International Journal of

Molecular Sciences

Review
Molecular Mechanisms of Hypothalamic
Insulin Resistance
Hiraku Ono
Department of Endocrinology, Hematology and Gerontology, Chiba University Graduate School of Medicine,
Chiba 260-8670, Japan; hono@chiba-u.jp; Tel.: +81-43-226-2091




Received: 31 January 2019; Accepted: 13 March 2019; Published: 15 March 2019


Abstract: Insulin exists in the central nervous system, where it executes two important functions
in the hypothalamus: the suppression of food intake and the improvement of glucose metabolism.
Recent studies have shown that both are exerted robustly in rodents and humans. If intact, these
functions exert beneficial effects on obesity and diabetes, respectively. Disruption of both occurs
due to a condition known as hypothalamic insulin resistance, which is caused by obesity and the
overconsumption of saturated fat. An enormous volume of literature addresses the molecular
mechanisms of hypothalamic insulin resistance. IKKβ and JNK are major players in the inflammation
pathway, which is activated by saturated fatty acids that induce hypothalamic insulin resistance.
Two major tyrosine phosphatases, PTP-1B and TCPTP, are upregulated in chronic overeating.
They dephosphorylate the insulin receptor and insulin receptor substrate proteins, resulting in
hypothalamic insulin resistance. Prolonged hyperinsulinemia with excessive nutrition activates the
mTOR/S6 kinase pathway, thereby enhancing IRS-1 serine phosphorylation to induce hypothalamic
insulin resistance. Other mechanisms associated with this condition include hypothalamic gliosis
and disturbed insulin transport into the central nervous system. Unveiling the precise molecular
mechanisms involved in hypothalamic insulin resistance is important for developing new ways of
treating obesity and type 2 diabetes.

Keywords: hypothalamus; insulin resistance; inflammation; obesity; food intake; glucose metabolism

1. Introduction
Obesity is a common problem worldwide, as it contributes to type 2 diabetes and other life
style-related diseases in susceptible people with genetic predispositions. In addition to the easy
access to calorie-dense foods and the predominance of lifestyles with little or no physical exercise
in modern society, a common cause of obesity is the lack of effective drugs against obesity that are
free of unacceptable side effects [1]. Exploring medications that are effective in treating obesity by
suppressing food intake and/or enhancing energy expenditure is among the most important research
goals in modern medicine.
Insulin, the pancreatic hormone secreted to maintain normal blood glucose levels, has been
recognized to suppress food intake and weight gain when injected into cerebral ventricles [2].
More recently, insulin has been found to improve peripheral glucose metabolism in the brain [3],
independent of its effects on food intake and body weight. Therefore, targeting insulin in the brain
could be a valid approach for treating obesity and type 2 diabetes, provided that its functions
in the brain remain intact. These beneficial effects are severely disturbed by excessive nutrition,
the consumption of fatty foods, and obesity itself, a condition referred to as brain insulin resistance.
Obesity induces brain insulin resistance, which blunts the suppressive action of insulin on food intake,
thus inducing more severe obesity. In other words, a vicious cycle develops and persists between
obesity and brain insulin resistance. Therefore, clarifying the mechanism by which brain insulin

Int. J. Mol. Sci. 2019, 20, 1317; doi:10.3390/ijms20061317 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 1317 2 of 16

resistance occurs, and devising strategies for breaking this vicious cycle, are important for developing
new medications for the effective treatment of obesity and type 2 diabetes.

2. Two Major Insulin Functions in the Hypothalamus: Suppression of Food Intake and
Endogenous Glucose Production
When insulin is injected into the cerebral ventricles of rodents, food intake [2] and endogenous
glucose production are both suppressed [4]. When insulin is sprayed into the nostrils of humans,
food intake [5] and endogenous glucose production [6] are both suppressed. Brain-specific insulin
receptor (IR)-knockout (NIRKO) mice are an animal model of both obesity and insulin resistance [7].
Deletion of IR in the hypothalamus using an antisense oligonucleotide induced hyperphagia and
insulin resistance [8]. These data consistently demonstrate that insulin in the central nervous system
(CNS) stimulates insulin signaling in some hypothalamic cell types, thereby suppressing food intake
and regulating glucose metabolism.
However, the question remains as to which cell types in the hypothalamus are involved in these
effects. In NIRKO, where Cre recombinase is driven by nestin, IR is deleted in neurons and glial
cells, suggesting that insulin may act on both cell types by transducing their effects [9]. Neurons are
an intensively studied cell type, and studies have shown that insulin signaling initiated by insulin
receptor (IR) activation ultimately results in electrophysiological and/or transcriptional changes in
neurotransmitters that are within or released by neurons. More recent studies have also revealed the
involvement of non-neuronal cells. Most notably, insulin was found to work in astrocytes, transporting
glucose from peripheral blood into the CNS [10,11]. Astrocyte-specific deletion of IR disturbed
glucose sensing in the hypothalamus, resulting in impaired glucose tolerance and systemic insulin
resistance [11]. Moreover, insulin receptors on vascular endothelial cells are reportedly involved in
insulin transport from the periphery to the brain [12–14]. Tanycytes, a special cell type lining the third
ventricle, have recently attracted attention as being responsible for the transport of hormones and
nutritional signals crossing the blood–brain barrier (BBB) [15]. While tanycytes have been shown to
transport leptin [16,17] via its receptor, the role of these cells in insulin transport requires further study.
The most important point regarding the effects of insulin on food intake and glucose metabolism
is that these functions are not always independent of each other. If blocking hypothalamic insulin
signaling induces significant changes in food intake—which would chronically result in obesity or
leanness—then glucose metabolism would be impaired or improved due to the resulting obesity and
leanness, respectively. This could lead to misunderstanding the primary effects on glucose metabolism.
Therefore, the primary effect of intervening in hypothalamic insulin signaling on glucose metabolism
can be demonstrated only by: (1) The lack of a significant effect on body weight; (2) an acute-phase
intervention such as 1–3 days of a high fat diet (HFD), during which it is still too early for obesity to
occur; or (3) food restriction in the orexigenic or to-be-obese group to match body weights between
groups (pair-feeding).
The molecular mechanism by which insulin signaling in the hypothalamus suppresses food
intake and mediates systemic glucose metabolism has been intensively studied [18]. Specific IR
tyrosine residues are phosphorylated by IR itself when it binds insulin, thereby inducing tyrosine
phosphorylation of insulin receptor substrate (IRS) proteins. This process results in the activation of PI
3-kinase, which in turn produces phosphatidylinositol (3,4,5) triphosphate (PIP3) [19]. When inhibitors
of PI 3-kinase are injected into the ventricle, insulin neither suppresses food intake [20] nor enhances
glucose metabolism [21], showing that PI 3-kinase activation is necessary for both effects of central
insulin. We bidirectionally modulated PTEN, the negative regulator of PI 3-kinase signaling, in the rat
hypothalamus and showed that hypothalamic PIP3 is responsible for the regulation of food intake
and glucose metabolism [22]. These studies indicate that the effects on both food intake and glucose
metabolism occur via a common pathway from IR to PI 3-kinase (Figure 1).
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Int. J. Mol. Sci. 2019, 20, x 3 of 16

Figure
Figure 1. Molecular
1. Molecular mechanisms
mechanisms of insulin
of insulin functionsfunctions and associated
and associated modifications
modifications in the
in the hypothalamus.
hypothalamus. Insulin enters the hypothalamus and suppresses food intake and hepatic glucose
Insulin enters the hypothalamus and suppresses food intake and hepatic glucose production. Many
production. Many molecules disturb hypothalamic insulin signaling at several sites in response to
molecules disturb hypothalamic insulin signaling at several sites in response to saturated fatty acids
saturated fatty acids (SFA), inflammation, and excess nutrition. Hypothalamic insulin also affects
(SFA), inflammation, and excess nutrition. Hypothalamic insulin also affects metabolic processes
metabolic processes in white adipose tissue (WAT) and brown adipose tissue (BAT). The dot line
in white
showsadipose tissue
a pathway less(WAT) andand
confirmed, brown adipose
the dot tissue
line with “?” is(BAT). The dotunconfirmed
a hypothetical line shows pathway.
a pathway less
confirmed, and the dot line with “?” is a hypothetical unconfirmed pathway.
Int. J. Mol. Sci. 2019, 20, 1317 4 of 16

Elevated PIP3 induces Akt phosphorylation and activation in insulin-sensitive tissues.

Neuronal-specific deletion of Rictor, the key component of mTORC2, which activates Akt by
phosphorylating its serine 473 residue, induces obesity and impairs glucose tolerance [23]. Consistently,
the proopiomelanocortin (POMC) neuron-specific deletion of Rictor enhances food intake [23]. Among
Akt’s many substrates, transcription factor FoxO1 is phosphorylated by Akt and inactivated by
nuclear exclusion upon insulin stimulation. Without insulin, FoxO1 transcriptionally increases
orexigenic neuropeptide AGRP via GPR17 [24] and decreases anorexigenic neuropeptide POMC
via carboxypeptidase E (CpE) [25]. Therefore, insulin presumably suppresses food intake via the
IR–IRS–PI3k–PIP3–Akt–FoxO1–GPR17–AGRP/CpE–POMC pathway. Notably, deleting hypothalamic
FoxO1 via the Nkx2.1 promoter only resulted in a mild food intake- and glucose metabolism-related
phenotype, implicating extrahypothalamic FoxO1 in the observed effects on neuropeptides [26].
Furthermore, the role of hypothalamic Akt itself in food intake has yet to be fully explored,
making it a topic for future investigations. On the other hand, the effects of insulin on glucose
metabolism are mediated by the ATP-sensitive potassium (KATP ) channel [3], which is activated
by PI 3-kinase and its downstream molecule, PIP3. Hypothalamic activation of the KATP channel
transduces this signal to Kupffer cells in the liver via electrical inactivation of the hepatic branch
of the efferent vagus nerve [4]. Acetylcholine signaling via the α7-nicotinic acetylcholine receptor
(α7nAchR) in Kupffer cells is suppressed when hypothalamic insulin signaling is activated and
the hepatic branch of the vagus is electrically suppressed, resulting in the stimulation of hepatic
IL-6/STAT3 signaling, which ultimately transcriptionally suppresses key gluconeogenic enzymes
and hepatic glucose production [27]. Therefore, insulin suppresses hepatic glucose production via
the IR–IRS–PI3k–PIP3–KATP channel–vagal efferent-α7nAchR–IL6–STAT3 pathway. While the KATP
channel is thought to be downstream of PIP3 but not Akt, recent findings have indicated that Rictor
deletion in AGRP neurons induces mild glucose intolerance without changing body weight, indicating
that at least a portion of hypothalamic insulin’s glucose-regulatory mechanism is located downstream
of Akt [23].
Phosphodiesterase-3B (PDE3B), another mediator of insulin signaling in the hypothalamus, has
also been recently identified. Intra-cerebro-ventricular (ICV) insulin activates PDE3B. Hypothalamic
inactivation of PDE3B by a specific inhibitor or a genetic deletion blunts central insulin-induced
anorexia or weight gain, respectively [28,29]. PDE3B is activated by PI 3-kinase and decreases the
intracellular cAMP level [30], which is another potential pathway by which insulin suppresses food
intake by acting on the hypothalamus. Interestingly, while PDE3B is downstream from PI 3-kinase,
it is independent of Akt phosphorylation [31].
Hypothalamic insulin has recently been shown to be involved not only in glucose, but also in fat
metabolism. Insulin acts on POMC neurons to suppress lipolysis, thereby enhancing lipogenesis in
adipose tissue, which transduce the signal via the sympathetic nerves [32,33]. Moreover, the acute
activation of AGRP neurons, by optogenetic or designer receptors exclusively activated by designer
drugs (DREADD) technologies, has been shown to suppress glucose uptake in brown adipose tissue
by upregulating myostatin [34]. While it is not clear whether this effect depends on hypothalamic
insulin, taken together with the observation that insulin inactivates AGRP neurons, the mechanism by
which hypothalamic insulin improves systemic glucose metabolism may involve the suppression of
liver glucose production and glucose uptake into brown adipose tissue.
While the hypothalamus has been most intensively studied as an insulin-sensitive site involved in
regulating food intake and glucose metabolism in the CNS, extrahypothalamic regions have also been
implicated. Nasal insulin administered to human’s decreases blood flow in the hypothalamus and
prefrontal cortex, a phenomenon that is blunted in overweight people [35]. Since the prefrontal cortex
has a crucial role in decision making, including feeding behavior, this area may be an extrahypothalamic
target by which insulin regulates food intake. Moreover, food palatability is reduced by CNS insulin
via suppression of mesolimbic pathways in both human [36] and animal [37].
Int. J. Mol. Sci. 2019, 20, 1317 5 of 16

However, the central effects of insulin should not be overstated. To our knowledge,
there have been no studies showing that CNS insulin exerts effects strong enough to induce
hypoglycemia, indicating that its effect on glucose metabolism of central insulin is relatively moderate,
and presumably easily compensated for by counter-regulatory hormones. Several animal studies using
super-physiological doses of insulin have demonstrated its suppressive effects on food intake [38].
Inconsistent results regarding the effects of insulin on food intake have been obtained using animal
models [39]. Central insulin suppression of hepatic glucose production has not been observed in
dogs [40]. The effects of intranasal insulin administration in humans remain controversial due to
its spillover into the bloodstream [41]. These observations indicate that further intensive studies are
required to clarify the roles of species and timing that would provide robustly beneficial effects of
central insulin [42,43].
Compared to other insulin-sensitive tissues such as the liver, muscle, and adipose tissue, it is
interesting that the hypothalamus uses a common proximal signaling cascade from the IR to PI
3-kinase/Akt for glucose metabolism regulation. However, the distal signaling pathways are more
specific to each tissue. Furthermore, the existence of the BBB makes insulin transport a unique potential
blockage point for the CNS when considering insulin resistance in target tissues.

3. Hypothalamic Insulin Resistance Induced by Excessive Nutrition

While administering insulin as a nasal spray suppresses food intake and endogenous glucose
production in normal-weight humans, these phenomena are lost in obese individuals [6,44]. Similarly,
while nasal insulin suppresses hypothalamic blood flow in lean people, as demonstrated by functional
magnetic resonance imaging, this suppression is blunted in obese individuals [35]. This observation
of “brain insulin resistance” has been mirrored in rodent studies: ICV insulin does not suppress food
intake in high-fat-diet (HFD)-fed rats [45], and even a single day of HFD abolishes the suppressive
effects of hypothalamic insulin on hepatic glucose production [46]. These data show that both
beneficial effects of central insulin are disturbed by obesity. An important area of research focuses
on which part of the insulin transport and/or signaling pathway is blocked in the hypothalamus.
Some studies have indicated that insulin delivery from the bloodstream to the CNS is disturbed
by HFD feeding [12,13]. However, higher insulin concentrations—even in the cerebrospinal fluid
(CSF) in obese individuals [47–49]—indicate the involvement of mechanisms other than disturbed
insulin delivery to the CNS. Several studies have shown a decrease in tyrosine phosphorylation
of IR and IRS [46,50], which can be partly explained by the increase in two tyrosine phosphatases,
PTP-1B [51] and TCPTP [52,53], which are detailed below. However, only long-term, i.e., not short-term,
HFD feeding has been shown to increase both phosphatases. Another mechanism underlying the
decrease in the tyrosine phosphorylation of IRS-1 involves the serine phosphorylation of this protein,
which inhibits the former. P70 S6 kinase [46] and JNK [54] are known to phosphorylate the serine
residues of IRS-1, mediating the inhibition of insulin signal transduction. In our study, the suppression
of hypothalamic PTEN in HFD-fed rats reversed insulin resistance without exerting effects on food
intake [22], indicating that the HFD-induced blockage of hypothalamic insulin signals, such as IRS-1
serine phosphorylation, exist upstream from PI 3-kinase. Another blockage point exists downstream
PI 3-kinase and impacts the regulation of food intake. Insulin activates the hypothalamic KATP channel
in lean but not obese rats [55], showing that a site between PIP3 and the KATP channel may be blocked
by HFD feeding. Such blockages impact the anorexic effect of hypothalamic insulin.
How rapidly does this hypothalamic insulin resistance occur? Hypothalamic insulin resistance
caused by excessive nutrition occurs more rapidly than that in other insulin-sensitive tissues.
Three days of HFD feeding sufficiently blunt the suppressive effects of insulin on food intake in rats [56].
We found that one day of HFD feeding was enough to blunt the suppressive effects of hypothalamic
insulin on glucose production [46]. This one-day HFD also decreased tyrosine phosphorylation of
IRS-1 and Akt phosphorylation in the hypothalamus, but not in the liver. Hepatic insulin resistance,
which blunts endogenous glucose production, occurred within three days of initiating HFD feeding,
Int. J. Mol. Sci. 2019, 20, 1317 6 of 16

while insulin resistance developed later in muscle and adipose tissues [57]. In contrast, hepatic insulin
signaling, such as that involving PI 3-kinase activity, is somewhat upregulated by HFD feeding [58].
This controversy is explained by blunting the hypothalamic pathway, which potentially induces
compensatory upregulation of hepatic insulin signaling [59].

4. Inflammation with ER Stress Induces Hypothalamic Insulin Resistance

Inflammation is an important pathway responsible for hypothalamic insulin resistance [60].
Inflammation induces biphasic effects on food intake. While high-level inflammation such as
adenovirus infection in the hypothalamus [22] or ICV injection of high dose TNFα [61] suppresses food
intake, low-level inflammation induced by ICV injection of low dose TNFα instead blocks the anorexic
effects of ICV insulin as well as insulin signaling in the hypothalamus [61]. It is conceivable that after
severe inflammation associated with a life-threatening infection, systemic recovery is permitted by
higher nutritional intake, which would be evolutionarily programmed as an orexigenic reaction caused
by low-grade inflammation. Long-chain saturated fatty acids (SFAs) cross the BBB, accumulate in the
hypothalamus [45], and induce acute hypothalamic inflammation via microglial activation [62,63].
SFA binds to Toll-like receptor 4 (TLR4) [64], activates the IKKβ/NFκB pathway by activating the
myeloid differentiation primary response gene 88 (MyD88) [64,65], and ultimately enhances the
expressions of pro-inflammatory genes such as TNFα, IL-1β, and IL-6 in the hypothalamus [45,50].
Endoplasmic reticulum (ER) stress is a condition in which the ER cannot carry out normal
protein folding and assembly. ER stress is also responsible for hypothalamic insulin resistance [66].
Glucose regulated protein 78 kDa/binding immunoglobulin protein (GPR78/Bip) has recently been
found to reverse ceramide-induced hypothalamic ER stress [67]. SFA-induced ER stress [64] in the
hypothalamus also contributes to activation of the IKKβ/NFκB pathway [68].
It is not caloric excess but rather SFA that initiates this inflammation signal, because even when the
same number of calories in a fat-rich diet and normal chow are provided, activation of the IKKβ/NFκB
pathway can still be observed [45]. In contrast, a recent finding that excess carbohydrate, not fat,
induces hypothalamic inflammation via advanced glycation end products [69] challenges the canonical
SFA-induced inflammation theory. Unlike SFA, unsaturated fatty acids such as oleic acid in the
hypothalamus exert a relatively anorectic effect and enhance insulin sensitivity [70].
Another inflammation pathway starting from TLR4 activation is the MAP kinase pathway,
including p38 and JNK. HFD enhances JNK phosphorylation, which then phosphorylates IRS-1
at serine 307, and inhibits insulin signaling at the IRS-1 level. IKK and JNK reportedly have different
roles: JNK activation is mainly related to leptin resistance to food intake, while IKK activation is more
related to glucose metabolism, at least in AGRP neurons [54].
Another pathway by which SFA induces hypothalamic insulin resistance is the ceramide–PKC
pathway. SFA increases the plasma membrane association of PKCθ in the hypothalamus, which
inhibits insulin signaling [67]. On the other hand, inhibiting de novo ceramide synthesis, as well as
PKCζ inactivation in hypothalamic neurons, normalize insulin signaling [71,72].
Reactive oxygen species (ROS) have dual roles in hypothalamic insulin signaling. Hypothalamic
insulin triggers the transient production of ROS to enhance insulin signaling [73]. Activation of
NADPH oxidase [73] and the mitochondrial respiratory chain [74] are reportedly mechanisms by
which insulin produces ROS as the signaling molecule. In contrast, obesity and/or diabetes are related
to ROS overproduction, which in turn induces inflammation, thereby blunting insulin signaling [75].

5. Involvement of Phosphatases and SOCS3 in Hypothalamic Insulin Resistance

Tyrosine phosphorylation of IR is the first step in insulin signaling. Protein phosphatase 1B
(PTP-1B) and T-cell protein tyrosine phosphatase (TCPTP) are two major phosphatases involved in the
regulation of hypothalamic insulin signaling via dephosphorylation of IR tyrosine residues [53]. PTP-1B
dephosphorylates IR and IRS proteins at their tyrosine residues, and thus negatively regulates insulin
signaling [76]. Hypothalamic PTP-1B expression increases in the hypothalamus of rats fed HFD for 2–4
Int. J. Mol. Sci. 2019, 20, 1317 7 of 16

months [77,78]. TNF-α upregulates PTP-1B expression in the hypothalamus [78], indicating chronic
inflammation as the mechanism inducing PTP-1B upregulation. On the other hand, POMC-specific
deletion of PTP-1B and PTP-1B knockdown in the hypothalamus by antisense oligonucleotide protects
animals from HFD-induced obesity and insulin resistance [51,77]. Moreover, insulin sensitivity
improved in POMC-specific PTP-1B knockout mice without changes in body weight, suggesting the
impact on insulin sensitivity as the primary effect. TCPTP was also upregulated in the hypothalamus
over three months of HFD feeding [52]. Neuron-specific deletion of TCPTP protects mice from
developing HFD-induced obesity [52]. Its deletion in AGRP neurons enhanced the suppression of
hepatic glucose production and glucose uptake in brown adipose tissue [79]. PTP-1B and TCPTP
increases are initially observed after six and nine weeks of HFD feeding, respectively, suggesting
that their upregulation does not trigger hypothalamic insulin resistance, which can be observed from
day 1 of HFD feeding. However, these phosphatases contribute to the maintenance of hypothalamic
insulin resistance.
The phosphatase and tensin homolog in chromosome 10 (PTEN) is a phosphatase that mainly
dephosphorylates PIP3 and antagonizes PI 3-kinase. Constitutive activation of hypothalamic PTEN
induces weight gain and insulin resistance, mimicking HFD feeding. In contrast, suppression of
hypothalamic PTEN by overexpressing its dominant-negative form suppresses food intake, while
this effect is blunted by HFD feeding [22]. However, even in HFD-fed animals, hypothalamic PTEN
suppression reversed HFD-induced insulin resistance. During pregnancy, hypothalamic PTEN is
less inactivated by low phosphorylation than during non-pregnancy periods, thereby inducing
hypothalamic insulin resistance, which protects animals from insulin-induced anorexia [80]. To our
knowledge, there have been no reports describing whether HFD feeding induces any changes in PTEN
expression and/or its phosphorylation level.
Suppressor of cytokine signaling 3 (SOCS3) is another molecule responsible for hypothalamic
insulin signaling induced by obesity. When leptin binds to its own receptor, the Janus-activated
kinase-2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway is activated,
and ultimately increases SOCS3 transcription. SOCS3 in turn suppresses insulin signaling by binding
to and enhancing the degradation of IRS proteins. SOCS3 also suppresses tyrosine phosphorylation.
Obesity-induced hyperleptinemia enhances SOCS3 expression, resulting in hypothalamic insulin
resistance. Interestingly, the deletion of SOCS3 in leptin receptor-expressing cells protects mice from
HFD-induced systemic insulin resistance, without significant weight changes [81]. This observation
suggests that SOCS3 is a negative regulator of hypothalamic insulin signaling only for glucose
metabolism, and not for food intake regulation.

6. Involvement of the mTOR-S6 Kinase Pathway in Hypothalamic Insulin Resistance

mTOR and its downstream effector, p70 S6 kinase (S6K), are activated by chronic hyperinsulinemia
and excess nutrition. mTOR/S6K pathway activation leads to phosphorylation of IRS-1 at serine
residues, inducing negative feedback inhibition of insulin signaling. Systemic deletion of S6K protects
mice from diet-induced obesity and insulin resistance [82]. We reported that one-day HFD feeding
induced S6K activation, downregulation of IRS-1 tyrosine phosphorylation, and downregulation
of Akt phosphorylation in the rat hypothalamus [46]. Constitutive activation of hypothalamic S6K
using viral vectors induces hypothalamic and systemic insulin resistance. Conversely, hypothalamic
mTOR/S6K pathway suppression reverses HFD-induced insulin resistance. These changes in glucose
metabolism are independent of body weight changes. Interestingly, the role of S6K in food intake does
not parallel its effect on glucose metabolism. In contrast to its negative effect on glucose metabolism,
the hypothalamic mTOR/S6K pathway suppresses food intake, which appears to be a mechanism by
which the hypothalamus senses how nutrition inhibits food intake, independently of its role in the
negative feedback input to insulin signaling [83].
Several reports have focused on the contradictory roles of the hypothalamic mTOR/S6K pathway.
Overexpression of DEPTOR (DEP-domain containing mTOR-interacting protein), a negative regulator
Int. J. Mol. Sci. 2019, 20, 1317 8 of 16

of the mTOR/S6K pathway, specifically using a viral vector in the mediobasal hypothalamus, prevents
HFD-induced obesity and improves glucose metabolism [84]. This is consistent with our glucose
metabolism observations [46], but not with reports showing that hypothalamic mTOR/S6K inhibits
food intake [85,86]. Genetic deletion of S6K in POMC neurons does not affect food intake or weight,
and surprisingly induces insulin resistance [87], which contradicts the results obtained in postnatal
overexpression models using viral vectors [46]. Insulin resistance reported in Reference [87] was
measured using a hyperinsulinemic–euglycemic clamp in anesthetized mice, where endogenous
glucose production (EGP) was not fully suppressed by ~3 mU/kg/min of insulin. In clamp studies
performed on non-anesthetized and non-restrained mice [88,89] or rats [46] using arterial catheter
blood sampling, this level of insulin infusion was usually sufficient to completely suppress endogenous
glucose production, suggesting that anesthesia-induced hepatic insulin resistance might have altered
the physiological hypothalamic effect of insulin on glucose metabolism [87]. Another study showed
that transgenic overexpression of DEPTOR in POMC neurons did not lead to weight changes,
instead it induced slight insulin resistance [90]. These seemingly contradictory reports indicate
that (1) POMC is not the main neuronal cell type mediating mTOR/S6K signals that regulate food
intake or glucose metabolism, and that (2) the genetic phenotype and postnatal intervention models
do not match, presumably due to the prolonged effects during the developmental period in the
former. The inconsistency between a POMC-specific genetic model and postnatal intervention in the
hypothalamus has also been observed for PTEN. While postnatal suppression of hypothalamic PTEN
using a viral vector suppresses food intake [22]—consistent with the theory that hypothalamic insulin
suppresses food intake via the PI 3-kinase pathway—the genetic deletion of PTEN in POMC neurons
tends to induce weight gain instead [91]. Similarly, postnatal deletion of the leptin receptor in AGRP
neurons produced very different phenotypes when compared to genetic knockout models [92,93].
ATF4 (activating transcription factor 4) is reportedly an ER stress-responsive target, which
induces leanness and enhanced insulin sensitivity when deleted [94]. Hypothalamic overexpression
of ATF4 induces hepatic insulin resistance, which is reversed by the inhibition of hypothalamic
S6K. On the other hand, suppression of hypothalamic ATF4 reverses ER stress-induced hepatic
insulin resistance. This report shows that the hypothalamic ATF4–S6K pathway is responsible
for ER stress-induced hypothalamic insulin resistance, which results in hepatic insulin resistance.
A chemokine, CCL5/RANTES, activates CCR5 and reportedly decreases serine phosphorylation of
IRS-1 in the hypothalamus by suppressing S6K [95]. Blocking CCL5/RANTES–CCR5 by genetic
deletion or ICV injection of an antagonist inhibited hypothalamic insulin signaling and insulin
resistance, indicating that this chemokine has a role in suppressing S6K-mediated negative feedback
input to insulin signaling.

7. Cell Populations Involved in Hypothalamic Insulin Functions and Insulin Resistance

Insulin has been detected in the CSF at concentrations 10–25% of those in the bloodstream.
When plasma insulin increases, CSF insulin levels also rise [96]. While some brain insulin may be
synthesized in the CNS [97], most is thought to come from the bloodstream [98]. Obesity increases
CSF insulin levels in rodents [47], sheep [48], and humans [49]. However, the transport ratio of insulin
from the periphery to the brain is blunted by HFD feeding [99,100]. Insulin-resistant individuals have
a lower CSF/blood insulin concentration ratio [101]. Therefore, hypothalamic insulin resistance is
partly explained by disrupted insulin transport into the brain. A portion of insulin in the brain is
transported from the bloodstream into the brain by IR-expressing brain endothelial cells [14,38,96,98].
Insulin transport by brain endothelial cells reportedly decreases by HFD feeding [12], while insulin
transport via receptor-mediated endothelial transcytosis remains controversial [96]. IR deletion in
astrocytes blunted insulin transport into the brain, demonstrating that astrocytes are also involved in
this transport [10,11].
In addition to neurons, glial and brain endothelial cells are also present in the hypothalamus,
where research has been focused on determining which cell types are involved in insulin functions and
Int. J. Mol. Sci. 2019, 20, 1317 9 of 16

hypothalamic insulin resistance [102,103]. IR expression levels are higher in neurons compared to glial
cells. Among neuronal cells, AGRP-expressing neurons are the primary site in which insulin acts to
suppress hepatic glucose production [33,79,104]. POMC-expressing cells are responsible for the central
insulin functions of suppressing lipolysis and promoting lipogenesis in adipose tissue [33]. However,
the role of POMC neurons in suppressing glucose production is controversial. POMC-specific genetic
deletion of IR does not significantly affect the ability of insulin to suppress glucose production [33].
It was recently found that TCPTP expression is upregulated by fasting and downregulated by feeding,
and this upregulation of TCPTP in POMC neurons during fasting masks the suppressive effects of
insulin on glucose production [105]. When TCPTP expression in POMC neurons is suppressed by
genetic deletion or feeding, the ability of insulin to suppress glucose production becomes apparent.
Non-AGRP-expressing, neuropeptide Y (NPY)-expressing neurons have been recently shown to be
responsible for the suppressive action of insulin on food intake [106].
Cultured hypothalamic neuronal cells are resistant to SFA-induced inflammation and insulin
resistance [107], suggesting that SFA mainly affects non-neuronal cells, leading to neuronal insulin
resistance. HFD feeding for one day is enough to induce hypothalamic gliosis, including both
microgliosis and astrogliosis [108]. Microgliosis is induced by HFD feeding, but not obesity [63],
and contributes to hypothalamic inflammation [62,109]. In contrast, astrogliosis is recognized as a
protective reaction of the brain responding to acute excess nutrition [110]. The roles of inflammation,
including that of the IKKβ/NFκB pathway in astrocytes, are controversial, because while one report
has shown that the inhibition of NFκB in astrocytes enhanced food intake [111], another demonstrated
astrocytic-mediated inhibition of NFκB to protect animals from HFD-induced obesity [112,113].
Astrocyte-specific IR deletion was shown to disturb glucose sensing, in addition to insulin and glucose
transport from the bloodstream into the brain. The animal model used also showed impaired glucose
tolerance and insulin resistance [11]. Brain endothelial cells are another cell type responsible for the
transport of circulating insulin into the brain, where the uptake of insulin was downregulated after
weeks of HFD feeding, showing an increase in NFκB binding activity [12]. Insulin transport by brain
endothelial cells is not dependent of PI3k signaling, and the mechanism of the insulin resistance in
brain endothelial cells induced by HFD feeding merits further study.
Long-term hypothalamic inflammation results in hypothalamic angiogenesis [114] and loss of
POMC neurons. Moreover, long-term HFD induces expansion of the macrophage pool, which normally
resides in the median eminence, to the arcuate nucleus of the hypothalamus. Inhibition of inducible
nitric oxide synthase in these hypothalamic macrophages not only abrogates macrophage activation,
but also improves glucose metabolism [115].

8. Concluding Remarks
Given the current global pandemic of obesity and related diseases such as type 2 diabetes, and
the lack of effective treatments, understanding the molecular mechanisms of hypothalamic insulin
resistance is necessary for the development of safe and efficacious medications for the treatment of
these metabolic disorders. Insulin nasal sprays effectively stimulate hypothalamic insulin signaling to
suppress food intake as well as glucose production in lean but not obese men. Thus, the same approach
might be useful for administering novel drugs that reverse or bypass the blockage point of insulin
signaling as an obesity treatment, once the blockage mechanism and the responsible molecules have
been fully elucidated. We now know that numerous heterogeneous cell types in the hypothalamus
(POMC or AGRP neurons, other neurons, astrocytes, microglia, endothelial cells, macrophages in
median eminence, etc.) are related to hypothalamic insulin resistance. Since commonly used drugs are
not effective exclusively in one cell type, even one specific to the hypothalamus, when administered
nasally they might not exert the desired “total effect” on heterogeneous hypothalamic cells. Thus,
each candidate medication should be carefully studied and considered before its application to patients.

Author Contributions: H.O. wrote this manuscript and prepared the figures.
Int. J. Mol. Sci. 2019, 20, 1317 10 of 16

Funding: This research was funded by the Ministry of Education, Culture, Sports, Science, and Technology of
Japan, grant number 18K08502.
Conflicts of Interest: The author has no conflicts of interest to declare.

Abbreviations
AchR Acetylcholine receptor
AGRP Agouti-related protein
ATF4 Activating transcription factor 4
BAT Brown adipose tissue
BBB Blood–brain barrier
CCL5 C C motif chemokine 5
CCR5 C–C chemokine receptor type 5
CpE Carboxypeptidase E
CSF Cerebrospinal fluid
DEPTOR DEP domain-containing mTOR-interacting protein
DREADD Designer receptors exclusively activated by designer drugs
EGP Endogenous glucose production
ER stress Endoplasmic reticulum stress
FoxO1 Forkhead box protein O1
G6Pase Glucose 6-phosphatase
GPR G protein-coupled receptor
HFD High-fat diet
IκB Nuclear factor of kappa-light-chain-enhancer in B-cells inhibitor
IKKβ IκB kinase β
IL-6 Interleukin 6
IR Insulin receptor
IRS Insulin receptor substrate
JNK c-Jun-NH2-terminal kinase
mTORC1 Mechanistic target of rapamycin complex 1
Myd88 Myeloid differentiation primary response 88
NFκB Nuclear factor of kappa-light-chain-enhancer in B-cells
NPY Neuropeptide Y
PDE3B Phosphodiesterase 3B
PEPCK Phosphoenolpyruvate carboxykinase
PI3K Phosphoinositide 3-kinase
PIP3 Phosphatidylinositol 3,4,5-triphosphate
PKC Protein kinase C
POMC Proopiomelanocortin
PTEN Phosphatase and tensin homolog on chromosome 10
PTP-1B Protein tyrosine phosphatase 1B
RANTES Regulated on activation, normal T cell expressed and secreted
Rictor Rapamycin-insensitive companion of mammalian target of rapamycin
ROS Reactive oxygen species
S6K P70 S6-kinase
SFA Saturated fatty acids
STAT3 Signal transducer and activator of transcription 3
TCPTP T-cell protein tyrosine phosphatase
WAT White adipose tissue

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