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. 2018 Feb 8;159(4):1585–1594. doi: 10.1210/en.2017-03199

In Uncontrolled Diabetes, Hyperglucagonemia and Ketosis Result From Deficient Leptin Action in the Parabrachial Nucleus

Thomas H Meek 1, Miles E Matsen 1, Chelsea L Faber 1, Colby L Samstag 1, Vincent Damian 1, Hong T Nguyen 1, Jarrad M Scarlett 1, Jonathan N Flak 2, Martin G Myers Jr 2, Gregory J Morton 1,✉
PMCID: PMC5939636  PMID: 29438473

Abstract

Growing evidence implicates neurons that project from the lateral parabrachial nucleus (LPBN) to the hypothalamic ventromedial nucleus (VMN) in a neurocircuit that drives counterregulatory responses to hypoglycemia, including increased glucagon secretion. Among LPBN neurons in this circuit is a subset that expresses cholecystokinin (LPBNCCK neurons) and is tonically inhibited by leptin. Because uncontrolled diabetes is associated with both leptin deficiency and hyperglucagonemia, and because intracerebroventricular (ICV) leptin administration reverses both hyperglycemia and hyperglucagonemia in this setting, we hypothesized that deficient leptin inhibition of LPBNCCK neurons drives activation of this LPBN→VMN circuit and thereby results in hyperglucagonemia. Here, we report that although bilateral microinjection of leptin into the LPBN does not ameliorate hyperglycemia in rats with streptozotocin-induced diabetes mellitus (STZ-DM), it does attenuate the associated hyperglucagonemia and ketosis. To determine if LPBN leptin signaling is required for the antidiabetic effect of ICV leptin in STZ-DM, we studied mice in which the leptin receptor was selectively deleted from LPBNCCK neurons. Our findings show that although leptin signaling in these neurons is not required for the potent antidiabetic effect of ICV leptin, it is required for leptin-mediated suppression of diabetic hyperglucagonemia. Taken together, these findings suggest that leptin-mediated effects in animals with uncontrolled diabetes occur through actions involving multiple brain areas, including the LPBN, where leptin acts specifically to inhibit glucagon secretion and associated ketosis.

In uncontrolled diabetes, deficient leptin action in the lateral parabrachial nucleus contributes to increased glucagon levels and associated ketosis in this setting.

The recent finding that systemic (1, 2) or intracerebroventricular (ICV) (3–7) administration of the adipocyte hormone leptin can fully normalize blood glucose levels in rodent models of uncontrolled, insulin-deficient diabetes mellitus (uDM) suggests that mechanisms driving hyperglycemia in this setting are sensitive to leptin action in the brain. Among these mechanisms is increased hepatic glucose production (HGP) and reduced glucose utilization, both of which are reversed by ICV leptin (5). Also normalized in this setting are elevated plasma levels of glucagon, corticosterone, and ketone bodies (4, 5). Because this action of leptin occurs despite the absence of any detectable effect on pancreatic insulin synthesis or secretion, leptin action in the brain appears to have the capacity to regulate glucose metabolism independent of insulin action. A key priority for future work is to identify the neuronal substrates underlying this effect.

The biological actions of leptin are mediated through the long form of the leptin receptor (LepRb) (8) that is expressed widely in the brain, including both hypothalamic and extrahypothalamic areas (9, 10). Within the hypothalamus, leptin action in the ventromedial nucleus (VMN) appears to be sufficient but not required for leptin’s glucose-lowering effects in rodent models of type 1 diabetes (T1D) (11). Similarly, leptin receptor signaling in pro-opiomelanocortin (Pomc) neurons, situated in the adjacent arcuate nucleus, is also not required for the antidiabetic effect of leptin because (1) reactivation of leptin receptors in these neurons does not recapitulate the antidiabetic effects of leptin in rodent models of T1D, and (2) leptin’s glucose-lowering effects are only mildly attenuated in mice lacking leptin receptors in Pomc neurons (12). By comparison, leptin signaling in γ-aminobutyric acid–ergic neurons appears to mediate much of the glucose-lowering effects of leptin in insulin-deficient mice (12). Yet neither glutamate nor γ-aminobutyric acid release from LepRb (+) neurons is required for leptin’s antidiabetic effects, whereas STAT3, a key signal transduction molecule involved in leptin signaling, is required (13). Taken together, these observations suggest that leptin action within a distributed neuronal network, rather than in a particular brain area or neuronal subset, may mediate reversal of hyperglycemia in uncontrolled diabetes.

Among other leptin-regulated neurons implicated in glucose homeostasis are those situated in the lateral parabrachial nucleus (LPBN) that coexpress the leptin receptor and cholecystokinin (CCK) (14, 15). These LPBN LepRbCCK neurons project to the VMN, and activation of this LPBN→VMN neurocircuit is implicated in the counterregulatory response (CRR) to hypoglycemia responsible for returning low blood glucose levels (e.g., during insulin-induced hypoglycemia) back to the normal range. Specifically, LPBN neurons that express both CCK and LepRb are activated by hypoglycemia, and using a designer receptors exclusively activated by designed drugs–based pharmacogenetic approach, activation of LPBN LepRbCCK neurons is sufficient to activate the CRR and thereby raise blood glucose levels, whereas conversely, inhibition of the same neurons blunts the glycemic response to glucoprivation (14, 15). Because this circuit is engaged to increase blood glucose in response to noxious stimuli (16), its activation may serve to raise blood glucose levels across a wide range of physiological and pathophysiological conditions.

Many of the same behavioral, neuroendocrine, and autonomic responses are engaged in response to either deficiency of immediately available fuel (i.e., low glucose) or stored energy (i.e., low leptin) (13, 17). Because activation of this LepRbPBN→VMN circuit is both strongly implicated in the CRR to hypoglycemia and sufficient to raise blood glucose levels (18), its activation could also potentially contribute to hyperglycemia and associated neuroendocrine abnormalities characteristic of uDM. To test this hypothesis, we determined whether leptin action limited to the LPBN is capable of blunting either hyperglycemia or its underlying neuroendocrine mediators in rodent models of T1D. Our findings implicate deficient leptin action in this brain area in the genesis of increased glucagon levels (and associated ketosis) in this setting.

Research Design and Methods

Animals

All procedures were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee at the University of Washington. All studied animals were individually housed under specific-pathogen free conditions in a temperature-controlled room with a 12-hour light:12-hour dark cycle and provided with ad libitum access to water and chow unless otherwise stated (PMI Nutrition, St. Louis, MO).

Adult male Wistar rats were obtained from Harlan Laboratories (Indianapolis, IN). To determine if leptin’s antidiabetic effects in uDM are dependent on LepRb expression in the LPBN, we generated mice with deletion of leptin receptors from this brain region. Because a variety of cell types in the LPBN express LepRb (14), this goal was achieved by crossing Cckcre mice (which express cre recombinase in CCK neurons) with Leprflox/flox animals to generate mice lacking LepRb expression specifically in this neuronal subset [i.e., LepRbCCK knockout (KO) animals] as previously described (14). Cckcre mice and Leprflox/flox animals were kindly provided to us by Drs. Martin Myers Jr. (University of Michigan, Ann Arbor, MI) and Streamson Chua Jr. (Albert Einstein College of Medicine, New York, NY). Mice were genotyped for the Cckcre and Leprflox alleles as described previously (14).

Surgery

Rats underwent implantation of a bilateral cannula directed to the LPBN (Plastics One, Roanoke, VA) under isoflurane anesthesia at stereotaxic coordinates: 9.4 mm posterior to bregma; ±2.1 mm lateral, and 4.8 mm below the skull surface. The cannula was secured to the skull with stainless steel screws and dental cement. Buprenorphine hydrochloride (0.3 mg/kg; Reckitt Benckiser Healthcare, Richmond, VA) was administered perioperatively, and animals were allowed to recover for at least a week prior to experimentation.

For mouse studies, both LepRbCCK KO animals and their littermate controls were implanted with a single cannula placed in the lateral ventricle (Alzet; DURECT Corp., Cupertino, CA) at stereotaxic coordinates: 0.7 mm posterior to bregma, 1.3 mm lateral, and 2.3 mm below the skull surface as previously described (11, 19). After allowing the animals at least 1 week to recover from surgery, streptozotocin-induced diabetes mellitus (STZ-DM) animals were implanted subcutaneously (SC) with an osmotic minipump that was connected to the lateral ventricle cannula to enable direct infusion of either vehicle [veh; phosphate-buffered saline (PBS), pH 7.9] or leptin (1 μg/d) directly to the brain.

Effect of intra-LPBN leptin on diabetic hyperglycemia in STZ-DM rats

Following a recovery period, adult male Wistar rats bearing a cannula directed to the LPBN received either two consecutive daily SC injections of STZ (40 mg/kg body weight) to induce uDM or veh (NaCit, pH 4.5) as previously described (5, 20). Five days later, animals received daily bilateral intraparenchymal injections into the LPBN of either veh (PBS, pH 7.9) or leptin (0.1 µg/d; Dr. Parlow; National Hormone Peptide Program) using a microinjection needle that extended 1 mm beyond the tip of the cannula. In this way, the following three groups of rats were studied: (1) SC veh/LPBN veh, (2) SC STZ/LPBN veh, and (3) SC STZ/LPBN leptin. Intraparenchymal injections were performed over a 60-second period, with leptin or its veh dissolved in a final volume of 500 nL (5, 11, 19). Body weight, food intake, and blood glucose were measured daily in the fed state at 10 am.

Role of leptin receptors in the LPBN on diabetic hyperglycemia in STZ-DM mice

Both male Cre (+) (LepRbCCK KO) and Cre (−) LepRflox/flox [LepRb wild-type (WT)] mice were allowed to recover for at least 1 week following cannulation of the lateral ventricle. Each animal subsequently received two SC injections spaced 3 days apart of STZ (150 mg/kg body weight) to induce uDM (11, 19). Animals were defined as diabetic when, after 2 consecutive days, the fed blood glucose level was >200 mg/dL. Animals meeting this criterion were then implanted SC with an osmotic minipump connected to the lateral ventricle cannula to enable continuous infusion of either veh (PBS, pH 7.9) or leptin (1 μg/d) directly into the brain. Body weight, food intake, and blood glucose were measured daily in the fed state at 10 am.

Blood collection and tissue processing

Daily blood glucose levels were measured using a handheld glucometer (Accu-Chek, Corydon, IN) on blood obtained from tail capillary samples. At study completion, blood samples for plasma hormonal measures was collected from trunk blood into appropriately treated tubes (5) and centrifuged with the plasma removed, aliquoted, and stored at −80°C for subsequent assay. Plasma insulin levels were determined by enzyme-linked immunosorbent assay (ELISA) (Crystal Chem, Elk Grove Village, IL), ketone bodies using a colorimetric kit (Wako Chemicals, Richmond, VA), thyroxine (T4) levels using an enzyme immunosorbent assay (MP Biomedicals, Solon, OH), plasma corticosterone (Alpco, Salem, NH) levels using an ELISA, and glucagon levels for rat and mouse using a RIA kit (Linco Research, St. Charles, MO) and an ELISA (Mercodia, Winston-Salem, NC), respectively.

For immunohistochemical studies to verify cannula placement and injectate spread in rats, anesthetized animals were perfused with ice-cold PBS (Diamedix, Miami Lakes, FL) followed by 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M PBS. Brains were removed, postfixed in 4% paraformaldehyde overnight, and transferred to a 25% sucrose solution in PBS for 48 hours at 4°C before being frozen in isopentane cooled on dry ice. Coronal sections (14 μM) were taken throughout the hindbrain and hypothalamus, slide-mounted, and stored at −80°C for immunohistochemical staining and cannula verification.

Verification of cannula and injection site

Cannula placement in rats was confirmed postmortem using standard histological techniques in which Chicago Sky Blue ink was microinjected in the LPBN of euthanized animals, and the brains were removed, sectioned on a cryostat, mounted on glass slides, and analyzed for placement. To further verify the spread of injectate, we examined leptin-induced activation of pSTAT3, a marker of leptin receptor activation in a subset of animals that received an intraparenchymal injection of leptin directly into the parabrachial nucleus. Immunoreactivity for pSTAT3 was visualized in the hypothalamus and LPBN using a Nikon Eclipse E600 microscope fitted with a grid reticule with the investigator blinded to study conditions. A representative image of immunostaining of pSTAT3 in the LPBN and adjacent arcuate nucleus (as a negative control) is depicted in Fig. 1. Rats with injection sites located outside of the LPBN were excluded from further analysis.

Figure 1.

Figure 1.

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Validation of lateral parabrachial nucleus (lPBN) injection site. Schematic and representative image of immunostaining for pSTAT3, a downstream marker of leptin receptor activation following intraparenchymal injection of leptin into the lateral parabrachial nucleus in the (A, C) hypothalamic arcuate nucleus (negative control) and (B, D) lateral parabrachial nucleus. 3V, third ventricle; 4V, fourth ventricle; Arc, arcuate nucleus.

Immunohistochemistry

Immunohistochemical identification of pSTAT3 was used to measure direct leptin signaling in the brain using a protocol described previously (11, 21). Briefly, sections were washed in PBS at room temperature and were subsequently incubated 0.1% hydrogen peroxide for 1 minute. Sections were then washed and permeabilized for 10 minutes with 0.5% Triton in PBS, containing 0.1% BloxAll (Vector Laboratories, Burlingame, CA), and subsequently incubated in 1% normal serum blocking solution plus rabbit anti-pSTAT3 antibody [1:1000; Sigma-Aldrich, St. Louis, MO; Research Resource Identifier (RRID): AB_10620527] overnight at 4°C. Sections were then washed and blocked for 1 hour in blocking solution containing biotinylated antirabbit antibody (Vectastain Elite ABC kit; RRID: AB_2336820) and incubated for 30 minutes with Vector avidin/biotin complex reagent (Vector Laboratories). Slides were washed in PBS and incubated with Streptavidin Alexa 488 (RRID: AB_2336881) for 30 minutes and then mounted with Fluoromount-G (Thermo Fisher Scientific, Waltham, MA). Confocal images were taken on a Leica SP8X (Leica Microsystems, Inc., Buffalo Grove, IL).

Statistical analysis

All results are expressed as mean ± standard error of the mean. For rat studies, a one-way analysis of variance (ANOVA) with Tukey post hoc tests were used to compare mean values between multiple groups. For mouse studies, two-way repeated-measures ANOVA with Tukey post hoc tests were used. Hormonal data were analyzed by standard two-way ANOVA with Tukey post hoc tests. Statistical analyses were performed using Statistica (version 7.1; StatSoft, Inc., Tulsa, OK) or Prism (version 7.4; GraphPad Software, Inc., La Jolla, CA). In all instances, probability values of less than 0.05 were considered significant.

Results

Intra-LPBN leptin administration is not sufficient to reverse diabetic hyperglycemia in STZ-DM rats

As expected, relative to nondiabetic controls, all animals that received STZ exhibited marked reductions of both plasma insulin and leptin levels (Fig. 2A), consistent with previous evidence that STZ-DM is characterized by insulin and leptin deficiency (22). Consequently, STZ-DM animals that received veh in the LPBN exhibited a profound hyperglycemia that was maintained throughout the duration of the study, and leptin administration directly into the LPBN failed to attenuate this effect (Fig. 2B). Although a higher dose may have had a greater effect, the dose of leptin we used was previously demonstrated to normalize blood glucose levels when administered directly into the VMN of STZ-DM rats while having no effect when administered into the lateral ventricle (11). As expected, STZ-veh–treated rats were also characterized by increased food intake relative to nondiabetic controls, and this diabetic hyperphagia was similarly not attenuated by intra-LPBN leptin (Fig. 2C). Despite the increase of food intake, however, STZ-veh–treated animals lost body weight, because the excess calories cannot be stored into adipose tissue as result of the insulin deficiency and are lost in the urine. Because STZ-DM rats that received intra-LPBN leptin also exhibited similar weight loss (Fig. 2D), these data suggest that leptin signaling in the LPBN is insufficient to reverse the hyperphagia, weight loss, and hyperglycemia characteristic of STZ-DM rats.

Figure 2.

Figure 2.

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Intra-LPBN leptin is not sufficient to ameliorate diabetic hyperglycemia or hyperphagia in STZ-diabetic rats. (A) Plasma insulin and leptin levels, mean daily (B) fed blood glucose levels, (C) food intake, and (D) body weight change in either nondiabetic (veh-veh) or STZ-induced diabetic animals receiving daily intraparenchymal injections of either veh (STZ-veh) or leptin [STZ-lep parabrachial nucleus (PBN)] administered directly into the PBN (n = 6 to 7 per group). The arrow represents the start of daily leptin injections. STZ was administered on days 0 and 1 to induce uDM. Data represent mean ± standard error of the mean. *P < 0.05 vs veh-veh.

Effect of intra-LPBN on metabolic and neuroendocrine parameters in STZ-DM rats

Although leptin administration directly into the LPBN failed to attenuate the diabetic hyperglycemia in STZ-DM rats, it significantly blunted the increase of plasma glucagon levels characteristic of STZ-DM (Fig. 3A), and this effect was accompanied by reduced plasma ketone body levels (Fig. 3B). These findings are consistent with previous evidence that (1) leptin action in the brain of rats with uDM ameliorates hyperglucagonemia, and (2) ketosis, but not hyperglycemia, in this setting is dependent on elevated plasma glucagon levels (5, 11, 19, 23). Further support for this concept is found in the observation that variation in plasma glucagon levels across study groups was strongly predictive of plasma ketone body levels but not blood glucose levels among diabetic rats (Fig. 3C and 3D) (23). In contrast, leptin microinjection into the LPBN had no effect on either plasma corticosterone or T4 levels (Fig. 3E and 3F). This finding, like the absence of an effect on hyperglycemia, stands in contrast to the effect of ICV leptin in STZ-DM (5, 24).

Figure 3.

Figure 3.

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Intra-LPBN leptin is sufficient to ameliorate diabetic hyperglucagonemia in STZ-diabetic rats. (A) Plasma glucagon, (B) plasma ketone bodies, correlations between (C) plasma glucagon and blood glucose levels and (D) plasma glucagon and ketone bodies, (E) plasma corticosterone, and (F) plasma T4 levels in either nondiabetic (veh-veh) or STZ-induced diabetic animals receiving daily intraparenchymal injections of either veh (STZ-veh) or leptin [STZ-lep parabrachial nucleus (PBN)] administered directly into the PBN (n = 6 to 7 per group). Data represent mean ± standard error of the mean. *P < 0.05 vs veh-veh; #P < 0.05 vs STZ-veh.

Role of leptin receptor-expressing LPBNCCK neurons in the control of diabetic hyperglycemia in mice

To determine whether leptin signaling in LPBNCCK neurons is required for leptin’s glucose-lowering effects in uDM, we studied mice with selective deletion of leptin receptors from this neuronal subset (14). Consistent with available evidence (14), we found no differences in body weight (29.36 ± 0.38 vs 28.60 ± 0.46 g), mean daily food intake (4.98 ± 0.63 vs 4.85 ± 0.37 g/d), or blood glucose levels (118.1 ± 2.8 vs 117.7 ± 2.7 mg/dL) in LepRbCCK KO mice relative to their littermate controls at baseline (P = not significant for each) (Fig. 4). Similarly, all animals were characterized by markedly reduced plasma insulin levels, elevated blood glucose levels, and hyperphagia following STZ administration (Fig. 5A–5C). Specifically, because the increases of both blood glucose and food intake induced by STZ did not differ between LepRbCCK KO and littermate controls (LepRb WT mice), we infer that leptin signaling in LPBNCCK neurons is not required for diabetic hyperglycemia and hyperphagia (Fig. 5B and 5C). The more relevant question for this study is whether leptin signaling in this neuronal subset is required for the ability of ICV leptin to ameliorate hyperglycemia, ketosis, and other neuroendocrine manifestations of uDM. Here, we found that continuous ICV infusion of leptin normalized both blood glucose levels (Fig. 5B) and food intake (Fig. 5C) to a similar extent and at a similar rate (days 12 to 21; P < 0.05 for each) in both LepRb WT and LepRbCCK KO mice. These data suggest that leptin action in LPBNCCK neurons is not required for leptin’s ability to normalize both glycemia and food intake in STZ-DM.

Figure 4.

Figure 4.

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Baseline phenotype of mice deficient in leptin receptor in LPBNCCK neurons. (A) Body weight, (B) mean daily food intake, and (C) blood glucose levels in mice with leptin receptors deleted from LPBNCCK neurons (LepRbCCK KO) or their littermate controls (LepRb WT) (n = 7 to 10 per group). Data represent mean ± standard error of the mean.

Figure 5.

Figure 5.

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Antidiabetic effects of leptin in STZ-DM do not require leptin signaling through LPBNCCK neurons. (A) Plasma insulin and leptin levels, mean daily (B) fed blood glucose levels, (C) food intake, and (D) body weight in STZ-induced diabetic mice with leptin receptors deleted from LPBNCCK neurons (LepRbCCK KO) or their littermate controls (LepRb WT) treated with either ICV veh or leptin (n = 7 to 10 per group). Data represent mean ± standard error of the mean. *P < 0.05 vs STZ-veh WT; #P < 0.05 vs STZ-veh KO.

Our findings also showed a significant main effect of ICV leptin to lower plasma glucagon levels (P < 0.05). Further analysis revealed that ICV leptin infusion significantly reduced plasma glucagon in diabetic controls (LepRb WT; P < 0.05) but not in STZ-DM LepRbCCK KO mice (P = not significant) (Fig. 6A), although there was no significant interaction between leptin and genotype. Taken together, these observations suggest that leptin action in LPBNCCK neurons is required for the full effect of leptin to suppress hyperglucagonemia in uDM, although leptin action in other brain areas also likely contributes. Our findings also revealed that although there was a significant main effect of ICV leptin to markedly reduce plasma ketone body levels in both STZ-DM LepRbCCK KO and LepRb WT mice (P < 0.05 for each) (Fig. 6B), the variation in plasma glucagon levels was predictive of plasma ketone bodies (r = 0.471; P < 0.05). In a similar manner, we found that the effect of continuous ICV leptin infusion on plasma levels of corticosterone and T4 did not differ by genotype (Fig. 6C and 6D).

Figure 6.

Figure 6.

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Leptin-mediated normalization of hyperglucagonemia in STZ-DM mice requires leptin signaling through LPBNCCK neurons. (A) Plasma glucagon levels, (B) ketone bodies, (C) plasma corticosterone, and (D) plasma T4 levels in STZ-induced diabetic mice with leptin receptors deleted from LPBNCCK neurons (LepRbCCK KO) or their littermate controls (LepRb WT) treated with either ICV veh or leptin (n = 7 to 10 per group). Data represent mean ± standard error of the mean. *P < 0.05 vs STZ-veh WT; #P < 0.05 vs STZ-veh KO.

Discussion

Since its discovery nearly a century ago, insulin has remained the cornerstone of medical management of T1D, including the prevention of acute (e.g., ketoacidosis) and chronic diabetes complications (e.g., nephropathy, neuropathy, retinopathy, and cardiovascular disease) (25). However, recent studies have demonstrated that leptin action in the central nervous system (CNS) can restore euglycemia in rodent models of T1D via mechanisms that are independent of insulin action (3–7). Based on the recent identification of a leptin-regulated circuit involved in glucose control (14, 15), which appears to involve projections from neurons in the LPBN to the VMN and subsequently to the anterior bed nucleus of the stria terminalis (aBNST) (25), we hypothesized that leptin action on discrete components of this circuit underlies leptin’s antidiabetic action in T1D. Our data in STZ-DM rats show that whereas leptin action limited to the LPBN is insufficient to reverse the effect of uDM on levels of blood glucose, corticosterone, and T4 levels, it nevertheless attenuated the associated hyperglucagonemia and ketosis. We also show that although the antidiabetic effects of leptin remain intact in mice in which the leptin receptor was deleted from LPBN CCK neurons (LepRbCCK KO), leptin failed to normalize elevated plasma glucagon levels in these mice but did so effectively in controls. Collectively, these findings suggest that in uDM, reduced leptin action in the LPBN drives hyperglucagonemia in a selective manner, without substantially altering hyperglycemia or associated neuroendocrine defects.

To place these findings in context, consider that in response to conditions of either acute or chronic energy deficit, the brain engages a series of autonomic, behavioral, and neuroendocrine responses designed to increase the availability of fuel to the CNS while simultaneously conserving fuel stores. These responses include an increase in both the drive to eat and HGP, with the latter being mediated through increased secretion of the counterregulatory hormones, glucagon and corticosterone. Because fasting and STZ-DM are each characterized by deficiency of both insulin and leptin, we and others have argued that the adaptive CNS response to the two conditions overlaps in ways that resemble the CRR to hypoglycemia (13, 17). Inherent in this concept is the possibility that neurocircuits that drive the response to hypoglycemia are tonically inhibited by input from leptin, and recent work supports this possibility (13, 17).

Also consistent with this concept is the observation that hyperglucagonemia is a feature of both uDM (26, 27) and the CRR to hypoglycemia (28). In T1D, hyperglucagonemia contributes to both ketosis and diabetic hyperglycemia by driving hepatic production of both glucose and ketones (29). Conversely, suppression of glucagon hypersecretion (e.g., with somatostatin) lowers HGP, reduces blood glucose levels, and reverses diabetic ketoacidosis in uDM (26, 29–31) and similarly blunts the increase of HGP characteristic of the response to hypoglycemia (32). Moreover, because the antidiabetic effects of leptin in uDM are associated with normalization of plasma glucagon levels (4, 5), and leptin blunts the effect of neuroglucopenia to stimulate glucagon secretion (26, 27), it suggests that leptin’s glucose-lowering effects in uDM might involve inhibition of a neurocircuit normally activated by hypoglycemia.

This hypothesis is consistent with our finding that leptin administration directly into the LPBN of STZ-DM rats attenuates the hyperglucagonemia of uDM and thereby attenuates diabetic ketosis. That this intervention fails to attenuate diabetic hyperglycemia suggests further that leptin-responsive LPBN neurons are but one node in a complex and distributed circuit that, when activated, drives the complex but highly integrated behavioral, metabolic, autonomic, and neuroendocrine manifestations of uDM.

In support of this concept, evidence suggests that leptin acts through a distributed network, whereby multiple leptin-responsive neurons carry out different aspects of the overall leptin effect rather than leptin acting on one specific brain area or neuronal subset. Consistent with this model, we found that repeated daily microinjection of a low dose of leptin directly into the VMN is sufficient to ameliorate hyperglycemia in uDM, in effect mimicking the antidiabetic effect of ICV leptin (11). However, we also observed that ICV leptin fully reverses hyperglycemia in STZ-DM mice lacking leptin receptor specifically in VMNSF1 neurons (11, 12). These findings suggest that leptin signaling in the VMN is not required for leptin’s antidiabetic effects and implicate leptin-responsive neurocircuits outside the VMN in this effect.

Given that VMN neurons receive excitatory axonal projections from leptin-sensitive LPBN LepRbCCK neurons (14, 15, 18) as part of a LPBN→VMNSF1→aBNST neurocircuit, we hypothesized in the current work that leptin action in the LPBN, as well as in the VMN, underlies its antidiabetic actions, and our data support this hypothesis. Specifically, our data suggest that leptin action in the LPBN plays a specific role to inhibit glucagon secretion, and because uDM is a state of leptin deficiency, activation of leptin-inhibited LPBN neurons drives hyperglucagonemia in this setting. However, additional leptin-responsive neurocircuits are likely to be involved as glucagon secretion was not fully normalized following LPBN leptin administration. Furthermore, although leptin significantly reduced plasma glucagon levels in WT but not LepRbCCK KO mice, the difference between groups did not reach statistical significance. One possibility is that hypothalamic Pomc neurons play a role because most of these neurons express leptin receptors (33), and we have previously shown that a melanocortin-dependent mechanism contributes to leptin-mediated inhibition of glucagon secretion in uDM (19).

We note that unlike what has been previously reported following ICV leptin administration in uDM, leptin microinjection into the LPBN was without effect on plasma levels of either T4 or corticosterone in rats with STZ-DM (5, 24). This outcome is not surprising given that leptin regulation of the thyroid axis is believed to involve both a direct effect on thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus and an indirect effect mediated via activation of Pomc neurons that project onto thyrotropin-releasing hormone neurons (34–36). Similarly, the effect of leptin to regulate the hypothalamic-pituitary-adrenal axis involves a melanocortin-dependent pathway, and we are unaware of links between leptin-responsive neurons in the LBPN and the melanocortin system (37).

These considerations add to evidence that leptin’s capacity to reverse hyperglycemia in uDM involves a distributed system of leptin-responsive neurocircuits, each of which subserves a specific aspect of the associated behavioral, neuroendocrine, or autonomic phenotype. Thus, we propose that the effect of CNS leptin action to restore euglycemia in STZ-DM rats involves (1) leptin-mediated normalization of hypothalamic-pituitary-adrenal axis activation (38, 39) via effects that we anticipate are localized to hypothalamic arcuate and paraventricular nuclei; (2) leptin action of VMN neurons, which likely promotes insulin-independent glucose lowering via effects on both HGP and glucose utilization (11, 40, 41); (3) leptin-mediated amelioration of diabetic hyperphagia (20, 42), which likely involves both inhibition of Agrp neurons and activation of Pomc neurons in the arcuate nucleus (43); and (4) leptin inhibition of LPBNCCK neurons, which suppresses glucagon secretion. According to this model, leptin deficiency contributes to hyperglycemia and other manifestations of uDM via the loss of these leptin effects and perhaps others as well.

In summary, our data suggest that the hyperglucagonemia characteristic of STZ-DM arises, in part, via deficient leptin action specifically within the LPBN. This finding is consistent with a model in which (1) leptin deficiency activates the LPBN→VMN→aBNST neurocircuit, which is designed to respond to a critical deficiency of available fuel and is known to play a crucial role in glucose counterregulation, and (2) this activation, in concert with the peripheral and central consequences of severe insulin deficiency, drives hyperglycemia, hyperphagia, and other manifestations of uDM. Additional studies to test this working model have important potential to shed new light on both the mechanisms underlying leptin regulation of glucose homeostasis and the pathogenesis of diabetic hyperglycemia.

Acknowledgments

We thank Michael W. Schwartz (University of Washington) for scientific discussions and for carefully reading this manuscript. We also thank Dr. Gerald Taborsky Jr. from the Puget Sound Health Care System (Department of Veterans Affairs Medical Center, Seattle, WA) for performing plasma glucagon measurements, as well as Jennifer Deem and Nathaniel Peters, who provided technical and imaging support.

Financial Support: This work was supported by National Institutes of Health (NIH) Grants DK089056 (to G.J.M.), F32 DK097859 (to T.H.M.), and F31 DK113673 (to C.L.F.); the National Institute of Diabetes and Digestive and Kidney Diseases–funded Nutrition Obesity Research Center Grant DK035816; Diabetes, Obesity and Metabolism Training Grant T32 DK0007247 at the University of Washington (to T.H.M.); and University of Washington W. M. Keck Microscopy Center (NIH Grant S10 OD016240).

Current Affiliation: T. H. Meek’s current affiliation is the Obesity Research Unit, Novo Nordisk, Seattle, Washington 98109.

Disclosure Summary: The authors have nothing to disclose.

Glossary

Abbreviations:

aBNST

anterior bed nucleus of the stria terminalis

ANOVA

analysis of variance

CCK

cholecystokinin

CNS

central nervous system

CRR

counterregulatory response

ELISA

enzyme-linked immunosorbent assay

HGP

hepatic glucose production

ICV

intracerebroventricular

KO

knockout

LepRb

leptin receptor

LPBN

lateral parabrachial nucleus

PBS

phosphate-buffered saline

Pomc

pro-opiomelanocortin

RRID

Research Resource Identifier

SC

subcutaneous

STZ-DM

streptozotocin-induced diabetes mellitus

T1D

type 1 diabetes

T4

thyroxine

uDM

uncontrolled, insulin-deficient diabetes mellitus

veh

vehicle

VMN

ventromedial nucleus

WT

wild-type

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