Biology
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The two distinct mTOR complexes (TORC1 and TORC2)
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Model of the mTOR Signaling Network in Mammalian
Cells
Cell. 2006 Feb 10124(3)471-84.
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(No Transcript)
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in vivo imaging of adenoviral CRE-luciferase
activity
Ad-CRE-Luc or Ad-CRE-Luc Ad-RNAi-TORC2 and
Ad-RNAi-SIK2 or Ad-COP1, Ad-TORc2 and Ad-SIK2
(normal and mutant)
blood-glucose levels
Fasting or Re-feeding
mice
HEK293T cells
gluconeogenic gene expression
forskolin (adenylate cyclase activator) Insulin G
lucagon
Protein-Protein interaction Phosphorylation Ubiqut
ination) degradation
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They monitored the activity of the TORC2 pathway
in liver by in vivo imaging with a
cAMP-responsive adenoviruscontaining cAMP
responsive elements (CREs) upstream of a minimal
promoterCRE-luciferase reporter (Ad-CRE-Luc).
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FIGURE 1. Insulin inhibits TORC2 activity during
re-feeding.
Fasting increased amounts of de-phosphorylated
TORC2 which is an active form, whereas
re-feeding promoted TORC2 re-phosphorylation and
degradation
Fasting increased hepatic Ad-CRE-Luc activity
20-fold over that of re-fed mice.
RNAi-mediated knockdown of TORC2 reduced
Ad-CRE-Luc but not CREB-independent reporter
activity in the liver
gluconeogenic gene Pepck expression and fasting
blood-glucose levels were decreased in Ad-TORC2i
(Ad-Rsv(Rous sarcoma virus)-Luc)
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Hepatic TORC2 protein levels are downregulated
during re-feeding, they wondered whether TORC2
activity is increased in insulin resistance.
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FIGURE 1. Insulin inhibits TORC2 activity during
re-feeding. (Cont.)
db/db diabetic mice had higher levels of
Ad-CRE-Luc reporter activity and TORC2 protein in
the liver, and re-feeding did not trigger TORC2
phosphorylation or degradation
IP administration of GLU stimulated hepatic
Ad-CRE-Luc activity in mice these effects were
reversed by subsequent exposure to INS, which
also promoted the phosphorylation and degradation
of hepatic TORC2 in fasted mice
glucagon (GLU)
GLU then insulin (GLU/INS)
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If insulin inhibits TORC2 activity by Ser 171
phosphorylation, then mutant TORC2(S171A) should
be resistant to these effects.
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FIGURE 2. SIK2 promotes TORC2 phosphorylation
during re-feeding.
Ad-CRE-Luc activity, gluconeogenic gene
expression, and blood-glucose concentrations were
elevated in Ad-TORC2(S171A) mice relative to
Ad-TORC2 mice during re-feeding
Ad-TORC2(S171A) was not degraded during
re-feeding
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The salt inducible kinase 2 (SIK2 also known as
SNF1LK2) has been found to inhibit TORC2 activity
by promoting its Ser 171 phosphorylation,
prompting them to test the role of this kinase in
the liver.
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RNAi-mediated knockdown of SIK2 (Ad-SIK2i)
increased Ad-CRE-Luc activity and blood glucose
concentrations during re-feeding
TORC2 (Ser 171) phosphorylation was also
disrupted in re-fed Ad-SIK2i mice, and TORC2
protein levels were consequently elevated
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They noticed that SIK2 contains a single
conserved AKT phosphorylation site (RQRRPS) at
Ser 358.
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in vitro kinase assays
HEK293T cells
Insulin increased the phosphorylation of
wild-type but not the SIK2(S358A) mutant, by
western blot assay with phospho-AKT substrate
antiserum and by metabolic labelling with
inorganic 32P these effects were blocked by
addition of LY294002 inhibitor
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Because phosphorylation often targets nuclear
proteins for degradation by triggering their
cytoplasmic export and polyubiquitination, they
examined effects of fasting and feeding signals
on TORC2 shuttling in primary hepatocytes.
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FIGURE 3. COP1 promotes the ubiquitination and
degradation of TORC2.
primary hepatocytes
Exposure to FSK promoted TORC2 de-phosphorylation
and nuclear translocation. Exposure to insulin
alone increased TORC2 phosphorylation.
sequential exposure of primary hepatocytes to
glucagon and insulin stimulated TORC2
poly-ubiquitination in the cytoplasm, but only
mono-ubiquitination in the nucleus. the
constitutively nuclear and degradation-resistant
TORC2(S171A) protein was selectively
mono-ubiquitinated.
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In proteomic studies to identify
TORC2-interacting proteins, they recovered
constitutive photomorphogenic protein-1 (COP1,
also known as RFWD2) and DNA damage binding
protein 1 (DDB1), components of an E3 ubiquitin
ligase complex that recognizes substrates
containing the consensus motif (D/E-D/E-X-X-X-V-P)
.
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TORC2COP1 interaction in co-IP of primary
hepatocytes exposed to FSK/INS. Indeed, mutation
of Val and Pro residues in COP1 recognition motif
(V214A, P215A) blocked the association of TORC2
with COP1. Ad-COP1 expression triggered TORC2
degradation and reduced glucose output from
primary hepatocytes exposed to FSK.
IPHA
IPCOP1
HEK293T cells
in vitro ubiquitination
In the presence of relevant cofactors (DDB1,
DET1), COP1 promoted the ubiquitination of
wild-type but not COP1-interaction defective
VP/AA TORC2. COP1 also stimulated the
ubiquitination of TORC2(K213R) but had no effect
on TORC2(K628R), suggesting an important role for
Lys 628 in this regard. COP1 enhanced the
degradation inHEK293T cells.
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They suspected that ubiquitin-defective TORC2
proteins would also be resistant to degradation
by re-feeding signals.
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TORC2 degradation by COP1 during re-feeding is
required for glucose homeostasis.
control medium (G5)
exposure of primary hepatocytes to FSK/INS
decreased amounts of wild-type but not
TORC2(K628R) or TORC2(V214A, P215A) protein
TORC2(K628R) and TORC2(V214A, P215A) were also
more active in stimulating Ad-CRE-Luc activity
from primary hepatocytes relative to wild-type
TORC2.
RNAi-mediated knockdown of COP1 reduced TORC2
ubiquitination and correspondingly increased
amounts of TORC2 protein in primary hepatocytes
exposed sequentially to glucagon and insulin
Ad-CRE-Luc activity was elevated in Ad-COP1i
hepatocytes, particularly in cells that had been
previously treated with FSK/INSwhen TORC2 is
degraded and then re-exposed to FSK
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They examined the role of COP1 in promoting
glucose homeostasis in vivo.
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TORC2 degradation by COP1 during re-feeding is
required for glucose homeostasis.
Ad-COPi mice had higher total amounts of hepatic
TORC2 protein compared with Ad-USi mice.
Gluconeogenic gene expression was also elevated
in Ad-COP1i mice, revealing the importance of
COP1 in this setting.
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MODEL PhosUb?translocation?Poly Ub
E3 ligase
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Editor's Summary
20 September 2007
TORC2 and diabetes
A previously unrecognized pathway by which
insulin modulates hepatic glucose production is
described in this issue. Glucose levels are
maintained within a narrow range in mammals
through the effects of pancreatic hormones on
liver gluconeogenesis. This new pathway
facilitates the inhibition of the expression of
gluconeogenic genes by insulin by promoting the
phosphorylation and ubiquitin-dependent
degradation of the CREB coactivator TORC2. The
signalling pathway involves the kinase SIK2 and
the E3 ligase COP1. The findings point to TORC2
and SIK2 as potential therapeutic targets in type
II diabetes.
http//www.nature.com/nature/journal/v449/n7160/ed
summ/e070920-14.html
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Glowing Mice Light The Way To Understanding
Type 2 Diabetes