TCPTP regulates insulin signalling in AgRP neurons to ... · 27/04/2018 · TCPTP regulates insulin signalling in AgRP neurons to coordinate glucose metabolism with feeding Garron

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: [email protected]

Garron Dodd (+61) 3 9902 9345

Email: [email protected]

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,


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


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.


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


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),


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.


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


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


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.


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).


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-


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.


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


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


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


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


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.


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-


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.


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.


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.


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