Insulin Regulation of Gluconeogenesis 2018

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Ann N Y Acad Sci. Author manuscript; available in PMC 2019 January 01.
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Published in final edited form as:

Ann N Y Acad Sci. 2018 January ; 1411(1): 21–35. doi:10.1111/nyas.13435.
Insulin regulation of gluconeogenesis

Maximilian Hatting, Clint D. J. Tavares, Kfir Sharabi, Amy K. Rines, and Pere Puigserver
Department of Cancer Biology, Dana-Farber Cancer Institute and Department of Cell Biology,
Harvard Medical School, Boston, Massachusetts
Abstract
The coordinated regulation between cellular glucose uptake and endogenous glucose production is
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indispensable for the maintenance of constant blood glucose concentrations. The liver contributes
significantly to this process by altering the levels of hepatic glucose release, through controlling
the processes of de novo glucose production (gluconeogenesis) and glycogen breakdown
(glycogenolysis). Various nutritional and hormonal stimuli signal to alter hepatic gluconeogenic
flux, and suppression of this metabolic pathway during the postprandial state can, to a significant
extent, be attributed to insulin. Here, we review some of the molecular mechanisms through which
insulin modulates hepatic gluconeogenesis, thus controlling glucose production by the liver to
ultimately maintain normoglycemia. Various signaling pathways governed by insulin converge at
the level of transcriptional regulation of the key hepatic gluconeogenic genes PCK1 and G6PC,
highlighting this as one of the focal mechanisms through which gluconeogenesis is modulated. In
individuals with compromised insulin signaling, such as insulin resistance in type 2 diabetes,
insulin fails to suppress hepatic gluconeogenesis, even in the fed state; hence, an insight into these
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insulin-moderated pathways is critical for therapeutic purposes.
Keywords
glycogenolysis; glucose; regulation; gluconeogenesis; insulin
Introduction
Glucose is a major metabolic fuel that serves the energetic demands of mammalian tissues.
During periods of starvation, glucose can be generated through the gluconeogenesis
pathway, which is highly evolutionarily conserved from microorganisms to vertebrates. In
the human body, the liver is the main site of gluconeogenesis. Increased gluconeogenesis in
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the liver of patients with type 2 diabetes is considered a major contributor to hyperglycemia
and subsequent diabetic organ damage. Insulin is a key hormone that inhibits
gluconeogenesis, and insulin resistance is a hallmark of type 2 diabetes. Understanding the
regulation of gluconeogenesis and the role of insulin signaling in this pathway is important
Address for correspondence: Dr. Pere Puigserver, Dana-Farber Cancer Institute, 450 Brookline Av. CLSB-11144, Boston, MA 02215.
[email protected].
Competing Interests
The author declares no competing interests
Hatting et al. Page 2
to developing new therapies for type 2 diabetes. Here, we aim to depict the role of insulin in
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the framework of hepatic gluconeogenesis.
Gluconeogenesis contributes to hepatic glucose production

Hepatic glucose production is a sum of gluconeogenesis, which is the formation of glucose
from pyruvate or other 3- or 4-carbon compounds, and glycogenolysis, which is the
breakdown of glycogen to glucose. The main substrates of gluconeogenesis in humans are
lactate, glycerol, alanine, and glutamine. Together, these account for 90% of gluconeogenic
substrates; however, other amino acids and citric cycle intermediates can also serve as
substrates for gluconeogenesis.1,2 Starting from lactate or an α-keto acid derived from
amino acid breakdown, pyruvate can be generated for gluconeogenesis. Pyruvate is
converted via carboxylation to oxaloacetate in the mitochondria. This reaction is stimulated
by high levels of acetyl-CoA, which is produced via β-oxidation of fatty acids in the liver,
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and inhibited by high levels of ADP and glucose. After an intermediate step that allows
oxaloacetate to leave the mitochondria via malate, oxaloacetate is decarboxylated and then
phosphorylated to form phosphoenolpyruvate by phosphoenolpyruvate carboxykinase
(PEPCK). This enzyme is a regulator of the rate of gluconeogenesis, and its transcription is
targeted by multiple factors, including glucagon and insulin.3 After several steps of reverse
glycolysis, fructose 1,6-bisphosphatase converts fructose 1,6-bisphosphate to fructose 6-
phosphate. Fructose 2,6-bisphosphate and AMP inhibit this reaction, while citrate activates
the fructose 1,6-bisphosphatase enzyme. Glucose-6-phosphate (G-6-P) is formed from
fructose 6-phosphate by phosphoglucoisomerase. G-6-P can be used in other metabolic
pathways or dephosphorylated by glucose-6-phosphatase (G-6-Pase) to form free glucose.
Whereas free glucose can easily diffuse into and out of the cell, the phosphorylated form
(G-6-P) cannot, providing a mechanism by which intracellular glucose levels are controlled.
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The final reaction of gluconeogenesis––the formation of glucose––occurs in the lumen of

the endoplasmic reticulum, where G-6-P is hydrolyzed by G-6-Pase to produce glucose and
release an inorganic phosphate. Like the two previous step, this step is a reversal of
glycolysis, in which hexokinase catalyzes the conversion of glucose and ATP into G-6-P and
ADP. Glucose is then shuttled into the cytoplasm by glucose transporters located in the
endoplasmic reticulum membrane.
Enhanced hepatic glucose production leads to increased glucose release to the blood, which
can cause hyperglycemia. Type 2 diabetes is characterized by persistent hyperglycemia. In
patients with type 2 diabetes, gluconeogenesis has been identified as the primary source of
glucose production, while glycogenolysis was found not to contribute.4 These findings
underscore the importance of maintaining normal gluconeogenic rates to avoid disease
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pathophysiology. Insulin is a major hormone regulator of gluconeogenesis, so understanding

its role in determining gluconeogenesis rates is essential to understanding the cause of and
potential treatments for type 2 diabetes.
Insulin action on gluconeogenesis is both direct and indirect

A role for insulin in the regulation of hepatic glucose output is widely accepted. In healthy
individuals, physiological hyperinsulinemia suppresses gluconeogenesis by 20%, while
glycogenolysis is completely suppressed.5 Hyperglycemia alone suppresses hepatic

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glycogenolysis with only minimal effects on glycogen storage. Only the combination of
hyperglycemia and hyperinsulinemia has a significant effect on hepatic glycogen synthesis.6
Thus, insulin plays a crucial role in hepatic glucose metabolism.
The dominant mechanism of insulin-mediated regulation of hepatic gluconeogenesis is not

clear. Insulin exerts direct control of gluconeogenesis by acting on the liver, but also
indirectly affects gluconeogenesis by acting on other tissues. The direct effect of insulin was
demonstrated in fasted dogs, where portal plasma insulin suppressed hepatic glucose
production. even without changes in glucagon or gluconeogenic precursors.7 However, in
mouse models, insulin was found to have more potent effects on hepatic glucose production
in vivo rather than in vitro.8–10 Moreover, indirect effects of insulin on extrahepatic tissues
have been shown to be sufficient to maintain normal glucose metabolism, suggesting an
important role for indirect insulin regulation of gluconeogenesis.11
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Suppression of gluconeogenesis through indirect effects of insulin is known to involve

multiple tissues and cell types, with pancreatic α cells, adipose tissue, skeletal muscle, and
the brain exerting known effects on hepatic gluconeogenesis. In pancreatic α cells, insulin
inhibits the secretion of glucagon, which can indirectly lead to suppression of hepatic
glucose production by reducing hepatic glucagon signaling.12 Glucagon has effects on the
transcriptional regulation of gluconeogenesis, primarily through the transcription factor
CREB, but also through metabolite flux by affecting the activity of phosphofructokinase 1
(PFK1) in a PKA-dependent manner.13,14 Insulin decreased plasma glucagon levels in vivo
and inhibited glucagon secretion from pancreatic αcells in vitro.15 However, in mice lacking
the insulin receptor in the liver, insulin did not suppress glucagon secretion or hepatic
glucose production, highlighting the importance of intrahepatic insulin signaling.16
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Other indirect mechanisms by which insulin suppresses hepatic gluconeogenesis are through
reducing gluconeogenic substrate release from adipose tissue and skeletal muscle or by
acting on the brain. Insulin has inhibitory effects on lipolysis and proteolysis and thus
decreases plasma levels of non-esterified fatty acids (NEFAs) and glycerol derived from
adipose tissue, as well as amino acids from skeletal muscle.10,14 A reduction of free fatty
acid delivery to the liver has been shown to decrease hepatic glucose output.17 However,
NEFA failed to reduce hepatic glucose production in liver-specific insulin receptor gene
knockout mice, suggesting that NEFAs are substrates that depend on intrahepatic insulin
signaling to regulate hepatic gluconeogenesis.16 The role of the central nervous system in
gluconeogenesis is complex and has been recently reviewed,18 but insulin has been found to
inhibit gluconeogenesis by acting on the brain in an insulin receptor–dependent manner.19
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The idea that extrahepatic insulin signaling can control hepatic glucose production (HGP) is
supported by the fact that insulin can suppress HGP in mice where the canonical hepatic
insulin signaling components Akt and FOXO1 are depleted.20 Moreover, acute depletion of
the insulin receptor and FOXO1 in liver does not prevent insulin from suppressing HGP.21 It
is important to mention, however, that in these experiments insulin can still signal through
the IGF receptor, which might be sufficient to suppress HGP. A recent study further supports
the idea that insulin’s main effect on HGP is through suppression of lipolysis in white
adipose tissue.22 Here, intrahepatic acetyl-CoA levels were shown to be elevated in HFD-fed
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rodents, resulting in an increase in pyruvate carboxylase activity and gluconeogenesis. The

increase in hepatic acetyl-CoA is a result of increased lipolysis due to insulin resistance in
fat. In support of the importance of the fat lipolysis–hepatic acetyl-CoA axis in controlling
HGP, reducing lipolysis by inhibition of adipose triglyceride lipase or by neutralizing
interleukin (IL)-6, a cytokine known to promote lipolysis in fat, normalizes hepatic acetyl-
CoA levels and pyruvate carboxylase activity as well as HGP.22
Insulin regulation of hepatic gluconeogenesis through transcriptional

modulation
Insulin can regulate hepatic gluconeogenesis via transcription of genes involved in
gluconeogenic control, including PCK1 and G6PC.23 Changes in transcription may not
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contribute to acute regulation of hepatic glucose output, but they determine the
gluconeogenic capacity of the liver and may have long-term effects, especially in
pathological states.
Whether transcriptional regulation of gluconeogenic enzymes can be observed in human

livers is controversial. A recent study showed no induction of PCK1 and G6PC in liver
biopsies of patients with type 2 diabetes.24 However, another study in patients found a clear
correlation between insulin resistance and PCK1, G6PC, and FOXO1 mRNA levels.25 An
explanation for these findings may be that lesions in nonalcoholic steatohepatitis are
unequally distributed over the liver, and significant sampling errors might occur.26 Also, the
degree of liver damage and other parameters, such as total body weight and tissue cross talk,
may affect whether changes in gene expression are observed. Nonetheless, since
gluconeogenesis is a primary driver of hepatic glucose production in type 2 diabetic
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patients4,27 and insulin affects gluconeogenesis through transcription, determining the

insulin signaling pathways that alter gluconeogenic gene transcription and ultimately
gluconeogenesis can contribute to our understanding of type 2 diabetes pathophysiology.
Insulin activates several signaling pathways that regulate gluconeogenesis

Insulin binds to and initiates signaling through the insulin receptor, which is a tyrosine
kinase that is activated upon ligand binding. This activation leads to phosphorylation of a
variety of intracellular substrates. Two major downstream pathways of insulin action are the
PI3K and MAPK pathways.28 However, the activities of several other pathways are
modulated by insulin action, with implications for transcriptional control of gluconeogenesis
(Fig. 1).
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IRS1 and IRS2

Among the numerous downstream signaling components of the insulin receptor, the insulin
receptor substrates (IRS) play a key role in the regulation of hepatic glucose production.
Mice lacking the IRS2 protein exhibited diabetes-like symptoms and increased hepatic
glucose production.29 Mice with knockout of Irs1 had decreased insulin sensitivity, but not
diabetes per se.30 Double knockout of Irs1 and Irs2 in the liver leads to severe
hyperglycemia, hyperinsulinemia, and induction of gluconeogenic genes, such as Pck1 and

G6pc.31 However, the double knockout also had severe growth defects, so interpretation of
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data from this animal model must be cautious. Taken together, these data demonstrate that
IRS proteins are important for insulin signal transduction, and that IRS1 and IRS2 can
compensate for each other.
PI3K
The activation of IRS proteins results in the recruitment of the lipid kinase PI3K to the
plasma membrane, where it phosphorylates PI-(4,5)-bisphosphate (PtdIns(4,5)P2/PIP2) to
generate PtdIns(3,4,5)P3 (PIP3), an important second messenger of several growth factor
receptors and mediators of PEPCK and G-6-Pase expression levels. Adenoviral
overexpression of the dominant-negative mutant of the PI3K regulatory subunit p85α, which
lacks the binding site for the PI3K catalytic subunit, increased Pck1 and G6pc gene
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expression, as well as hepatic glucose production in vivo.32 Pharmacological inhibition of

PI3K also inhibited the insulin-mediated suppression of Pck1 and G6pc expression.33
Therefore, there is evidence that PI3K is a necessary downstream component of insulin-
suppressed gluconeogenesis.
PDK1
Downstream of PI3K, the generation of PIP3 is known to increase the activity of 3-
phosphatidylinositol-dependent kinase-1 (PDK1).28 Mice deficient for PDK1 in the liver
have decreased glucose tolerance, decreased pyruvate tolerance, and fail to normalize blood
glucose levels 2 h after insulin administration,34 probably because livers from these mice
display a massive defect in glycogen storage. Furthermore, hepatic glucose output and
gluconeogenic gene expression were not suppressed by feeding in these mice. Acute insulin-
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triggered Akt signaling was also impaired in mice lacking PDK1 in the liver. These findings
suggest that PDK1 is important for insulin signaling but also plays a major role in glucose
metabolism under physiological conditions.
Akt
PIP3 generated by PI3K allows Akt/PKB to bind and be phosphorylated by PDK1.35
Insulin-stimulated PI3K-mediated phosphorylation of Akt at Ser473 activates the kinase.36
Akt kinases control diverse functions, including cell growth, survival, proliferation, and
metabolism. However, the mechanisms through which Akt activity is specified to particular
cellular functions in response to extracellular stimuli are not fully understood. Studies of Akt
isoform–specific gene knockout mice suggested that Akt signaling diversity might in part be
due to the different functions of the three Akt family members AKT1, AKT2, and AKT3.37
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Among the three, AKT2 appears to play the major role in glucose metabolism; however,
redundancy in Akt functions also appear to occur. Akt2 knockout mice developed a type 2
diabetes–like phenotype, and cells derived from these mice had impaired glucose utilization,
suggesting a central role for AKT2 in the maintenance of glucose homeostasis.38
Simultaneous deletion of Akt1 and Akt2 caused lethality shortly after birth,39 and Akt1 and
Akt3 double-knockout mice were embryonic lethal.40 However, mice with knockout of Akt2
and Akt3 with a single functional allele of Akt1 (Akt1+/−Akt2−/−Akt3−/−) were viable
despite reduced body weight and insulin and glucose intolerance.37 While knockout of Akt2
in mice caused insulin resistance and diabetes-like symptoms, including increased hepatic
glucose production, AKT1 was dispensable for glucose homeostasis.38,39 Of note, a role for
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increased Pck1 and G6pc transcription was not demonstrated in these mouse models,
suggesting regulation of acute glucose output in vivo rather than of chronic transcriptional
changes. However, overexpression of Akt in hepatoma cells and primary hepatocyte cultures
decreased Pck1 and G6pc gene transcription, demonstrating that Akt can regulate
transcription of gluconeogenic enzymes.41
Several proteins have been identified to mediate suppression of Pck1 and G6pc by Akt, such
as FOXO1, a master regulator of gluconeogenesis.42 Akt seems to be the key regulator of
FOXO1 activity.43 Ablation of Akt1 and Akt2 in the livers of mice leads to insulin resistance
and diabetes and increased expression of several FoOXO1 target genes.20 Pck1 and G6pc
expression levels in the knockout livers were not different from that in the control livers
during fasting, but the normal suppression of these genes after feeding was lost. Deletion of
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Foxo1 in these mice normalized the hyperglycemia, glucose intolerance, hyperinsulinemia,

and the response to feeding despite defective insulin signaling.20 This study elegantly
showcased the regulatory effect of the Akt–FOXO1 network on hepatic gluconeogenesis.
Activated Akt can phosphorylate and inhibit glycogen synthase kinase-3 (GSK-3), which
inhibits glycogen synthase and therefore enhances the release of G-6-P from glycogen.
However, whether this is a direct insulin-mediated effect remains unclear. Pharmacological
inhibition of GSK-3 was shown to suppress Pck1 and G6pc transcription, but to a lesser
extent than insulin.40 Furthermore, overexpression of GSK-3 had no effect on insulin-
mediated suppression of Pck1 and G6pc transcription, suggesting different mechanisms of
action for GSK-3 and insulin on Pck1 and G6pc expression.40
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MAPK
Insulin is a potent simulator of the Raf/MEK/ERK1/2 pathway, and this pathway might be
involved in the regulation of gluconeogenesis. Thus far, neither pharmacological inhibition
nor overexpression of components of the Raf/MEK/ERK1/2 pathway have been shown to be
efficient in suppressing Pck1 and G6pc transcription or hepatic glucose production.33,44,45
However, the downstream effector p38 has gained attention in the regulation of
gluconeogenesis. Inhibition of p38 by siRNA or a pharmacological inhibitor reduced
glycemia in mice and suppressed gluconeogenesis in liver, along with suppression of Pck1
and G6pc transcription.46 Activation of p38 by free fatty acids showed similar results on
Pck1 and G6pc transcription.47,48 Mice lacking MAPK phosphatase 1 (MKP-1), a negative
regulator of p38 and JNK activity, exhibited increased gluconeogenesis and hepatic insulin
resistance.49 P38 inhibition also decreased transcription of peroxisome proliferator–activated
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receptor γ coactivator 1α (Ppargc1a) and Creb, suggesting transcriptional control of these

major regulators of gluconeogenic gene expression.46 P38 can also activate PGC-1α by
phosphorylation.50 Along with PGC-1α and CREB, p38 also phosphorylates and activates
FOXO1, implicating a broader role for p38 in the transcriptional regulation of glucose
homeostasis and gluconeogenesis.46,47,51 Additionally, insulin promotes glycogen synthesis
through the activation of protein phosphatase-1.52 This is mediated through the Ras/MAPK
pathway, and protein phosphatase-1 in turn activates glycogen synthase while
simultaneously inactivating phosphorylase a and phosphorylase kinase to control glycogen

metabolism.52
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CDC-like kinase
CDC-like kinase 2 (CLK2) belongs to a large and highly conserved family of kinases.
Among the CLKs, CLK1–4 play roles in mRNA splicing and nuclear recruitment of
proteins.53 However, CLK2 is the only kinase known to be regulated by insulin and to have
an effect on hepatic gluconeogenesis. CLK2 is directly phosphorylated and thereby
stabilized via insulin-activated Akt.54 Overexpression of CLK2 decreased hepatic glucose
output in mice and corrected hyperglycemia in db/db mice.55 On the contrary, in liver-
specific Clk2 knockout mice, no diabetic phenotype was observed, suggesting a
compensatory adaptive mechanism during chronic CLK2 deficiency.56 CLK2 potentially
mediates its effects through modulation of the PP2A–phosphatase complex and
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phosphorylation of PGC-1α, leading to decreased transcriptional activity of PGC-1α.54,56
Cyclin-dependent kinases
Cyclin-dependent kinases (CDKs) are a family of protein kinases first characterized for their
roles in the cell cycle.57 They are also involved in regulating transcription, mRNA
processing, and differentiation.58 They are present in all known eukaryotes, and their
regulatory function in the cell cycle has been evolutionarily conserved.59 The mechanism by
which insulin affects the activity of CDKs is not completely understood. Reports suggested a
role for insulin in the activity of CDK regulatory subunit 1 (CKS1), CDK4, and CDK5.
CKS1 is upregulated by insulin via the insulin receptor to promote cell proliferation in
insects, but no effect on gluconeogenesis was reported.60 Two independent reports recently
showed the activation of CDK4 through refeeding and insulin in the liver. Both groups also
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observed profound effects on hepatic gluconeogenesis. This very likely occurs via the
inhibition of PGC-1α transcriptional activity.61,62 The role of CDK5 in insulin signaling is
unclear. CDK5 activity is increased by insulin, and this effect can be abolished by a PI3K
inhibitor in primary adipocytes.63 In adipose tissue, activated CDK5 promotes adipogenesis
through phosphorylation and activation of PPAR-γ.64 A role for CDK5 in regulating hepatic
glucose production remains elusive.
Transcriptional regulation of gluconeogenesis by insulin

FOXO1
In mammals, the Forkhead protein family comprises four proteins, FOXO1, FOXO3
(FOXO3a), FOXO4, and FOXO6. Among these, FOXO6 shows a high specificity to
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neuronal localization, while the other three factors are widely distributed and are present in
most tissues. There seems to be a considerable overlap in the transcriptional targets of the
first three, and there is also evidence that each of these can compensate for loss of the others
to some degree.65,66 While Foxo1 knockout is embryonically lethal owing a the failure of
angiogenesis, Foxo3 knockout produces premature ovarian failure, and Foxo4 knockout has
no obvious phenotype.28 Triple conditional knockouts resulted in lymphomas,
hemangiomas, and angiosarcomas, which did not occur with double-knockout combinations.
65 FOXO1 specifically plays a major role in the regulation of insulin sensitivity.66 FOXO1
transcriptional activity is regulated by a complex array of posttranslational modifications

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(PTMs). With regards to the gluconeogenic function of FOXO1, the primary regulatory
event is Akt-mediated phosphorylation of three conserved residues, one threonine and two
serines (Thr24, Ser253, and Ser316 in Mus musculus), that results in binding to 14-3-3
proteins and nuclear export of FOXO1.66
Mice with specific deletion of Foxo1 in the liver show reduced fasting glucose and
gluconeogenic gene expression, and glucose clamp studies demonstrate that Foxo1 ablation
impairs fasting- and cAMP-induced glycogenolysis and gluconeogenesis. Furthermore,
Foxo1 deletion prevents neonatal diabetes and steatohepatitis in insulin receptor gene
knockout mice.67 When FOXO1 is constitutively expressed in the murine liver, fasting blood
glucose increases.68 There is strong evidence that FOXO1 directly regulates Pck1 and G6pc
transcription. FOXO1 has been reported to bind to the insulin-responsive sequences of the
Pck1 and G6pc promoters in vitro.69,70 The importance of Akt phosphorylation on FOXO1
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regulation is striking. In the fed state, insulin signaling activates PI3K and subsequently Akt.
Akt then phosphorylates FOXO1, leading to its inactivation though its export from the
nucleus, with subsequent suppression of gluconeogenesis.20 Hyperglycemia following Akt
gene deletion can be corrected by concomitant hepatic deletion of Foxo1.20,38 These data
indicate that the regulation of hepatic glucose metabolism and the maintenance of glucose
homeostasis are strongly mediated though the Akt–FOXO1 axis. In this context, the main
function of insulin-mediated signaling is to counteract FOXO1 and thus reduce glucose
production during the fed state. However, FOXO1 does not inhibit the insulin-mediated
upregulation of anabolic processes, such as glycogen and lipid synthesis.20
As mentioned briefly earlier, the activity of FOXO1 is also modulated by processes other
than Akt-mediated phosphorylation. Its acetylation and deacetylation provide a second tier
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of regulation, and deacetylation is generally considered to enhance FOXO1 activity to

promote gluconeogenesis. FOXO1 can be deacetylated by SIRT1 under conditions of
cellular stress, in the process overriding the nuclear exclusion effect of Akt and causing
nuclear retention and expression of FOXO1 target genes, including Pck1 and G6pc.71 Class
IIa HDACs can also contribute to deacetylation of FOXO1 and have been shown to be
positive regulators of hepatic FOXO1 in response to glucagon signaling during fasting.72
Once these HDACs are activated through AMPK-dependent phosphorylation, they
translocate to the nucleus, where they deacetylate and activate FOXO1, inducing
transcription of gluconeogenic genes.66,71
Several other mechanisms of modulating FOXO1 activity, such as regulation of its

glycosylation, ubiquitination, and proteosomal degradation, have recently been described.
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The transcription factor XBP-1, involved in the unfolded protein response, has been shown
to increase insulin sensitivity. This activity is independent of its transcriptional effects, and
may be mediated through its indirect interaction with FOXO1, acting as a chaperone to
direct it toward proteosomal degradation.73 The herpesvirus-associated ubiquitin-specific
protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease
(HAUSP)) is capable of mono-deubiquinating FOXO1, resulting in suppression of FOXO1
transcriptional activity through decreased FOXO1 occupancy on the promoters of
gluconeogenic genes.74 Another mechanism that can be utilized to modulate FOXO1
activity is through the glucose-derived O-linked β-N-acetylglucosamine (O-GlcNAc)

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modification. In diabetes, this specific modification is increased on hepatic FOXO1. It

regulates FOXO1 activation in response to glucose, resulting in increased expression of
gluconeogenic genes, while concomitantly inducing expression of genes involved in the
ROS detoxification pathways.75,76 Paradoxically, it is induced by hyperglycemia and
appears to result from PGC-1α binding to O-GlcNAc transferase and targeting it to nuclear
FOXO1.77 Other signaling pathways, such as thyroid hormone signaling, retinoid signaling,
and p53-regulated pathways, have been linked to modulation of the FOXO1 transcriptional
activity on gluconeogenic genes.55,78,79 The physiological relevance of these findings has
yet to be determined.
CREB
CREB was first identified as a transcription factor that binds to cyclic AMP–binding element
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(CRE) on the promoter of the somatostatin gene.80 Mice that express a dominant-negative
inhibitor of CREB in the liver (alb-ACREB transgenic mice) showed decreased blood
glucose levels and decreased expression of gluconeogenic genes, emphasizing the role of
CREB in hepatic gluconeogenesis as a progluconeogenic factor.81 Since then, the role of
CREB as a crucial transcription factor in liver metabolism has been studied extensively. The
key event in CREB activation is its phosphorylation at Ser133 following an increase in
intracellular cAMP levels and subsequent activation of PKA. Activated PKA translocates to
the nucleus and phosphorylates CREB, which is critical for the interaction of CREB with
CBP/p300 to promote its transcriptional activity.13 Apart from PKA, other kinases have been
reported to phosphorylate CREB, including calmodulin-dependent kinases and MAPK
kinases, such as p38.13 Reversal of CREB phosphorylation can be mediated through PP1
and PP2 phosphatases, with potential tissue- and cell-specific regulatory patterns.13
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However, the interaction of CREB with its coactivators, rather than the phosphorylation of
Ser133, appears to be the critical event that promotes its transcriptional activity.13 CBP and
p300 are closely related histone acetyl transferase orthologs that catalyze the transfer of the
acetyl group from acetyl CoA to lysine residues of histone or non-histone proteins. The
formation of a transcriptional complex of CREB and CBP/p300 seems to be critical for the
selective transcriptional induction of cAMP-responsive genes.13,82 Insulin has also been
found to inhibit gluconeogenesis by selectively disrupting the CREB–CBP interaction.
Refeeding triggers the phosphorylation of CBP at Ser436 by the atypical PKC-ι/γ (aPKCι/
γ). This modification on CBP appears to block binding of CREB that is phosphorylated at
Ser133. However, the regulatory Ser436 site in CBP is not conserved in p300, suggesting
that CBP and p300 perform distinct roles in the liver.13,83 CBP/p300 can additionally be
phosphorylated and thereby inactivated by salt-inducible kinase (SIK).84 It has also been
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reported that active CBP/p300 can stabilize CRTC2 through acetylation of a lysine residue,
thereby increasing its activity.85 The CRTC family consists of three members (CRTC1,
CRTC2, and CRTC3) that have similar modular structures: they all contain an N-terminal
CREB-binding domain (CBD), a central regulatory domain (REG), a splicing domain (SD),
and a C-terminal transactivation domain (TAD).13 In the basal state, CRTCs are sequestered
in the cytoplasm through phosphorylation-dependent interactions with 14-3-3 proteins, such
as SIK.13 Exposure to cAMP and calcium, but not other signals, triggers the calcineurin-
mediated dephosphorylation and nuclear translocation of CRTCs, which then bind to CREB
over relevant gene promoters. Among the different family members, CRTC2 is highly
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expressed in the liver. In fasting conditions, PKA-dependent inactivation of SIK leads to

dephosphorylation of CRTC2 and its nuclear translocation.86 Mice with a Crtc2 knockout
have decreased circulating glucose concentrations during fasting, lowered circulating blood
glucose concentrations, and improved insulin sensitivity in the context of diet-induced
obesity.87 Hyperglycemia affects the activity of CRTC2 by enhancing O-glycosylation,
resulting in reduced phosphorylation at Ser171, nuclear retention, and subsequent increased
activity.13,88
CREBH
CREBH is an endoplasmic reticulum (ER)-bound transcription factor that is controlled by
regulated intramembrane proteolysis (RIP) of the ER, which is known to maintain sterol
homeostasis and to mediate the unfolded protein response (UPR). It is mainly expressed in
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the liver and is activated upon acute ER stress, when it promotes the transcription of acute
phase response genes, such as Crp (C-reactive protein) or Sap (serum amyloid P-
component). CREBH has been reported to induce the transcription of gluconeogenic genes,
and acute knockdown of CREBH reduces fasting blood glucose levels in mice.89 In mice
with hepatic knockout of CREBH, a decrease in hepatic lipid content was observed in those
on normal chow diet, while those on a high-fat diet displayed increased steatosis, suggesting
that CREBH is involved in lipogenesis and lipolysis; however, no abnormalities in blood
glucose were observed.90 The same study reported that insulin treatment promotes cleavage
and thereby activation of CREBH.90
PGC-1α
PGC-1α was initially identified in brown adipocytes as a coactivator of the transcription
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factor PPAR-γ, where it is critical for the control of thermogenesis and mitochondrial
biogenesis.91,92 The family of PGC-1 transcriptional coactivators includes various isoforms
of PGC-1α as well as PGC-1β and PGC-1–related co-activator (PRC). They share similar
structural features, comprising an N-terminal activation domain, a central regulatory domain,
and a C-terminal RNA-binding domain.93 Following PPAR-γ, PGC-1α has been shown to
interact with various transcription factors, such as PPAR-α, PPAR-δ, FOXO1, and SREBP,
and nuclear receptors, such as estrogen related receptors (ERRs), hepatocyte nuclear factor
4α (HNF4α), and glucocorticoid receptor (GR).93 In addition, PGC-1α interacts with
several other proteins, including HAT domain–containing proteins, such as CBP and p300,
via the N-terminal domain, and the mediator complex via the C-terminal region.94,95
Knockdown or knockout of Ppargc1a in primary hepatocytes results in decreased glucose
production and reduced expression of gluconeogenic genes, while Ppargc1a null mice show
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increased gluconeogenic gene expression insensitive to normal feeding controls.96

Overexpression of PGC-1α in the liver of mice promotes the expression of gluconeogenic
genes by enhancing the activity of various transcription factors, such as CREB, HNF4α, and
FOXO1.81,97,98 Insulin-stimulated Akt-mediated phosphorylation of FOXO1 obstructs its
interaction with PGC-1α and interferes with the activation of hepatic gluconeogenic gene
expression in mice.97 Insulin can also regulate PGC-1α activity via the alteration of its
acetylation and phosphorylation status. Acetylation of hepatic PGC-1α is controlled by
counteracting effects of the GCN5 acetyltransferase and the SIRT1 deacetylase, and this
PTM impairs the ability of PGC-1α to promote gluconeogenic gene expression.99,100 Insulin
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activates the cyclin D1/cyclin-dependent kinase 4 complex, which increases GCN5

acetyltransferase activity through phosphorylation, to promote the acetylation of PGC-1α
that results in the suppression of hepatic glucose production.62 The transcriptional co-
regulator CITED2 (CBP- and p300-interacting transactivator with glutamic acid– and
aspartic acid–rich COOH-terminal domain 2) activates PGC-1α by decreasing its acetylation
through blocking its interaction with GCN5, resulting in increased gluconeogenic gene
expression; however, this event is negatively regulated by insulin in an Akt-dependent
manner.101 Additionally, activation of Akt through insulin signaling enables it to
phosphorylate PGC-1α directly at Ser570, thereby inhibiting its ability to be recruited to
gluconeogenic gene promoters.102 The insulin-regulated CLK2 phosphorylates PGC-1α in
its serine- and arginine-rich (SR) domain, impairing its ability to function as a
transcriptional coactivator, specifically toward FOXO1.55 The insulin-inducible corepressor
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small heterodimer partner–interacting leucine zipper protein (SMILE) directly competes

with PGC-1α for HNF4α to suppress its hepatic transcriptional activity and
gluconeogenesis.103
Glucocorticoid receptor
GR has been linked to hepatic gluconeogenesis for a long time. It is activated by endogenous
cortisol as well as exogenous corticosteroids. Upon fasting, increased secretion of
glucocorticoids activates GR in the liver, which promotes transcription of gluconeogenic
genes.104 Even though insulin does not directly interact with GR, the transcriptional activity
of GR is important for modulating insulin-mediated signaling. GR increases the
transcription of Mapk14 (p38 MAPK) and thereby counteracts the insulin-mediated effects
on gluconeogenesis. Glucocorticoids also diminish IR and IRS1 phosphorylation in response
to insulin in liver.105
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Sterol response element–binding protein

Members of the sterol response element–binding protein (SREBP) family were initially
characterized as transcription factors that are activated by low cholesterol levels in the cell.
106 The SREBP-1c isoform in the liver has been demonstrated to be regulated by nutritional
stimuli in vivo, such as a carbohydrate-rich diet, and insulin has been found to rapidly
stimulate the transcription of Srebp1 in hepatocytes and adipose and muscle tissue. The
effect of insulin on the expression level of SREBP-1c protein in hepatocytes appears to be
mediated through activation of PI3K. In addition, by employing a version of Akt that can be
conditionally regulated, studies in hepatoma cells provide evidence that activation of Akt is
sufficient to mimic this biological response to insulin.107 While mice with siRNA-mediated
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knockdown of Srebp1 show decreased gluconeogenic gene expression, no effect was

observed on fasting glucose levels.108 A suggested mechanism of SREBP-1 action on
gluconeogenesis is its interference with the HNF4α-mediated recruitment of PGC-1α to
gluconeogenic gene promoters.109 Coimmunoprecipitation experiments demonstrate that
these two transcription factors (SREBP-1 and HNF4α) directly interact through the
transactivation domain of SREBP and the ligand binding/AF2 domains of HNF4α.109 In the
same study, SREBP-1 does not bind the Pck1 promoter, supporting an indirect effect of
SREBP-1 on gluconeogenic gene expression, possibly through altering the localization of
HNF4α and PGC-1α.109 Overall, the role of SREBP-1 in gluconeogenesis appears to be

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moderate, and the main functional role lies in the regulation of lipid metabolism.
STAT3
STAT3 is a member of the signal transducer and activator of transcription (STAT) protein
family. STAT3 is phosphorylated by receptor-associated Janus kinases (JAKs) in response to
cytokines and growth factors. Activated STAT3 proteins form homo- or heterodimers, and
translocate to the cell nucleus, where they act as transcription activators.110 In the liver,
STAT activation is mainly linked to inflammation and cancer. Interestingly, STAT3 has the
ability to directly bind to the promoters of Pck1 and G6pc to inhibit the promoter activity of
these gluconeogenic genes.111 In mice, liver-specific deletion of Stat3 increases the
expression of Pck1, G6pc, and Ppargc1a. Conversely, liver-specific overexpression of a
constitutively active form of STAT3 decreases hepatic glucose production and blood glucose
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levels in diabetic mice.112 A significant mechanism through which insulin is able to

modulate STAT3 activity is its hypothalamic action. Insulin action in the hypothalamus
stimulates IL-6 production in the liver, and IL-6 in turn suppresses gluconeogenesis by
activating STAT3.113 Another point of cross talk between insulin and STAT3 is through
GSK-3β. STAT3 suppresses the expression of GSK-3β, a negative regulator of the insulin
signaling pathway. Mice lacking STAT3 in the liver do not exhibit a physiological decrease
of Gsk3b mRNA or GSK-3β protein levels in response to feeding, which suggests that these
mice display an impaired response to insulin.114
DAX1
Dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X-
chromosome, gene 1 (DAX1) is an atypical nuclear receptor that lacks a classical DNA-
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binding domain. It was initially described in hypogonadotropic hypogonadism and is mainly

expressed in adrenal glands, the pituitary gland, and the testes.115 DAX1 has been shown to
be expressed in the liver, and its expression is increased by insulin and SIK1, whereas it is
decreased in diabetic and high-fat diet–fed mice.116 It has been shown that DAX1 blocks the
association of HNF4α and PGC-1α with the gluconeogenic gene promoters, suggesting a
novel mechanism for feeding-induced repression of gluconeogenesis.116 However, the
mechanism through which insulin or insulin-related signaling increases DAX1 is not clear.
Current data suggest an increase in Dax1 transcription. In testicular Leydig cells, Dax1
transcription is increased by insulin in an Akt-dependent manner. However, high-fat diet
suppresses hepatic DAX1 levels in mice, suggesting that DAX1 regulation is distinct in
different tissues.116,117
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Insulin signaling and its impact on hepatic gluconeogenesis in diabetes

and obesity
In obesity and diabetes, tissue homeostasis is disturbed in many compartments, such as
skeletal muscle, adipose tissue, liver, kidney, and connective tissues. Eventually, these
changes lead to tissue damage and remodeling, as seen in liver fibrosis, kidney fibrosis, or
atherosclerosis. Additionally, these changes are major drivers of insulin resistance and
thereby fuel a vicious cycle leading to organ damage, morbidity, and mortality.
Inflammation is a widely recognized occurrence in obesity and diabetes and has been linked
to the pathogenesis of diabetes, mainly through impaired insulin signaling.118 Chronic as
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well as acute inflammation have been clearly linked to insulin resistance and elevated levels
of cytokines, such as TNF-α, and have been described and extensively studied in this
context.119,120 TNF-α signaling can activate various intracellular pathways, such as those
involving JNK and IKK, and this inhibits insulin receptor signaling through serine
phosphorylation of IRS1.121 In contrast to the activating tyrosine phosphorylation, this
modification leads to inactivity of IRS1 and eventual degradation of the protein.122 Serine
phosphorylation of IRS1 can come about as a result of several events, including through
mTOR, S6 kinase, PKC-θ, and JNK signaling.123
Disturbed hepatic lipid metabolism is also known to affect insulin signaling and hepatic
glucose production. Several studies support a role for hepatic diacylglycerol (DAG)
accumulation and PKC-ε activation as factors for impaired hepatic insulin action. DAG has
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been shown to promote PKC activation with a subsequent decrease in insulin receptor
tyrosine kinase activity. Mice lacking PKC are protected from diet-induced insulin
resistance, even though hepatic lipid content is increased.124 In humans, FOXO1 expression
and transcriptional activity are increased in nonalcoholic steatohepatitis (NASH) liver,
emphasizing a role for inflammation and disturbed lipid metabolism in the dysregulation of
hepatic gluconeogenesis.25
Non-alcoholic fatty liver disease (NAFLD) is a growing subclass of liver disease, as it is

closely related to obesity, metabolic syndrome, and diabetes. Patients diagnosed with hepatic
steatosis have an increased risk for cardiovascular disease, as well as for diabetes and related
conditions.125 An important trigger may be hepatic insulin resistance, as it may be sufficient
to promote dyslipidemia and atherosclerosis.126 In addition, excess lipid accumulation in the
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liver has negative effects on the progression of liver disease. Interestingly, hepatic insulin
signaling is impaired, leading to not only an increase in HGP but also an ongoing hepatic
lipid production that results in hepatic steatosis, steatohepatitis, and cirrhosis. The
phenomenon where impaired insulin signaling blocks one of its actions (decrease of HGP)
while promoting another action (lipogenesis) is termed selective insulin resistance. A
potential mechanism leading to this paradox in insulin action in the brain and the adipose
tissue, which results in increased lipolysis and excess triglyceride synthesis in the liver, has
been recently reviewed elsewhere.127
Additionally, insulin action in the central nervous system can indirectly regulate metabolism
in the liver. Insulin signaling in the brain through hypothalamic PI3K and ATP-sensitive
potassium channels has been suggested to suppress Pck1 and G6pc expression in the liver,
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which results in the blunting of gluconeogenesis; activation of the hepatic IL-6/STAT3

pathway has been demonstrated to be mechanistically involved in this process.112,128–130
Insulin-mediated FOXO1 nuclear exclusion through the PI3K–Akt axis in pro-
opiomelanocortin (POMC) neurons has also been suggested to suppress basal HGP.131
However, a study by Ramnanan et al. indicated that, in dogs, regulation of hepatic
gluconeogenesis mediated by the action of insulin on the brain is debatable.132 Scherer et al.
additionally showed that insulin signaling in the rat brain is able to modulate triglyceride
secretion and lipid content in the liver.133 Thus, we highlighted the promise of potentially
targeting insulin resistance in the central nervous system for the treatment of type 2 diabetes.
134
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Concluding remarks
An intact insulin signaling system is paramount to maintaining blood glucose levels within a
narrow normal glycemic range during periods of fasting or excess nutrient availability, and
this is achieved in particular via regulation of metabolic flux through the hepatic
gluconeogenic pathway. This review summarizes some of the mechanisms through which
insulin can modulate hepatic gluconeogenesis. A significant node of control involves the
transcriptional regulation of expression of the key hepatic gluconeogenic genes Pck1 and
G6pc, which occurs primarily through the transcription factor FOXO1 and nuclear receptor
HNF4α and their transcriptional coactivator PGC-1α. Understanding these regulatory
pathways is of extreme importance in terms of identifying potential targets and devising
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treatments for disorders involving insulin resistance and dysregulated hepatic

gluconeogenesis, such as in type 2 diabetes.
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Figure 1.
Regulation of gluconeogenic gene expression by hepatic insulin signaling. Insulin action
regulates the activity of trancription factors controlling gluconeogenic gene expression.
AKT-mediated phosphorylation leads to nuclear export of FOXO1. Inhibitory
phosphorylation of CBP/p300 blocks trancription-complex formation of CREB.
Modification of PGC-1α by GCN5-mediated acetylation or AKT/CLK2-mediated
phosphorylation decreases PGC-1α transcriptional activity.
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Table 1
Extrahepatic effects of insulin that regulate hepatic gluconeogenesis.

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Organ/tissue Action Effect on liver Reference
Pancreatic alpha cells Secretion of glucagon ↓ Transcriptional regulation of gluconeogenic genes 12–14, 16
White adipose tissue Lipolysis ↓ Reduction of free fatty acid delivery to the liver 10,14,17
Skeletal muscle Proteolysis ↓ Reduction of amino acid flux to the liver 10,14
Central nervous system Pleiotropic manner Multiple effects 18
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Table 2
Animal models of targets of insulin signaling

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Insulin target Phenotype of (liver-specific) KO mice Potential mechanism of action Reference
IRS1, IRS2 Hyperglycemia, hyperinsulinemia Induction of gluconeogenic genes 29–31

PI3K (p110-α and p110-β Diet-induced liver steatosis ↓ Insulin-induced Akt phosphorylation at Ser473 ↓ 135
subunits) Glucose intolerance (p110-α) ↑
PDK1 Glucose tolerance ↓ Disruption of acute AKT-dependent insulin signaling 34
Pyruvate tolerance ↓
Insulin sensitivity ↑
Akt2 Insulin resistance ↑ Increased expression of FOXO1 target genes 20, 38, 39
Hepatic glucose production ↑
p38 MAPK Glycemia ↓ Decreased transcription of Pparg, Ppargc1a 46
Gluconeogenesis ↓
FoxO1 Fasting glucose ↓ Direct regulation of G6pc and Pck1 transcription 67–70
Gluconeogenic gene expression ↓
Gluconeogenic gene expression ↑
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PGC-1α Regulation of G6pc and Pck1 transcription 96

Loss of feeding-induced regulation
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Insulin Regulation of Gluconeogenesis 2018

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Insulin regulation of gluconeogenesis

insulin-moderated pathways is critical for therapeutic purposes.

the framework of hepatic gluconeogenesis.

Gluconeogenesis contributes to hepatic glucose production

The final reaction of gluconeogenesis––the formation of glucose––occurs in the lumen of

pathophysiology. Insulin is a major hormone regulator of gluconeogenesis, so understanding

Insulin action on gluconeogenesis is both direct and indirect

glycogenolysis is completely suppressed.5 Hyperglycemia alone suppresses hepatic

The dominant mechanism of insulin-mediated regulation of hepatic gluconeogenesis is not

Suppression of gluconeogenesis through indirect effects of insulin is known to involve

rodents, resulting in an increase in pyruvate carboxylase activity and gluconeogenesis. The

Insulin regulation of hepatic gluconeogenesis through transcriptional

Whether transcriptional regulation of gluconeogenic enzymes can be observed in human

patients4,27 and insulin affects gluconeogenesis through transcription, determining the

Insulin activates several signaling pathways that regulate gluconeogenesis

IRS1 and IRS2

hyperglycemia, hyperinsulinemia, and induction of gluconeogenic genes, such as Pck1 and

expression, as well as hepatic glucose production in vivo.32 Pharmacological inhibition of

Foxo1 in these mice normalized the hyperglycemia, glucose intolerance, hyperinsulinemia,

receptor γ coactivator 1α (Ppargc1a) and Creb, suggesting transcriptional control of these

simultaneously inactivating phosphorylase a and phosphorylase kinase to control glycogen

phosphorylation of PGC-1α, leading to decreased transcriptional activity of PGC-1α.54,56

Transcriptional regulation of gluconeogenesis by insulin

transcriptional activity is regulated by a complex array of posttranslational modifications

of regulation, and deacetylation is generally considered to enhance FOXO1 activity to

Several other mechanisms of modulating FOXO1 activity, such as regulation of its

activity is through the glucose-derived O-linked β-N-acetylglucosamine (O-GlcNAc)

modification. In diabetes, this specific modification is increased on hepatic FOXO1. It

expressed in the liver. In fasting conditions, PKA-dependent inactivation of SIK leads to

increased gluconeogenic gene expression insensitive to normal feeding controls.96

activates the cyclin D1/cyclin-dependent kinase 4 complex, which increases GCN5

small heterodimer partner–interacting leucine zipper protein (SMILE) directly competes

Sterol response element–binding protein

knockdown of Srebp1 show decreased gluconeogenic gene expression, no effect was

HNF4α and PGC-1α.109 Overall, the role of SREBP-1 in gluconeogenesis appears to be

levels in diabetic mice.112 A significant mechanism through which insulin is able to

binding domain. It was initially described in hypogonadotropic hypogonadism and is mainly

Insulin signaling and its impact on hepatic gluconeogenesis in diabetes

Non-alcoholic fatty liver disease (NAFLD) is a growing subclass of liver disease, as it is

which results in the blunting of gluconeogenesis; activation of the hepatic IL-6/STAT3

treatments for disorders involving insulin resistance and dysregulated hepatic

investigation. 1992; 90:1323–1327. [PubMed: 1401068]

32. Miyake K, Ogawa W, Matsumoto M, Nakamura T, Sakaue H, Kasuga M. Hyperinsulinemia,

chemistry. 2000; 275:30169–30175. [PubMed: 10913147]

regulate gluconeogenesis by promoting FoxO1 nuclear exclusion. Proceedings of the National

Young RA, Montminy M. Genome-wide analysis of cAMP-response element binding protein

protein-1c. The Biochemical journal. 2002; 366:377–391. [PubMed: 12061893]

114. Moh A, Zhang W, Yu S, Wang J, Xu X, Li J, Fu XY. STAT3 sensitizes insulin signaling by

133. Scherer T, Lindtner C, O’Hare J, Hackl M, Zielinski E, Freudenthaler A, Baumgartner-Parzer S,

Extrahepatic effects of insulin that regulate hepatic gluconeogenesis.

Organ/tissue Action Effect on liver Reference

Animal models of targets of insulin signaling

Insulin target Phenotype of (liver-specific) KO mice Potential mechanism of action Reference

IRS1, IRS2 Hyperglycemia, hyperinsulinemia Induction of gluconeogenic genes 29–31

PGC-1α Regulation of G6pc and Pck1 transcription 96

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