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2
Insulin-mediated Regulation
of Glucose Metabolism
Daniel Konrad, Assaf Rudich and Amira Klip
2.1 Introduction
Insulin was identified in the early 1920s as the major hypoglycaemic hormone,
capable of restoring normal blood glucose levels in pancreatectomized animals
and insulin-deficient humans. This physiological action of insulin is brought
about by its effects on glucose metabolism in its ‘classical’ target organs,
namely liver, skeletal muscle and adipose tissue. Decades of intense scientific
effort involving complementary disciplines have unravelled cellular mechanisms
whereby insulin regulates glucose metabolism in these tissues, resulting in the
control of blood glucose in diverse physiological states. In this chapter we first
outline the actions of insulin as the master switch for whole body glucose distri-
bution and summarize its effects on the main biochemical pathways of glucose
metabolism. We then focus on the effect of insulin on glucose disposal in muscle
and fat. As glucose uptake is rate limiting for glucose metabolism in these tis-
sues, we describe in more detail the complex mechanisms through which insulin
regulates this process.
2.2 Insulin as a master regulator of whole body glucose
disposal
Direct and indirect regulation of glucose metabolism by insulin
in its classical target tissues
Insulin is more than an endocrine messenger for the transition from fasted- to
fed-state metabolism; it is in fact a required regulator in all physiological states.
Insulin Resistance. Edited by Sudhesh Kumar and Stephen O’Rahilly
 2005 John Wiley & Sons, Ltd ISBN: 0-470-85008-6
64 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM
During fasting, circulating insulin levels are 5–20 per cent of those measured
after a meal. These low circulating concentrations of the hormone are required

to maintain the balance with counter-regulatory hormones to prevent ketoacido-
sis. The brain and cells such as erythrocytes that rely solely on glucose as their
energy source consume glucose evenly in the fed and overnight fasted states.
1
Therefore, maintaining normoglycaemia despite fluctuations in the availability
of exogenous glucose relies on a coordinated regulation of glucose disposal and
endogenous glucose production. During fasting, the liver, and to a lesser degree
the kidney, release glucose to the blood, matching its utilization by glucose-
dependent tissues such as the brain. Under these conditions, glucose disposal
into skeletal muscle and adipocytes is low, where lipids are consumed as the
major fuel. Upon a meal, when circulating glucose and insulin levels rise, glu-
cose is disposed of from the blood into muscle, fat and liver.
1
These tissues
therefore constitute the ‘classical target organs’ for insulin action through which
the hypoglycaemic response of the hormone is directly achieved (Table 2.1).
In addition to increasing glucose uptake into skeletal muscle and adipose tis-
sue, insulin promotes glucose storage as either glycogen (mainly in muscle
and liver) or lipids (mainly in fat and liver). To avoid futile metabolic cycles,
insulin simultaneously inhibits the breakdown of these macromolecules through
glycogenolysis and lipolysis, respectively. Similarly, in the liver and kidney
endogenous glucose production is curbed by insulin through the inhibition of
glycogenolysis and gluconeogenesis.
3, 4
Research of recent years utilizing tissue-specific gene deletions of the insulin
receptor in animal models largely confirmed the direct effects of insulin on
glucose metabolism in its classical target organs. Mice lacking the insulin recep-
tor in the liver (liver insulin receptor knock-out (LIRKO) mice) fail to sup-
press endogenous glucose production during hyperinsulinaemic clamps.
5

Like-
wise, ablation of the insulin receptor gene in skeletal muscle (MIRKO mice)
blunts insulin-stimulated glucose transport and glycogen synthesis in this tissue.
6
Remarkably, glucose uptake into adipose tissue is elevated in MIRKO mice,
suggesting that insulin’s direct effects on its classical target tissues are coordi-
nated, allowing for complex adaptive responses and balances in the regulation
of whole body glucose metabolism, as discussed further below. Hence, insulin
can be viewed as a glucose flux regulator, promoting peripheral glucose uptake
and hepatic glucose storage during a meal, and allowing hepatic glucose output
while preventing ketoacidosis between meals.
On top of the major direct effects of insulin on glucose fluxes in its classical
target organs (Table 2.1), the hormone also engages indirect mechanisms in the
regulation of glucose metabolism. These include insulin-mediated alterations in
lipid and protein metabolism (described in detail in Chapters 3 and 4 of this
book) that in turn impact on glucose metabolism, coordination of glucose fluxes
between its various target tissues, and regulation of circulating factors involved
in cross-talk between skeletal muscle, adipose tissue and liver.
INSULIN AS A MASTER REGULATOR OF WHOLE BODY GLUCOSE DISPOSAL 65
Table 2.1 ‘Classical’ and ‘non-classical’ target organs for insulin-regulated glucose
metabolism
Main effect of insulin on glucose metabolism
Organ/tissue Direct Indirect
‘Classical targets’
Skeletal and cardiac
muscle
↑ glucose uptake
↑ glucose oxidation
↑ glycogen synthesis
↓ NEFA availability and

oxidation
Liver ↓ glucose output –
•↓glycogenolysis
•↓gluconeogenesis
↑ glycogen synthesis
↑ glycolysis
↑ lipogenesis
↑ hexose monophosphate shunt
∗
↓ NEFA availability and
oxidation
Adipose tissue ↑ glucose uptake
↑ hexose monophosphate shunt
∗
↑ lipogenesis
Regulation of adipokines
synthesis and/or secretion
↓ lipolysis
‘Non-classical targets’
Pancreatic beta cells ? Permissive effect on
glucose-stimulated insulin
secretion (phase 1 release)
Brain/CNS ? ↓ Food intake
Vascular cells ? ↑ blood flow (vasodilatation)
↑ capillary recruitment
↑ NO secretion
A ‘direct effect’ of insulin on glucose metabolism is defined as an insulin-stimulated change in the flux
of glucose through a specific metabolic pathway that is initiated by the insulin receptor in the same
tissue. An ‘indirect effect’ is the regulation of glucose metabolism in one organ resulting from the effect
of insulin on other macronutrients (such as lipids) or in other organs.

∗
Insulin-stimulated lipogenesis consumes NADPH, and the resulting drop in NADPH/NADP
+
increases
the activity of G6PD, i.e. G6P flux through the shunt. In addition, insulin regulates the mRNA levels
of G6PD – the rate-limiting enzyme in this pathway.
2
Insulin is a key regulator of the interplay between glucose and fatty acids
metabolism, as follows. Insulin-mediated inhibition of lipolysis, i.e. the release
of non-esterified fatty acids (NEFAs) from adipose tissue, contributes to the
acute inhibition of hepatic glucose production induced by the hormone.
7, 8
This is brought about by lowering NEFA oxidation, since in this process high
intracellular ATP/ADP, NADH/NAD
+
and acetyl-CoA/CoA ratios are achieved,
providing the metabolite milieu required for gluconeogenesis. In addition, NEFA
availability to skeletal muscle also influences insulin-regulated glucose utiliza-
tion by this tissue. The original hypothesis of Randle
9
suggested that increased
NEFA availability as a fuel source to the muscle (as occurs physiologically dur-
ing fasting) blocks glycolysis through the elevated generation of acetyl-CoA and
66 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM
citrate, resulting in allosteric inhibition of pyruvate dehydrogenase and phospho-
fructokinase-1, respectively. The ensuing accumulation of glucose-6-phosphate
(G6P) would then secondarily diminish glucose uptake. However, while a glu-
cose–NEFA cycle probably exists, recent studies using nuclear magnetic res-
onance (NMR) spectroscopy demonstrate that, in skeletal muscle, increased
NEFA availability reduces, rather than elevates, intracellular G6P and free glu-

cose levels.
10, 11
These findings suggest that insulin-stimulated glucose uptake
is itself a primary site of inhibition by NEFA in skeletal muscle.
Further examples of the indirect actions of insulin emerge from knock-
out animal models. In particular, tissue-specific gene deletions have allowed
investigators to manipulate glucose flux in a single tissue and then assess the
ensuing changes in the non-targeted organs. As mentioned above, MIRKO mice
exhibited decreased insulin-stimulated glucose flux into the skeletal muscle,
accompanied by an increased glucose flux in adipose tissue.
6
Similarly, mice
lacking the insulin-responsive glucose transporter GLUT4 selectively in the adi-
pose tissue show not only reduced glucose uptake in fat cells but also blunted
insulin regulation of glucose metabolism in muscle and liver.
12
Such depen-
dency of glucose metabolism among the different organs suggests the existence
of mechanisms that mediate inter-organ cross-talk. An exciting development in
this regard has been the identification of adipose-derived factors (adipokines)
that modulate glucose metabolism and insulin responsiveness in muscle and the
liver.
13, 14
While factors such as tumour necrosis factor α and interleukin 6 are
not uniquely expressed in adipocytes, adiponectin and leptin are largely consid-
ered as adipose-specific gene products. Adiponectin (ACRP30) increases whole
body insulin sensitivity largely by suppressing glucose production in the liver, as
well as by increasing glucose uptake into skeletal muscle.
15, 16
Leptin, the prod-

uct of the obesity (ob) gene, signals through receptors in the hypothalamus to
decrease food intake and increase energy expenditure,
17
and may also act periph-
erally to regulate whole body insulin sensitivity.
18
Insulin positively regulates
both the gene expression and the secretion of leptin from adipose tissue,
19, 20
and may also regulate the gene expression of adiponectin.
21, 22
Affecting the cir-
culating concentrations of these regulatory factors provides a newly recognized
potent indirect mechanism through which insulin regulates glucose metabolism
in its classical target organs.
Non-classical targets of insulin in the regulation of total body glucose
metabolism
Although the major effect of insulin in controlling glucose metabolism is on its
classical target organs (muscle, liver and fat), virtually every cell type expresses
the insulin receptors. This raises the possibility that additional sites exist for
insulin action on carbohydrate metabolism. Tissue-specific ablation of the insulin
receptor has provided new insights about ‘non-classical’ target tissues for insulin
action (Table 2.1). Target tissues include pancreatic β-cells, the central nervous
INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLIC PATHWAYS 67
system and vascular cells. The findings suggest that these tissues may exert
important effects on glucose metabolism at the whole body level. For example,
mice lacking insulin receptors in β-cells manifest impaired glucose-mediated
insulin secretion. Conversely, mice in which the insulin receptor was ablated in
neuronal cells exhibit elevated food intake and diet-induced obesity, suggesting
that insulin delivers an anorexogenic input to the central nervous system.

23
Such
a role of insulin in the brain was suggested in early studies where the hormone
was administered intra-cerebroventricularly to monkeys.
24
Finally, vascular cells
are a target for insulin-induced vasodilatation and capillary recruitment that, by
enhancing glucose delivery, may complement the hormone’s direct stimulatory
effect on glucose uptake in muscle.
25
Given that quantitatively β-cells, neuronal
and vascular cells have only a minor contribution to insulin-stimulated whole
body glucose disposal, these studies suggest that the actions of insulin in ‘non-
classical insulin targets’ are indirectly involved in the regulation of total body
glucose metabolism. It will be interesting to see whether in addition insulin
regulates glucose metabolism in these sites.
In summary, insulin engages both ‘classical’ and ‘non-classical’ target organs
in orchestrating the control of glucose metabolism (Table 2.1). In its classical
target organs insulin directly modulates glucose uptake, metabolism and pro-
duction. In addition, the hormone affects glucose metabolism secondarily to
alterations in the metabolism of other macronutrients, and by utilizing complex
inter-organ cross-talk mechanisms.
2.3 Insulin-mediated regulation of glucose metabolic
pathways
Entry of the hydrophilic glucose molecule into the cell through a lipid mem-
brane requires a ‘gateway’ offered by glucose transporters (discussed later in
this chapter). Once in the cytosol, glucose is phosphorylated into glucose-6-
phosphate, and from this initial step its biochemical fate is diverse. Glucose-6-
phosphate is catabolized through glycolysis, the hexose monophosphate shunt
and mitochondrial oxidation, yielding high-energy compounds such as ATP and

NAD(P)H. Alternatively, glucose can be stored in polymer form (glycogen) or
converted to triglycerides. In addition, glucose can be metabolized through sev-
eral quantitatively minor pathways: it is a precursor for de novo synthesis of
nucleotides and certain amino acids, it can be converted to other sugars and alco-
hols (e.g. sorbitol) and it is required for the generation of complex compounds
such as glycoproteins and glycolipids.
At the cellular level, insulin regulates the flow of glucose through these
biochemical pathways by two basic mechanisms:
(a) by increasing uptake from the blood, as described in Section 2.4;
(b) by affecting regulatory enzymes in the various pathways of glucose
metabolism, as outlined next.
68 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM
Table 2.2 Major target proteins of insulin in the regulation of glucose metabolism, and the
main mechanism(s) of regulation
Biochemical
process Protein
Major mechanism of
regulation by insulin Reference
Stimulatory effect by insulin
Glucose uptake GLUT4 Translocation from intracellular
pools to the plasma membrane
26
Increased activity 27
Expressional regulation? 28
Glucose
phosphorylation
Glucokinase Transcriptional activation 29
Hexokinase II Transcriptional activation 30
Glycolysis Phospho-fructokinase 2 Reversible phosphorylation? 31
Allosteric activation

Phosphofructokinase 1 Allosteric activation
Phosphorylation and actin binding 32
Pyruvate kinase Transcriptional/post-transcriptional
activation
33
Dephosphorylation
Glycogenesis Glycogen synthase Dephosphorylation:
inhibited GS kinase (GSK3) 34
stimulated dephosphorylation
(PP1) 35
Glucose oxidation Pyruvate dehydrogenase Dephosphorylation by inhibition
of PDK-4/2 expression 36
Allosteric activation
Lipogenesis Acetyl CoA carboxylase Transcriptional activation 37
Allosteric activation
Reversible phosphorylation?
Inhibitory effect by insulin
Glycogenolysis Glycogen phosphorylase Dephosphorylation 38, 39
GP-kinase Dephosphorylation
Gluconeogenesis G6Pase Transcriptional repression 40
Acute inhibition by 3-PIPs?
PEPCK Transcriptional repression
Pyruvate carboxylase Transcriptional repression 41
GLUT4 – glucose transporter 4, GP-kinase – glycogen phosphorylase kinase, G6Pase –glucose-6-
phosphatase, GSK3 – glycogen synthase kinase 3, PDK – pyruvate dehydrogenase (PDH) kinase,
PEPCK – phosphoenolpyruvate carboxykinase, 3-PIPs –3

-phosphoinositide phosphates, PP1 –pro-
tein phosphatase 1.
? – ambiguity exists regarding the precise effect of insulin or its physiological relevance.

Enzyme regulation is achieved by changes in the phosphorylation state and/or
in expression levels. The result is a change in K
m
and/or V
max
in the first
case, or only V
max
in the second. In addition, the rise in glucose flux alters the
intracellular concentration of metabolites that in turn act as allosteric modulators,
and may regulate the expression levels of specific enzymes. Frequently, these
GLUCOSE UPTAKE INTO SKELETAL MUSCLE – THE RATE-LIMITING STEP 69
different mechanisms operate in a concerted fashion, translating metabolic and
hormonal information into both short and long term regulation of enzymatic
activity. Short term (seconds to minutes) regulation is frequently achieved by
allosteric modulation and reversible phosphorylation, whereas long term (hours
to days) regulation largely employs alterations in gene and protein expression.
Table 2.2 summarizes the major molecular mechanism(s) engaged by insulin in
regulating its major protein targets.
An example of combined allosteric modulation, covalent modifications and
expressional regulation is offered by the pyruvate dehydrogenase complex
(PDH). This large enzymatic complex catalyses the irreversible oxidative
decarboxylation of pyruvate to form acetyl CoA, linking glycolysis to the
mitochondrial citric acid cycle. PDH is negatively regulated allosterically mainly
by its products acetyl CoA and NADH, curbing energy production from
glucose when NEFA are abundant as a fuel source. In addition, reversible
phosphorylation is achieved by at least four PDH kinases and two phosphatases,
which respectively decrease or increase the overall catalytic activity of the
complex. Insulin rapidly decreases the expression level of PDH kinases 4 and
2, reducing the phosphorylation input on PDH and ultimately elevating its

catalytic activity.
The above sections discussed the complex mechanisms utilized by insulin to
regulate glucose metabolism and its homeostasis in the context of total body
fuel metabolism. Among these, the stimulation of glucose uptake by insulin
into skeletal muscle remains quantitatively at the centre of the hypoglycaemic
actions of this hormone under normal physiological conditions. Impairment of
this process calls into action extreme adaptive responses to maintain glucose
homeostasis, in the form of compensatory hyperinsulinaemia and/or glucose
funnelling into alternative sites (as demonstrated by the MIRKO mice). De-
compensation of these mechanisms results in pathological manifestations (e.g.
diabetes). We next focus on the cellular and molecular mechanisms by which
insulin stimulates the uptake of glucose into skeletal muscle.
2.4 Glucose uptake into skeletal muscle – the rate-limiting
step in glucose metabolism
As discussed above, glucose entering muscle cells encounters various fates, but
it rarely accumulates as free glucose in the sarcoplasm.
42–44
This observation
has led to the concept that, under both fasting and fed conditions, glucose
transport across the membrane of the muscle fibre is rate limiting for glucose
utilization. This notion was further confirmed by the use of magnetic resonance
spectroscopy, which allows detection of intracellular glucose-6-phosphate
43
as
well as intracellular glucose levels.
10
However, under certain physiological
conditions (e.g. exercise)
45
or in other tissues (e.g. cardiomyocytes)

46
steps
70 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM
beyond glucose transport such as glucose phosphorylation might become
rate limiting.
Does GLUT4 dictate glucose uptake in muscle and fat cells?
The GLUT family comprises 13 members, but only seven of these (GLUTs
1–4, 6, 8 and 11) have demonstrated glucose transport activity.
47
Muscle and
fat cells express predominantly GLUT4. From this expression pattern as well as
functional studies showing that GLUT4 translocates to the plasma membrane in
response to insulin, GLUT4 is generally believed to mediate insulin-stimulated
glucose uptake. This notion was further confirmed recently by experiments using
GLUT4 gene ablation or a rather selective inhibitor of GLUT4 (the HIV protease
inhibitor indinavir), as outlined below. Mice lacking GLUT4 have diminished
glucose uptake in response to insulin or exercise,
48, 49
and mice lacking GLUT4
selectively in skeletal muscle
50
or in adipose tissues
12
have impaired glucose
and insulin tolerance. Similarly, heterozygous GLUT4-null mice are severely
insulin resistant and develop diabetes.
51
Overall, these findings underline the importance of GLUT4 for insulin-
and contraction-dependent glucose uptake. In addition, muscle-specific GLUT4
knockout mice demonstrate the important regulatory role of skeletal muscle

in glucose homeostasis.
50
Surprisingly, homozygous GLUT4-null mice had
normal glucose tolerance even though they showed impaired insulin tolerance,
suggesting insulin resistance. The mechanism by which homozygous GLUT4-
null mice are protected from diabetes is still unclear. The most likely explanation
is that compensatory mechanisms are induced.
52, 48
For example, other GLUT
isoforms may be expressed at higher levels, which then compensate for
GLUT4. Although no up-regulation of GLUT1, GLUT3 or GLUT5 could be
detected,
52, 48
glucose uptake into isolated soleus muscle was mediated by a
saturable glucose-transport process and blocked by the classical inhibitor of
GLUTs, cytochalasin B.
48
Thus glucose uptake into soleus muscle from GLUT4-
null mice appears to be mediated by some other member of the GLUT family
(potentially GLUT8).
Genetic ablation of GLUT4 has confirmed its importance for normal insulin
sensitivity; however, it does not reveal the quantitative contribution of GLUT4
to glucose influx into normal tissues, since compensatory mechanisms in the
affected tissue are induced. Until recently, glucose uptake through a specific
GLUT isoform could not be directly assessed given the lack of inhibitors with
sufficient selectivity for any given GLUT. The HIV protease inhibitor indinavir
was recently found to selectively block GLUT4-mediated glucose uptake into
Xenopus laevis oocytes exogenously expressing different GLUTs.
53
The IC

50
of indinavir on glucose uptake was much lower in oocytes expressing GLUT4
compared with those expressing GLUT2, GLUT1, GLUT3 or a GLUT8 mutant
directed to the cell surface.
54
Using this approach, we studied the contribution