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TCPTP regulates insulin signalling in AgRP neurons to coordinate glucose metabolism
with feeding
Garron T. Dodd1, 2
*, Robert S. Lee-Young1, 2, 3
, Jens C. Brüning4
and Tony Tiganis1, 2, 3
*
1Metabolism, Diabetes and Obesity Program, Monash Biomedicine Discovery Institute,
Monash University, Victoria 3800, Australia.
2Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800,
Australia.
3Monash Metabolic Phenotyping Facility, Monash University, Victoria 3800, Australia.
4Max Plank Institute for Metabolism Research, Department of Neuronal Control of
Metabolism, Gleueler Str. 50, 50931 Cologne, Germany; Center for Endocrinology, Diabetes,
and Preventive Medicine (CEDP), University Hospital Cologne, Kerpener Str. 26, 50924
Cologne, Germany; Excellence Cluster on Cellular Stress Responses in Aging Associated
Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of
Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany; National Center for Diabetes
Research (DZD), Ingolstädter Land Str. 1, 85764 Neuherberg, Germany.
* To whom correspondence should be addressed:
Tony Tiganis (+61) 3 9902 9332
Email: Tony.Tiganis@monash.edu
Garron Dodd (+61) 3 9902 9345
Email: Garon.Dodd@monash.edu
Page 1 of 37 Diabetes
Diabetes Publish Ahead of Print, published online April 30, 2018
ABSTRACT
Insulin regulates glucose metabolism by eliciting effects on peripheral tissues as well as the
brain. Insulin receptor (IR) signalling inhibits AgRP-expressing neurons in the hypothalamus
to contribute to the suppression of hepatic glucose production (HGP) by insulin, whereas
AgRP neuronal activation attenuates brown adipose tissue (BAT) glucose uptake. The
tyrosine phosphatase TCPTP suppresses IR signalling in AgRP neurons. Hypothalamic
TCPTP is induced by fasting and degraded after feeding. Here we assessed the influence of
TCPTP in AgRP neurons in the control of glucose metabolism. TCPTP deletion in AgRP
neurons (Agrp-Cre;Ptpn2fl/fl
) enhanced insulin sensitivity as assessed by the increased
glucose infusion rates and reduced HGP during hyperinsulinemic-euglycemic clamps,
accompanied by increased [14
C]-2-deoxy-D-glucose uptake in BAT and browned white
adipose tissue. TCPTP deficiency in AgRP neurons promoted the intracerebroventricular
insulin-induced repression of hepatic gluconeogenesis in otherwise unresponsive food-
restricted mice yet had no effect in fed/satiated mice where hypothalamic TCPTP levels are
reduced. The improvement in glucose homeostasis in Agrp-Cre;Ptpn2fl/fl
mice was corrected
by IR heterozygosity (Agrp-Cre;Ptpn2fl/fl
;Insrfl/+
), causally linking the effects on glucose
metabolism with the IR signalling in AgRP neurons. Our findings demonstrate that TCPTP
controls IR signalling in AgRP neurons to coordinate HGP and brown/beige adipocyte
glucose uptake in response to feeding/fasting.
Page 2 of 37Diabetes
INTRODUCTION
Insulin is secreted by pancreatic β cells in response to elevated blood glucose levels
and signals via the insulin receptor (IR) in peripheral tissues, including skeletal muscle and
fat to promote glucose uptake and storage, and in the liver to repress hepatic glucose
production (HGP) to prevent postprandial hyperglycemia. Insulin can also promote glucose
uptake in brown adipose tissue (BAT) (1) where glucose can be stored as glycogen, used for
fatty acid esterification and triglyceride synthesis, or converted to lactate by anaerobic
glycolysis during non-shivering thermogenesis to produce ATP (2; 3). The latter may
compensate for the reduced ATP production that occurs during thermogenesis where fatty
acid oxidation is uncoupled from ATP production to generate heat (2; 3). The importance of
BAT in glucose metabolism is highlighted by studies demonstrating that greater BAT
abundance is accompanied by decreased glycaemic variability (4; 5). Beyond insulin’s roles
in the periphery, a large body of evidence now also supports a role for insulin action in the
CNS in the regulation systemic insulin sensitivity and glucose homeostasis (6; 7).
Mice in which IR was deleted in neurons and astroglia using the Nestin Cre transgene
become obese and develop systemic insulin resistance (8). Although the glucoregulatory
effects of the IR can be ascribed to different regions of the brain, several nuclei within the
hypothalamus are especially important. Neurons in the arcuate nucleus (ARC) of the
hypothalamus, residing at the base of the third ventricle, are positioned to readily sense
peripheral substances such as insulin that signal the nutritional and energy state of the
organism (6; 7; 9). These include two molecularly-defined neuronal populations, the
anorexigenic POMC neurons that repress feeding and increase energy expenditure, and the
orexigenic AgRP/NPY neurons that can antagonise the actions of POMC neurons to promote
feeding and decrease energy expenditure (6; 7; 9). Insulin signals via phosphatidylinositol 3-
kinase (PI3K) to activate the Ser/Thr protein kinase AKT, as well as other signaling cascades,
Page 3 of 37 Diabetes
to elicit discordant effects in POMC and AgRP/NPY neurons (6; 7; 9). Insulin regulates
POMC neuronal excitability and promotes POMC expression, which is processed into the
neuropeptide α-MSH; α-MSH agonises post-synaptic melanocortin-4 receptors (MCR4) on
neurons in other regions of the brain to repress feeding and increase ambulatory activity and
thermogenesis (6; 7; 9). By contrast, insulin hyperpolarises AgRP neurons and inhibits their
firing by opening KATP channels (10). Insulin also inhibits the expression of the neuropeptide
AgRP that functions post-synaptically to antagonise α-MSH/MCR4 interactions (6; 7; 9).
The inhibition of AgRP/NPY neurons by insulin can alleviate inhibitory constraints on
POMC neurons and the melanocortin response (6; 7; 9). However, AgRP neurons can also
elicit melanocortin-independent effects and signal through parallel and redundant neural
circuits to affect feeding (11; 12). Although the precise neuronal populations regulating
glucose homeostasis remain to be defined, there is evidence that these can be distinct from
those regulating feeding. For example, recent studies have shown that the acute
pharmacogenetic activation of AgRP neurons represses BAT glucose uptake via projections
to the bed nucleus of the stria terminalis that are not involved in the induction of feeding (13).
The infusion of insulin into the brain (lateral ventricle) results in the suppression of
HGP and lowers blood glucose even in the context of diabetes (14-16). The CNS effects on
HPG have been ascribed to AgRP neurons, as the specific deletion of IR in AgRP but not
POMC neurons, results in the defective repression of HGP (10; 17). The CNS-mediated
repression of HGP is orchestrated by vagal efferents and α7-nicotinic acetylcholine receptors
(18) that promote the expression and release interleukin-6 (IL-6) by Kupffer cells in the liver
(15; 19). IL-6 in turn acts on hepatocytes via STAT-3 to repress the expression of
gluconeogenic enzymes such as glucose-6-phosphatase (encoded by G6pc) and
phosphoenolpyruvate-carboxykinase (encoded by Pck1) (20; 21). Recent studies using
pharmacogenetic approaches have shown that the acute activation of AgRP neurons promotes
Page 4 of 37Diabetes
systemic insulin resistance by repressing the activity of sympathetic fibres supplying BAT
and thereby BAT glucose uptake, without affecting HGP (13). These effects appear to be
independent of the melanocortin system as the acute activation of POMC neurons has no
effect on BAT glucose uptake (13). The extent to which hormones such as insulin influence
BAT glucose uptake via AgRP neurons remain to be determined.
Previously we identified the tyrosine phosphatase TCPTP (encoded by Ptpn2) as a
key negative regulator of IR signalling in the periphery and in the ARC (22-26). TCPTP
dephosphorylates the IR and attenuates insulin-induced PI3K/AKT signalling in AgRP
neurons (24; 26; 27). TCPTP deletion in AgRP neurons exacerbated the insulin-mediated
inhibition of AgRP neurons to increase the sympathetic output to BAT and inguinal white
adipose tissue (26). This increased BAT activity and promoted the conversion of white
adipocytes to brown-like or beige adipocytes (browning) and increased their thermogenic
activity and energy expenditure to render mice resistant to diet-induced obesity (26).
Importantly, we reported that TCPTP abundance in the hypothalamus exhibited diurnal
fluctuations linked to feeding (26); fasting increased hypothalamic TCPTP to repress IR
signalling to facilitate AgRP neuronal activation, and feeding resulted in TCPTP being
degraded so that IR signalling was exacerbated and AgRP neurons inhibited to promote white
adipose tissue browning and energy expenditure (26). In this study we explored the role of
TCPTP in AgRP neurons on glucose metabolism and the extent to which TCPTP fluctuations
may integrate feeding with the CNS control of glucose homeostasis.
Page 5 of 37 Diabetes
MATERIALS AND METHODS
Mice
Ptpn2fl/fl
, Agrp-Ires-Cre;Ptpn2fl/fl
(AgRP-TC), Npy-hrGFP, Agrp-Ires-Cre;Ptpn2fl/fl
;
Npy-hrGFP (AgRP-TC:Npy-GFP), Pomc-eGFP and Agrp-Ires-Cre;Ptpn2fl/fl
;Insrfl/+
(AgRP-
TC-IR) mice have been described previously (26; 28-30). To generate Agrp-Ires-
Cre;Ptpn2fl/fl
;Insrfl/+
;Npy-hrGFP (AgRP-TC-IR;Npy-GFP) or Agrp-Ires-Cre;Insrfl/+
(AgRP-
IRfl/+
) mice, AgRP-TC:Npy-GFP or Agrp-Ires-Cre mice were mated with Insrfl/fl
mice (31)
respectively. Mice were maintained on a 12 h light-dark cycle in a temperature-controlled
high barrier facility with free access to food and water. Mice were fed a standard chow (8.5%
fat; Barastoc, Ridley AgriProducts, Australia). Experiments were approved by the Monash
University School of Biomedical Sciences Animal Ethics Committee.
Immunohistochemistry
Mice were injected intraperitoneally with either vehicle or human insulin (0.85, 2.5, 5
mU/g, Actrapid, Denmark) then transcardically perfused with 4% w/v paraformaldehyde and
post-fixed brains processed for p-AKT (Ser-473) or GFP immunohistochemistry as described
previously (26).
Quantitative PCR
Quantitative real time PCR as performed as described previously (25; 26). The
following TaqMan gene expression assays were used: Pomc (Mm00475829_g1), Npy
(Mm03048253_m1), Agrp (Mm00475829_g1), Gapdh (Mm99999915_g1), Pck1
(Mm01247058_m1), G6pc (Mm00839363_m1) and Il6 (Mm00446190_m1).
Metabolic Measurements
Page 6 of 37Diabetes
Unless otherwise indicated insulin, glucose and pyruvate tolerance tests were
performed on fasted (fasting from 9 am) conscious mice by injecting human insulin (0.5 mU
insulin/g body weight, 4 h fasted), D-glucose (2 mg/g body weight, 6 h fasted), or sodium
pyruvate (1 mg/g body weight, 6 h fasted) respectively into the peritoneal cavity. Glucose
levels in tail blood were measured using an Accu-Check glucometer (Roche, Germany). For
the determination of fed and fasted blood glucose and corresponding plasma insulin levels,
submandibular blood was collected at 9 am following an overnight fast (food removed at 7
pm the previous day). Plasma insulin levels were determined using a Rat insulin RIA kit
(Linco Research, St. Charles, MO) or an in house enzyme-linked immunosorbent assay
(Monash Antibody Technologies Facility). Body composition was measured by dual energy
X-ray absorptiometry (DEXA) as described previously (25). Lateral ventricle cannulations
were performed as described previously (25; 26).
Hyperinsulinemic-euglycaemic clamps
For hyperinsulinemic-euglycemic clamps, 8-10-week-old Ptpn2fl/fl
, AgRP-TC, or
AgRP-TC-IR mice were anesthetized under 2% (v/v) isoflurane in 250 ml/min oxygen and
the left common carotid artery and the right jugular vein catheterized for sampling and
infusions, respectively, as previously described (32). Lines were kept patent by flushing daily
with 10–40 µl saline containing 200 units/ml heparin and 5 µg/ml ampicillin. Animals were
housed individually after surgery and body weights recorded daily. On the day of the
experiment food was removed at between 7-8am. After 3.5 h fasting, a primed (2 min, 0.5
µCi/min) continuous infusion (0.05 µCi/min) of [3-3H]-glucose was administered to measure
whole-body glucose turnover, as described previously (32). After 5 h fasting, mice received a
continuous insulin infusion (4 mU/kg/min), and blood glucose was maintained at basal levels
by a variable infusion of a 50% (w/v) glucose solution. Arterial blood samples were collected
during steady state conditions (rate of glucose appearance = rate of glucose disappearance),
Page 7 of 37 Diabetes
and at 80, 90, 100, 110, and 120 min for determination of Rd and Ra as described above. At
120 min, a 13 µCi bolus of [14
C]-2-deoxy-D-glucose was injected into the jugular vein and
arterial blood sampled at 122, 125, 130, 135, and 145 min and mice were then culled and
tissues extracted and frozen for subsequent gene expression and glucose uptake
determinations.
Statistical Analyses
Statistical significance was determined by a one-way or two-way ANOVA with
multiple comparisons or repeated measures, or a two-tailed paired Student’s t-test as
appropriate. p < 0.05 was considered significant: * p<0.05, ** p<0.01 and *** p<0.001.
Page 8 of 37Diabetes
RESULTS
TCPTP regulates AgRP neuronal insulin sensitivity and glucose metabolism
To determine the extent to which TCPTP deficiency in AgRP/NPY neurons may
influence whole-body glucose metabolism, we crossed Ptpn2fl/fl
mice with Agrp-Ires-Cre
transgenic mice to excise Ptpn2 in AgRP-expressing neurons (Agrp-Ires-Cre;Ptpn2fl/fl
:
AgRP-TC). TCPTP deletion in AgRP neurons (26) did not influence the overall number of
AgRP/NPY neurons (Fig. S1), as defined by the Npy-rGFP reporter that marks >85% of
AgRP neurons (33). To specifically assesses the influence of TCPTP-deficiency on insulin
signalling in AgRP/NPY neurons (marked by Npy-rGFP) we monitored for AKT Ser-473
phosphorylation (p-AKT) by immunofluorescence microscopy. In particular, we monitored
for differences in p-AKT at intraperitoneal insulin doses where p-AKT predominated in
AgRP/NPY neurons, as opposed to higher insulin doses where p-AKT was also evident in
POMC neurons (Fig. 1; Fig. S2). As noted previously (26), basal p-AKT in AgRP/NPY
neurons (as reflected by the increased number of p-AKT-positive AgRP/NPY neurons) was
enhanced in AgRP-TC mice (Fig. 1). Importantly, insulin-induced p-AKT in AgRP/NPY
neurons was significantly increased in AgRP-TC mice (Fig. 1). Thus, TCPTP deficiency in
AgRP/NPY neurons enhances insulin signalling.
To explore the impact of TCPTP-deficiency and the promotion of IR signalling in
AgRP/NPY neurons on glucose metabolism we utilised 8-10-week old chow-fed Ptpn2fl/fl
and
AgRP-TC mice prior to any overt differences in body weight (Fig 2a) and whole-body
adiposity, as measured by DEXA (Fig 2b). We found that TCPTP deficiency in AgRP/NPY
neurons enhanced whole-body insulin sensitivity and glucose metabolism, as reflected by the
improved glucose handling in response to boluses of insulin or glucose (Fig. 2c-d) and by the
reduced fasted blood glucose and plasma insulin levels (Fig. 2e-f). To explore the extent to
which the enhanced insulin signalling in AgRP/NPY neurons in AgRP-TC mice might be
Page 9 of 37 Diabetes
responsible for the improved whole-body glucose metabolism we bred AgRP-TC mice onto
an Insrfl/+
background so that Insr gene expression in AgRP/NPY neurons would be reduced
by 50%; we have shown previously that this largely corrects the otherwise enhanced
hypothalamic insulin signalling, BAT activity and white adipose tissue browning in AgRP-
TC mice (26). We found that the enhanced insulin-induced p-AKT in AgRP/NPY ARC
neurons in AgRP-TC mice was attenuated by 67% in AgRP-TC-IR (Fig. 3a). Importantly,
glucose responses and fasted blood glucose and insulin levels in AgRP-TC mice were
corrected so that AgRP-TC-IR mice resembled Ptpn2fl/fl
controls (Fig. 3c-f). By contrast IR
heterozygosity alone (Agrp-Ires-Cre;Insrfl/+
: AgRP-IRfl/+
) repressed insulin-induced p-AKT
signalling in AgRP/NPY neurons when compared to Insrfl/+
controls (Fig. S3a), however,
this was not sufficient to overtly affect glucose metabolism (Fig. S3b-g), precluding any
effects of IR heterozygosity in AgRP-TC mice being due to baseline effects on glucose
homeostasis. Therefore, TCPTP-deficiency and the promotion of IR signalling in AgRP/NPY
neurons improves whole-body glucose metabolism independent of any effects on body
weight.
TCPTP-deficiency improves systemic insulin sensitivity.
To determine how TCPTP-deficiency and the promotion of IR signalling in
AgRP/NPY neurons might improve glucose metabolism we subjected mice to pyruvate
tolerance tests as a means of assessing effects on hepatic glucose production; administration
of the gluconeogenic substrate pyruvate can increase blood glucose levels by promoting
gluconeogenesis. We found that glucose excursions in response to pyruvate (mice fasted for 6
h from 9 am) were significantly attenuated in AgRP-TC mice and reversed by Insr
heterozygosity in AgRP-TC-IR mice (Fig. 4a). Moreover, genes encoding glucose-6
phosphatase (G6pc) and phosphoenolpyruvate-carboxykinase (Pck1), enzymes involved in
Page 10 of 37Diabetes
the rate-limiting steps of gluconeogenesis, were decreased in AgRP-TC mice (Fig. 4b). The
decreased glucose excursions in response to pyruvate in AgRP-TC mice were corrected by
Insr heterozygosity, so that AgRP-TC-IR mice were indistinguishable from Ptpn2fl/fl
controls
(Fig. 4a). These results are consistent with TCPTP deficiency and the promotion of IR
signalling in AgRP/NPY neurons repressing hepatic gluconeogenesis.
As the transformation of exogenous pyruvate into hepatic glucose is highly dependent on
insulin sensitivity, we further explored the effects of TCPTP-deficiency in AgRP/NPY
neurons on whole-body insulin sensitivity and glucose metabolism using hyperinsulinemic-
euglycemic clamps. The glucose-infusion rate (GIR) needed to maintain euglycemia during
the clamp was markedly increased in AgRP-TC versus Ptpn2fl/fl
mice, consistent with TCPTP
deficiency in AgRP/NPY neurons improving whole-body insulin sensitivity (Fig. 4c; Fig.
S4). The improved insulin sensitivity was accompanied by both an increased repression of
endogenous glucose production (EGP; a measure of HGP; Fig. 4d-e) decreased hepatic
expression of the gluconeogenic genes G6pc and Pck1 and increased hepatic expression of
Il6 (Fig. 4f) and an increased rate of glucose disposal (Rd; a measure of glucose uptake; Fig.
4g). Basal endogenous glucose production and glucose disposal were not altered in AgRP-
TC versus Ptpn2fl/fl
mice in keeping with the improved glucose metabolism being a specific
response to insulin (Fig. 4d, g). Furthermore, the increased GIR, decreased hepatic
gluconeogenic gene expression, increased hepatic Il6 expression, decreased EGP and
increased Rd in clamped mice were completely corrected in AgRP-TC-IR mice (Fig. 4c-g),
causally linking the improved systemic insulin sensitivity and glucose metabolism in AgRP-
TC mice with the promotion of insulin signalling in AgRP/NPY neurons.
TCPTP-deficiency represses hepatic glucose production.
Page 11 of 37 Diabetes
Our studies point towards the improved glucose homeostasis in AgRP-TC mice
resulting from both the enhanced repression of HGP and increased Rd. Therefore, the effects
on the liver may be secondary to the overall improvement in systemic insulin sensitivity, or
attributable to exacerbated hypothalamic-liver axis responses. To test the influence on the
hypothalamic-liver axis, overnight fasted Ptpn2fl/fl
versus AgRP-TC mice were
intracerebroventricular (ICV)-administered vehicle or insulin and then processed for pyruvate
tolerance tests, or hypothalami and livers were extracted and processed for quantitative real
time PCR (Fig. 5a-d). Although at the insulin doses chosen, glucose excursions in response
to pyruvate were not altered in Ptpn2fl/fl
mice, pyruvate responses were significantly
repressed in AgRP-TC mice (Fig 5b). Moreover, in AgRP-TC mice, ICV administered
insulin significantly repressed the hypothalamic expression of Agrp and Npy without altering
Pomc expression (Fig. 5c) and increased hepatic Il6 expression while repressing Pck1 and
G6pc expression (Fig 5d). Although basal hepatic Pck1 and G6pc expression in AgRP-TC
mice was increased (Fig. 5d), this is likely a compensatory response to prevent overt
hypoglycaemia after the overnight fast. Taken together these results are consistent with the
enhanced insulin signalling in AgRP/NPY neurons acting through the hypothalamic-liver
axis to directly repress hepatic glucose production and improve glucose metabolism.
TCPTP-deficiency increases BAT glucose uptake
Acute changes in AgRP neuronal activation may elicit effects on glucose metabolism
by specifically influencing glucose uptake in BAT (13). Our studies indicate that the
enhanced IR signalling in AgRP/NPY neurons in AgRP-TC mice increases systemic insulin
sensitivity, at least in part by improving Rd. To determine the extent to which this may
involve BAT glucose uptake we administered mice [14
C]-2-deoxy-D-glucose at the end of
hyperinsulinemic-euglycemic clamps and assessed uptake in varied tissues (Fig. 4 h).
Page 12 of 37Diabetes
Although glucose uptake was not altered in the brain (hypothalamus), where glucose uptake
is not insulin-responsive, nor in skeletal muscle or epididymal white adipose tissue, where
insulin promotes glucose uptake, we found that glucose uptake was overtly increased in the
BAT of AgRP-TC mice (Fig. 4 h). Moreover, glucose uptake was increased in the inguinal
fat pads of AgRP-TC mice (Fig. 4 h), where we have shown previously there is an increased
abundance of thermogenically active beige adipocytes (26). By contrast we did not note any
increase in glucose uptake in epididymal white adipose tissue in AgRP-TC mice (Fig. 4 h).
As the epididymal fat pad does not undergo browning in AgRP-TC mice (26), these results
are consistent with TCPTP-deficiency in AgRP/NPY neurons specifically increasing glucose
uptake in brown and beige adipocytes. Moreover, the selective increase in BAT and inguinal
white adipose tissue glucose uptake points towards this being a direct response to TCPTP-
deficiency in AgRP/NPY neurons, rather than being an outcome of the systemic increase in
insulin sensitivity. Importantly, the increased glucose uptake in BAT and inguinal white
adipose tissue glucose were reduced to normal levels by Insr heterozygosity, so that AgRP-
TC-IR mice were indistinguishable from Ptpn2fl/fl
controls (Fig. 4 h). Our studies point
towards TCPTP-deficiency in AgRP/NPY neurons promoting IR signalling to improve
whole-body glucose metabolism via the repression of HGP and the promotion of glucose
uptake in brown/beige adipocytes.
TCPTP regulates feeding-associated hepatic glucose metabolism
We have shown recently that TCPTP levels in the ARC are coordinated by diurnal
feeding rhythms (26). Accordingly, we asked whether diurnal fluctuations in TCPTP might
also help regulate the hypothalamic control of hepatic glucose production. To explore this,
we determined whether TCPTP-deficiency in AgRP neurons might differentially influence
the hypothalamic-liver axis in response to feeding and fasting. To test this we ICV-
Page 13 of 37 Diabetes
administered vehicle or insulin to mice where food was withheld (food-restricted) at the start
of the dark cycle (Fig. 6a-f) versus ad libitum fed mice 4 h after the start of the dark cycle
(Fig. 6g-k), when we have shown previously mice are satiated (26); under these conditions
hypothalamic TCPTP levels are high and low respectively (26). To explore the influence on
hepatic glucose metabolism we subsequently subjected mice to pyruvate tolerance tests, or
extracted livers for gene expression analyses. In food-restricted Ptpn2fl/fl
control mice, when
hypothalamic TCPTP levels are high, we found that ICV insulin had no effect on glucose
excursions in response to pyruvate (Fig. 6b, e). By contrast, in fed Ptpn2fl/fl
mice, when
hypothalamic TCPTP levels are low, ICV insulin effectively repressed pyruvate responses
(Fig. 6h, k). Strikingly, in food-restricted AgRP-TC mice, pyruvate responses were not only
lower than Ptpn2fl/fl
controls but reduced further in response to ICV insulin (Fig. 6c, e),
whereas in fed mice, responses were similar to those of controls (Fig. 6i, k). Moreover, the
precocious ICV insulin-mediated repression of pyruvate responses accompanying AgRP
TCPTP deficiency in otherwise unresponsive food-restricted mice were corrected by Insr
heterozygosity (Fig. 6d, e), whereas pyruvate responses in fed mice were not altered by Insr
heterozygosity (Fig. 6i, k). Similarly, in food-restricted Ptpn2fl/fl
mice, hepatic Il6, Pck1 and
G6pc expression was unaltered by ICV insulin, whereas in AgRP-TC mice ICV insulin
increased hepatic Il6 expression and repressed Pck1 and G6pc expression and these responses
were corrected in AgRP-TC-IR mice (Fig. 6f). These results point towards diurnal feeding-
associated fluctuations in TCPTP in AgRP neurons serving to coordinate hepatic glucose
metabolism.
Page 14 of 37Diabetes
DISCUSSION
Gene deletion studies in rodents have established that insulin action on AgRP neurons
in the hypothalamus is important for insulin’s ability to suppress hepatic glucose production
(10). Moreover, recent human studies exploring the utility of intranasal insulin administration
have substantiated insulin’s ability to act via the CNS to suppress hepatic glucose production
and promote glucose uptake in lean, but not overweight individuals (34; 35). However, the
mechanisms by which hypothalamic insulin action is coordinated to influence whole-body
glucose metabolism remain unclear.
We have shown previously that the abundance of the IR phosphatase TCPTP in
hypothalamic neurons including POMC and AgRP neurons is altered by diurnal feeding
rhythms in mice (26). Fasting increases TCPTP expression, whereas feeding both represses
TCPTP expression and promotes its rapid degradation (26). Increases in TCPTP serve to
attenuate IR signalling in AgRP neurons after a fast to facilitate AgRP neuronal activation by
hormones such as ghrelin (26), whereas the postprandial elimination of TCPTP helps
promote IR signalling in AgRP neurons to facilitate AgRP neuronal inhibition by circulating
insulin (26). We have shown that the regulation of IR signalling by TCPTP in AgRP neurons
coordinates the browning of white adipose tissue and the expenditure of energy with feeding,
so that fasting and AgRP neuronal activation repress browning and feeding and AgRP
neuronal inhibition promotes browning and the expenditure of energy (26). In this way
diurnal fluctuations in hypothalamic TCPTP associated with feeding and fasting help
maintain energy balance.
In this manuscript we report that the regulation of IR signalling by TCPTP in AgRP
neurons is also important in coordinating whole-body glucose metabolism. We demonstrate
that TCPTP deletion in AgRP neurons (emulating the fed state when hypothalamic TCPTP is
eliminated) promotes IR signalling to enhance whole-body insulin sensitivity and glucose
Page 15 of 37 Diabetes
homeostasis. Mice lacking TCPTP in AgRP neurons show improved responses to glucose,
pyruvate and insulin, reduced fasted blood glucose and plasma insulin levels and reduced
glucose infusion rates during hyperinsulinemic-euglycemic clamps, independent of any
differences in body weight/adiposity. Importantly, the enhanced glucose metabolism could be
corrected by Insr heterozygosity in AgRP neurons, which largely corrected insulin-induced
PI3K/AKT signalling in AgRP neurons. In part, the improved glucose metabolism was
attributable to the enhanced suppression of HGP. The enhanced suppression of HGP was a
direct consequence of the hypothalamic-liver axis (7; 10; 15; 16; 18-21), as the CNS insulin-
induced promotion of hepatic Il6 expression and STAT3 signalling, and consequent
suppression of gluconeogenic genes and glucose excursions in response to pyruvate, were
exacerbated by TCPTP deficiency in AgRP neurons. Although TCPTP deficiency in AgRP
neurons attenuated hepatic gluconegenic gene expression, we cannot rule out a contribution
of glycogenolysis to the overall suppressed HGP in AgRP-TC mice, as previous studies have
indicated that AgRP neurons may regulate glycogenolysis (36).
The extent to which feeding/fasting-associated TCPTP fluctuations in AgRP neurons
might help coordinate HGP was highlighted by the lack of any overt effect of ICV insulin on
pyruvate-induced glucose excursions and hepatic gluconeogenic gene expression in food-
restricted control mice, where hypothalamic TCPTP levels would be high (26). This was
contrasted by the striking ability of TCPTP deletion in AgRP neurons to reinstate such
responses. By comparison, ad libitum fed and satiated mice readily responded to ICV insulin
by repressing hepatic gluconeogenic gene expression and pyruvate-induced glucose
excursions and these responses were unaltered by TCPTP deletion. Our results point towards
the control of IR signalling by TCPTP in AgRP neurons serving to coordinate hepatic
glucose metabolism, so that fasting is accompanied by elevated HGP to prevent
hypoglycaemia. In obesity, where we have shown hypothalamic TCPTP levels are high and
Page 16 of 37Diabetes
remain elevated even after feeding (26; 28), the resulting sustained repression of IR
signalling in AgRP neurons would be expected to contribute to the elevated HGP and
hyperglycemia characteristic of the obese and insulin resistant state. However, the decreased
weight gain evident in high fat fed AgRP-TC mice prohibited us from testing this directly.
Beyond influencing glucose production, our studies indicate that the regulation of IR
signalling in AgRP neurons might also impact on glucose clearance by specifically
influencing glucose uptake in BAT and inguinal white adipose tissue, where browning in
mice predominates (37-39). Activated brown and beige adipocytes contain high amounts of
the uncoupling protein-1 (UCP-1) allowing for the uncoupling of fatty acid oxidation from
ATP production to generate heat (2; 3; 39). Although largely ignored as a tissue involved in
glucose homeostasis, early hyperinsulinemic-euglycemic clamp studies in rodents highlighted
BAT as a major insulin-responsive depot for glucose uptake, exceeding on a per unit mass
basis glucose uptake in muscle or white adipose tissue (1). Consistent with this we found that
when normalised for tissue mass, BAT was more effective than muscle or epididymal
adipose tissue in taking up glucose under hyperinsulinemic euglycemic conditions. In
humans, brown and beige adipocytes are found interdispersed in different white fat depots,
including the supraclavicular depot, as well as in the supraspinal, pericardial, and neck
regions (2; 39-45). Implantation of human brown/beige adipocytes into normal chow-fed,
high fat-fed or glucose-intolerant NOD-scid IL2(null)
mice, dramatically enhances systemic
glucose tolerance (46). Moreover variables such as high BMI, increased age or type 2-
diabetes have been shown to correlate with attenuated brown/beige glucose uptake at least in
some studies (41; 47-49). Noteworthy, Lee et al., (5) recently highlighted the importance of
brown/beige fat in humans in systemic glycemic control demonstrating that higher
brown/beige fat activity results in lesser glycemic variability. Moreover the same study
demonstrated that the thermogenic activity of brown/beige fat in humans was increased in
Page 17 of 37 Diabetes
response to glucose challenge, which increases circulating insulin (5). Our recent studies
have shown that enhanced leptin plus insulin signalling in POMC neurons, or insulin
signalling in AgRP neurons can function to promote BAT activity and the browning of
inguinal white adipose tissue in rodents (25; 26). In particular, we reported that in
fed/satiated mice when TCPTP was degraded, the enhancement of insulin signalling and
resultant inhibition of AgRP neurons increased the sympathetic innervation and browning of
white adipose tissue to promote the expenditure of energy (26). Mice lacking TCPTP in
AgRP neurons were remarkably resistant to weight gain due to the increased white adipose
tissue browning as well as BAT activity (26). In this study we assessed the influence of
TCPTP loss and the promotion of insulin signalling in AgRP neurons on glucose metabolism.
For these studies we used 8-10 week old mice prior to any overt differences in adiposity/body
weight resulting from the increased browning and BAT activity. Our studies indicate that the
control of insulin signalling by TCPTP in AgRP neurons influences glucose metabolism at
least in part through the promotion of BAT and beige adipocyte glucose uptake. Steculorum
et al., (13) reported that the pharmacogenetic activation of AgRP neurons promotes insulin
resistance by repressing BAT glucose uptake. Our results demonstrate that the deletion of
TCPTP and inhibition of AgRP neurons (26) promotes systemic insulin sensitivity
accompanied by increased glucose uptake in BAT and beige adipocytes in inguinal fat depots.
Our findings are consistent with the TCPTP control of insulin-mediated AgRP neuronal
activation being instrumental in coordinating BAT/beige adipocyte activity with glucose
uptake. Consistent with this assertion, Lee et al (5) reported that BAT glucose uptake in
humans correlates with thermogenesis (as assessed by measuring heat production with
infrared thermography). As fatty acid oxidation is essential for BAT thermogenesis (50-53),
it is likely that glucose indirectly contributes to BAT/beige adipocyte activity by promoting
lipogenesis and/or supporting ATP production during thermogenic responses through
Page 18 of 37Diabetes
anaerobic glycolysis (2; 3). By contrast, other studies argue that BAT activity and glucose
uptake can be dissociated. For example Blondin et al., (49) reported recently that despite
glucose uptake in BAT being diminished in older men with type 2 diabetes, cold-induced
BAT oxidative metabolism and thermogenesis were not altered. However, this does not
preclude feeding-induced beige adipocyte thermogenesis/WAT browning (26) normally
being coordinated with glucose metabolism. This would provide an effective mechanism for
coordinating both the removal and utilisation of glucose by beige adipocytes to prevent of
postprandial hyperglycemia.
Our results underscore the critical role of CNS insulin signalling in coordinating
peripheral glucose metabolism through effects on both HGP and BAT/beige adipocyte
glucose uptake. Taken together with our previous findings (26), our results point towards
feeding/fasting associated alterations in hypothalamic TCPTP integrating the systemic
control of glucose metabolism and energy expenditure with the nutritional state of organism
to maintain both glucose and energy homeostasis. Thus, the promotion of CNS insulin
sensitivity is likely to provide an important means by which to concomitantly promote weight
loss and improve whole-body glucose metabolism and glycemic control in obesity and type 2
diabetes.
Page 19 of 37 Diabetes
FIGURE LEGENDS
Figure 1. Deletion of TCPTP in AgRP neurons enhances insulin signalling. 8-10-week-old
AgRP-TC;Npy-hGFP or Ptpn2fl/fl
;Npy-GFP overnight fasted male mice were administered
(intraperitoneal) saline or 0.85 mU/g insulin for 15 min and paraformaldehyde-fixed brains
processed for immunofluorescence microscopy monitoring for p-AKT hypothalamic
immunoreactivity. Representative images and quantified (means ± SEM) results are shown
for the indicated number of mice.
Figure 2. TCPTP-deficiency in AgRP neurons improves whole body glucose metabolism.
a) Body weight and b) body composition of 8-week-old AgRP-TC or Ptpn2fl/fl
male mice. 8-
week-old male AgRP-TC or Ptpn2fl/fl
mice were subjected to c) insulin (0.5 mU/g), or d)
glucose (2 mg/g) tolerance tests; areas under curves were determined. Fed and fasted (14 h)
e) blood glucose and f) plasma insulin levels from 10-week-old AgRP-TC or Ptpn2fl/fl
male
mice. Results shown are means ± SEM for the indicated number of mice.
Figure 3. TCPTP-deficiency in AgRP neurons improves whole body glucose metabolism by
promoting insulin signalling in AgRP neurons. a) 8-10-week-old Ptpn2fl/fl
;Npy-GFP, AgRP-
TC;Npy-GFP or AgRP-TC-IR:Npy-GFP overnight fasted male mice were administered
(intraperitoneal) saline or 0.85 mU/g insulin for 15 min and paraformaldehyde-fixed brains
processed for immunofluorescence microscopy monitoring for p-AKT hypothalamic
immunoreactivity. b) Body weights in 8-week-old male Ptpn2fl/fl
, AgRP-TC or AgRP-TC-IR
mice. 8-week-old male Ptpn2fl/fl
, AgRP-TC or AgRP-TC-IR mice were subjected to c)
glucose (2 mg/g) or d) insulin (0.5 mU/g) tolerance tests; areas under curves were determined.
Fed and fasted e) blood glucose and f) plasma insulin levels in 10-week-old Ptpn2fl/fl
, AgRP-
Page 20 of 37Diabetes
TC or AgRP-TC-IR male mice. Representative images and quantified (means ± SEM) results
are shown for the indicated number of mice.
Figure 4. TCPTP-deficiency in AgRP neurons enhances the repression of hepatic glucose
production and BAT glucose uptake. a) 8-week-old male Ptpn2fl/fl
, AgRP-TC or AgRP-TC-
IR male mice were subjected to pyruvate tolerance tests (1 mg/g); areas under curves were
determined. b) 9-week-old Ptpn2fl/fl
, AgRP-TC or AgRP-TC-IR ad libitum fed male mice
were culled and livers extracted for quantitative PCR. c-h) Conscious unrestrained 8-10-
week-old Ptpn2fl/fl
, AgRP-TC or AgRP-TC-IR male mice were subjected to
hyperinsulinemic-euglycemic clamps. c) Glucose infusion rates (GIR), d-e) basal and
clamped endogenous glucose production (EGP; glucose appearance rate minus GIR), f) gene
expression in extracted livers and g) glucose disappearance rates (Rd). h) Hyperinsulinemic-
euglycemic clamped mice were administered a bolus of [14
C]-2-deoxy-D-glucose (13 µCi
intravenous) and tissue-specific insulin-stimulated glucose uptake was determined in brain
(hypothalamus), brown adipose tissue (BAT), gastrocnemius muscle, epididymal white
adipose tissue (WAT), and inguinal WAT. Results shown are means ± SEM for the indicated
number of mice.
Figure. 5. TCPTP-deficiency in AgRP neurons promotes the ICV insulin-mediated
repression of hepatic glucose production. a) Experimental paradigm schematic. 8-week-old
Ptpn2fl/fl
or AgRP-TC male mice were fasted overnight and administered
[intracerebroventricular (ICV)] saline or insulin (0.1 mU/animal, 5 injections over 5 h) as
indicated and subjected to either b) pyruvate tolerance tests (1 mg/g; areas under curves were
determined) or c) hypothalami and d) livers extracted for quantitative PCR. Results shown
are means ± SEM for the indicated number of mice.
Page 21 of 37 Diabetes
Fig. 6. TCPTP in AgRP neurons inhibits the ICV insulin-mediated repression of hepatic
glucose production in fasted mice. a, g) Experimental paradigm schematics. b-f) 8-10-week-
old Ptpn2fl/fl
, AgRP-TC or AgRP-TC-IR male mice were food-restricted (just prior to lights
out, 6:30pm) and administered ICV) saline or insulin (0.1 mU/animal, 5 injections over 5 h)
as indicated and subjected to either b-e) pyruvate tolerance tests (1 mg/g; areas under curves
were determined) or f) livers extracted for quantitative PCR. h-k) 8-10-week-old Ptpn2fl/fl
,
AgRP-TC or AgRP-TC-IR male mice were ad libitum fed until satiated (4 h after lights off)
and administered ICV saline or insulin (0.1 mU/animal, 5 injections over 5 h) as indicated
and subjected to pyruvate tolerance tests (1 mg/g; areas under curves were determined).
Results shown are means ± SEM for the indicated number of mice.
Page 22 of 37Diabetes
AUTHOR CONTRIBUTIONS
Conceptualization – T.T. and G.T.D.; Methodology, T.T., G.T.D., R.S.L.-Y. and J.C.B.;
Investigation – G.T.D, and R.S.L.-Y. Writing – Original Draft, T.T. and G.T.D.; Writing –
Review & Editing, T.T., G.T.D., R.S.L.-Y. and J.C.B.; Funding Acquisition, T.T. and G.D.
ACKNOWLEDGMENTS
We thank Sunena Bhandari for technical support and Herbert Herzog for access to Insrfl/fl
mice. This work was supported by the National Health and Medical Research Council
(NHMRC) of Australia (to T.T.) and the Diabetes Australia Research Trust (to G.D.). T.T. is
a NHMRC Principal Research Fellow. T.T. is the guarantor of the study.
CONFLICTS OF INTERTEST
None declared.
Page 23 of 37 Diabetes
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