Metabolic Sensors Mediate Hypoglycemic Detection at the Portal Vein
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糖尿病学杂志 2006年第5期
Department of Kinesiology and Integrative Biology, University of Southern California, Los Angeles, California
BHB, -hydroxybutyrate; CNS, central nervous system; DCA, dichloroacetic acid
ABSTRACT
The current study sought to ascertain whether portal vein glucose sensing is mediated by a metabolic fuel sensor analogous to other metabolic sensors presumed to mediate hypoglycemic detection (e.g., hypothalamic metabosensors). We examined the impact of selectively elevating portal vein concentrations of lactate, pyruvate, or -hydroxybutyrate (BHB) on the sympathoadrenal response to insulin-induced hypoglycemia. Male Wistar rats (n = 36), chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein (infusion), underwent hyperinsulinemic-hypoglycemic (2.5 mmol/l) clamps with either portal or jugular vein infusions of lactate, pyruvate, or BHB. By design, arterial concentrations of glucose and the selected metabolite were matched between portal and jugular (NS). Portal vein concentrations were significantly elevated in portal versus jugular (P < 0.0001) for lactate (5.03 ± 0.2 vs. 0.84 ± 0.08 mmol/l), pyruvate (1.81 ± 0.21 vs. 0.42 ± 0.03 mmol/l), or BHB (2.02 ± 0.1 vs. 0.16 ± 0.03 mmol/l). Elevating portal lactate or pyruvate suppressed both the epinephrine (64% decrease; P < 0.01) and norepinephrine (75% decrease; P < 0.05) responses to hypoglycemia. In contrast, elevating portal BHB levels failed to impact epinephrine (P = 0.51) or norepinephrine (P = 0.47) levels during hypoglycemia. These findings indicate that hypoglycemic detection at the portal vein is mediated by a sensor responding to some metabolic event(s) subsequent to the uptake and oxidation of glucose.
Hypoglycemia presents a major obstacle in the treatment of type 1 diabetes and late stage type 2 diabetes (1). Attributed primarily to imperfect insulin replacement therapy (2) and compromised glucagon secretion (3), hypoglycemia is further exacerbated in diabetes by the failure to mount an appropriate sympathoadrenal response, i.e., epinephrine and norepinephrine secretion (3,4). Patients demonstrating both suppressed glucagon and epinephrine secretion have been shown to be severalfold more likely to suffer a severe hypoglycemic episode than individuals with an uncompromised sympathoadrenal response (5). Because the defect in the sympathoadrenal response for type 1 diabetic patients is specific to hypoglycemia (6) and is often associated with hypoglycemic unawareness (1), impaired glucose sensing has been implicated as a cause. However, the mechanism(s) underlying hypoglycemic detection in vivo and the integration of that afferent input toward appropriate counterregulation remain largely unknown.
Hypoglycemic detection has often been attributed exclusively to the brain (7), more specifically the glucose-sensitive areas of the hypothalamus (8). However, our laboratory (9,10) and others (11) have provided evidence supporting the importance of portal vein glucosensing in mediating the full sympathoadrenal response to hypoglycemia. Normalizing portal vein glycemia has been shown to suppress the sympathoadrenal response under a variety of hypoglycemic conditions (12,13) for rats (9,10), dogs (13), and humans (14). Furthermore, ablation of portal vein afferents via total surgical hepatic denervation (11) or by the topical application of phenol (10) resulted in a blunting of the sympathoadrenal response to hypoglycemia comparable with that achieved via portal vein glucose normalization. More recently, we reported that sectioning the celiac ganglion (15), through which spinal afferents innervating the portohepatis pass, results in a similar suppression of the sympathoadrenal response to hypoglycemia.
Despite our improved insight into the neuronal axis of portal vein glucose sensing, the cellular mechanism(s) by which glycemic detection occurs in the portal vein remains obscure. Whole-body infusions of lactate and -hydroxybutyrate (BHB) have been shown to suppress the counterregulatory response to hypoglycemia (16,17). These findings suggest that those cells primarily responsible for hypoglycemic detection in vivo respond to a cellular metabolic event(s) subsequent to glucose uptake, i.e., metabolic sensors as opposed to specific glucose receptors. In vitro studies have demonstrated that glucose-sensing neurons of the hypothalamus respond to a variety of metabolic substrates, including lactate and BHB (18,19,20,21). Additionally, these neurons express characteristic components of -cell glucose-sensing "machinery," such as GLUT2 glucose transporter protein (22), the pancreatic form of glucokinase (23), and ATP-sensitive K+ channels (24). In the current study, we sought to ascertain whether hypoglycemic detection at the portal vein is also metabolic in nature using fuel-sensing mechanisms similar to those of the hypothalamus. This was achieved by examining the impact of selective portal vein elevations of substrates known to impact brain metabolic sensors, i.e., lactate and BHB, as well as pyruvate, a glycolytic intermediate that apparently fails stimulate glucose-sensitive neurons in the hypothalamus (20,21).
RESEARCH DESIGN AND METHODS
Male Wistar rats (241 ± 7 g, n = 36) in the conscious relaxed state were used in all experimental procedures. All animals were housed in individual cages, fed ad libitum, and subjected to standard 12-h light-dark cycle. The University of Southern California Institutional Animal Care and Use Committee approved all surgical and experimental procedures. One week before the study, rats were anesthetized (3:3:1 ketamine HCl:xylazine:acepromizine malate; 0.1 ml/0.1 kg body wt i.m.), and indwelling catheters were inserted into the portal vein (silastic tubing, 0.051 cm internal diameter [ID] with 0.03-cm-ID tip) for infusion of studied metabolites, into the carotid artery (polyethylene tubing, 0.058 cm ID) for arterial blood sampling, and into the jugular vein (dual silastic cannula, 0.03 cm ID) for peripheral insulin, glucose, and metabolite infusions. All cannulas were tunneled subcutaneously, exteriorized on the back of the neck, and incased in an infusion harness (Instech Laboratories, Plymouth Meeting, PA). All animals were allowed at least 6 days to recover from surgery and to regain their original body weight. All animals were fasted overnight before initiation of the experiment.
Hyperinsulinemic-hypoglycemic clamp.
All rats were exposed to a general protocol for the induction of hypoglycemia. On the day of the experiment, animals were placed in a metabolic chamber and allowed to acclimatize to the new environment for at least 60 min (eC90 to eC30 min). At min 0, whole-body hypoglycemia was induced via jugular vein insulin infusion (25 mU · kgeC1 · mineC1) and variable jugular glucose infusions (20% dextrose). Basal glucose concentrations were measured at eC30 and 0 min, and subsequent serial sampling for glucose was performed at 10-min intervals throughout the duration of the clamp (0eC105 min). Plasma concentrations of epinephrine, norepinephrine, and infused metabolites (lactate, pyruvate, or BHB) were determined at basal (eC30 and 0 min) and during sustained hypoglycemia (60, 75, 90, and 105 min). Plasma samples for the insulin assay were drawn during the basal period (eC30 min) and at the end of hypoglycemia (105 min).
Study design.
Experiments were subdivided according to the infused metabolite, i.e., portal lactate clamp (n = 13), portal pyruvate clamp (n = 11), and portal BHB clamp (n = 12). Animals were randomly selected to take part in one of six separate protocols distinguished by the site of infusion, portal vein versus jugular vein, and metabolite infused, lactate, pyruvate, and BHB.
Portal lactate clamp.
For portal-lactate animals (n = 7), whole-body hypoglycemia was induced as described above with a lactate (2.5 mol/l lactic acid in phosphate buffer, pH 4.5; Sigma, St. Louis, MO) infused via the portal vein at variable rates starting at 40 min to attain local portal vein hyperlactatemia (5 mmol/l) in the midst of systemic hypoglycemia. This lactate infusate has previously been shown to elevate lactate levels without impacting blood pH (25). Jugular-lactate control animals (n = 6) were exposed to an identical hyperinsulinemic-hypoglycemic clamp with lactate infused (variable rate) via the jugular vein beginning at 40 min to match arterial lactate concentrations in the portal lactate group. To isolate the increase in lactate levels to the portal vein, dichloroacetic acid (DCA; 300 mg/kg) was infused in both groups via the jugular vein to prevent any elevation in arterial lactate concentration above basal. Dichloroacetate selectively inhibits tissue lactate release via activation of pyruvate dehydrogenase and has been used in a number of animal and human studies without any adverse affects (26,27).
Portal pyruvate clamp.
Portal-pyruvate (n = 6) and jugular-pyruvate (n = 5) rats were exposed to a protocol identical to that above for the lactate clamp with the exception that animals received pyruvate infusions (1 mol/l sodium pyruvate in sterile 0.9 mol/l NaCl, pH 7.4; Sigma), without DCA.
Portal BHB clamp.
Portal-BHB (n = 5) and jugular-BHB (n = 7) animals were exposed to a protocol identical to that above for the lactate clamp with the exception that animals received BHB infusions (1 mol/l sodium BHB in sterile 0.9 mol/l NaCl, pH 7.4; Sigma), without DCA.
Analytical procedures.
Glucose and lactate were assayed by the glucose oxidase method (YSI, Yellow Springs, OH). Arterial BHB (28) and pyruvate (29) concentrations were assayed spectrophotometrically. Catecholamines were analyzed using a single-isotope radioenzymatic method (30). Insulin samples were assayed using a radioimmunoassay kit (Linco Research, St. Charles, MO).
Calculations and data analysis.
The estimated local portal vein metabolite concentrations (MPV) were calculated as MPV = MA+ (MINFPOR/PVPF), where MA is arterial metabolite concentration (in micromoles per milliliter), MINFPOR is the portal vein metabolite infusion rate (in micromoles per minute), and PVPF is portal vein plasma flow rate in (milliliters per minute). The portal vein plasma flow rate was assumed to be 80% of hepatic plasma flow rate, where hepatic plasma flow rate was estimated to be 1.3 ml · g liver wteC1 · mineC1 (31). Mean steady-state metabolite concentrations were determined as the average of at least four serial samples that differ nondirectionally by <0.5% per minute. Experimental results were expressed as means ± SE. A one-way ANOVA was used to compare characteristics between independent groups. Repeated measures ANOVAs were used to determine group differences over time with Tukey’s test for post hoc analysis. Significance was set at P < 0.05.
RESULTS
Portal lactate clamp.
Arterial plasma insulin increased from an average basal value of 98 ± 10.41 pmol/l (P = 0.76, between groups) to a hyperinsulinemic value of 5,246.34 ± 310.59 pmol/l (P = 0.52, between groups). Basal glucose concentrations for portal-lactate versus jugular-lactate (6.76 ± 0.18 vs. 7.10 ± 0.16 mmol/l, P = 0.12) and glucose concentrations at the hypoglycemic nadir (2.54 ± 0.14 vs. 2.51 ± 0.14 mmol/l, P = 0.81) were not significantly different between the groups (Fig. 1A). Furthermore, no significant differences were found between portal-lactate versus jugular-lactate protocols with respect to basal arterial lactate (0.88 ± 0.08 vs. 0.9 ± 0.09 mmol/l; P = 0.81; Fig. 1B) and arterial lactate during sustained (60eC105 min) hypoglycemia (1.01 ± 0.1 vs. 0.91 ± 0.09 mmol/l; P = 0.36; Fig. 1B). As expected, portal vein lactate infusions significantly elevated mean portal vein plasma lactate concentrations in the portal-lactate group, whereas portal vein lactate concentrations in the jugular-lactate group remained at basal levels (5.03 ± 0.20 vs. 0.91 ± 0.09 mmol/l, P < 0.0001; Fig. 1C)
In response to insulin-induced hypoglycemia, jugular-lactate animals demonstrated a robust sympathoadrenal response where plasma epinephrine concentrations increased from 1.68 ± 0.51 nmol/l at basal to 14.44 ± 2.70 nmol/l by 105 min (Fig. 2A). In contrast, selective elevation of portal vein lactate resulted in a 64% suppression of the epinephrine response (14.44 ± 2.70 vs. 5.26 ± 0.77 nmol/l, P < 0.01; Fig. 2A) at 105 min of hypoglycemia. Additionally, statistically significant suppressions in the epinephrine response to hypoglycemia were observed in the portal-lactate group at min 60, 75, and 90 of hypoglycemia. Correspondingly, the norepinephrine response above basal was also suppressed by 67% in the portal-lactate versus the jugular-lactate group (1.66 ± 0.57 vs. 5.05 ± 1.21 nmol/l, P < 0.01; Fig. 2B).
Portal pyruvate clamp.
Mean arterial glucose concentrations were not significantly different between the portal-pyruvate versus jugular-pyruvate groups at basal (5.77 ± 0.2 mmol/l, P = 0.69) nor were they statistically different during the hypoglycemic plateau (2.55 ± 0.12 mmol/l, P = 0.84; Fig. 3A). Furthermore, at basal (P = 0.76) and during sustained hypoglycemia (P = 0.17), no significant differences between the two groups were observed with respect to mean arterial pyruvate concentrations (Fig. 3B). On the other hand, as a result of the selective portal vein pyruvate infusion, mean portal vein pyruvate levels were significantly elevated (1.81 ± 0.21 vs. 0.42 ± 0.05 mmol/l, P < 0.0001; Fig. 3B) in portal-pyruvate versus jugular-pyruvate animals, respectively. In jugular-pyruvate animals, hypoglycemia elicited 16-fold increase in the epinephrine response from basal 1.00 ± 0.19 to 16.15 ± 3.21 nmol/l at min 105 of the hyperinsulinemic-hypoglycemic clamp. On the other hand, as a result of selective elevation of portal vein pyruvate concentrations, epinephrine response to whole-body hypoglycemia in the portal-pyruvate group was suppressed by 64% at min 105 compared with the jugular-pyruvate group (5.84 ± 0.71 vs. 16.15 ± 3.21 nmol/l, P < 0.01; Fig. 3C). In jugular-pyruvate animals, norepinephrine concentrations increased approximately threefold from the basal value of 2.25 ± 0.38 to 6.13 ± 0.77 nmol/l at the end point of hypoglycemia. Similar to the epinephrine response, elevation of portal vein pyruvate resulted in a 80% suppression of the norepinephrine response above basal at min 105 of hypoglycemia (0.78 ± 0.41 vs. 3.88 ± 0.69 nmol/l, P < 0.01; Fig. 3D).
Portal BHB clamp.
By design, there were no significant differences between the portal-BHB and jugular-BHB groups with respect to arterial glucose concentrations at basal (5.86 ± 0.17 vs. 5.61 ± 0.16 mmol/l; P = 0.45; Fig. 4A), during the fall in glycemia, and during sustained hypoglycemia (2.53 ± 0.14 vs. 2.52 ± 0.14 mmol/l; P = 0.91; Fig. 4A). Also, there were no significant differences between portal-BHB and jugular-BHB treatments with respect to mean arterial BHB levels at basal (0.55 ± 0.23 vs. 0.67 ± 0.10 mmol/l; P = 0.59, data not shown) and during hypoglycemia (0.12 ± 0.03 vs. 0.16 ± 0.03 mmol/l; P = 0.32; Fig. 4B). Portal vein BHB infusions elevated BHB concentrations in the portal vein 12-fold in the portal-BHB versus jugular-BHB groups (2.02 ± 0.03 vs. 0.163 ± 0.03 mmol/l; P < 0.0001; Fig. 4B). In contrast to selective elevation of portal vein lactate or pyruvate, no significant differences were observed between the portal-BHB versus jugular-BHB groups with to respect epinephrine (5.42 ± 0.92 vs. 6.84 ± 1.59 nmol/l; P = 0.51 at min 105; Fig. 4C) and norepinephrine (2.67 ± 0.49 vs. 3.45 ± 0.81 nmol/l; P = 0.47 at min 105; Fig. 4D) responses to hypoglycemia.
DISCUSSION
In the current study, we investigated the effects of selective portal vein elevation of lactate, pyruvate, or BHB on the sympathoadrenal response to insulin-induced systemic hypoglycemia. Clamping portal vein lactate or pyruvate concentrations at approximately fivefold above basal, while maintaining arterial concentrations during systemic hypoglycemia, resulted in a substantial suppression of the epinephrine and norepinephrine responses, 64 and 75%, respectively. In contrast, elevating portal vein BHB to levels 12-fold above basal failed to impact the sympathoadrenal response to hypoglycemia. The ability to detect alternative metabolic substrates known to enter the glycolytic pathway distal to the uptake of glucose is indicative of a portal vein metabolic sensor rather than a glucoreceptor specific for the glycosyl moiety. The metabolic nature of hypoglycemic detection at the portal vein is further underscored by the fact that portal elevation of either lactate or pyruvate led to a quantitative blunting of the sympathoadrenal response comparable with that achieved via portal glucose normalization (Fig. 5).
Whole-body infusions of lactate have been shown to blunt the sympathoadrenal response to insulin-induced hypoglycemia in humans (16,17). It is generally assumed that such lactate-induced suppression of the sympathoadrenal response is mediated via its action on brain metabolic sensors. Lactate can be readily used by nervous tissue (32) and has been shown, both in vitro and in vivo, to activate glucose-sensitive neurons in the brain (19eC21,33,34). The ability of lactate to alter the firing rate of glucose-responsive neurons has been demonstrated in the hypothalamus (19,20) and in the hindbrain (35). However, in vitro studies have not been uniform in their observations of the effect of lactate on glucose-inhibited hypothalamic neurons. Yang et al. (21), reported that the addition of lactate, similar to glucose, decreased the firing rate of hypothalamic glucose-inhibited neurons, whereas Song and Routh (19) observed an increased discharge from such neurons. Local microdialysis of the ventromedial hypothalamus with 100 mmol/l lactate has been shown to substantially suppress the sympathoadrenal response to systemic hypoglycemia in rats in vivo (33). Exogenous lactate delivery (50 e蘭ol/h) into the caudal fourth ventricle has also been observed to prolong recovery from insulin-induced hypoglycemia (34).
Although the abovementioned findings clearly demonstrate that elevated brain lactate levels can impact the counterregulatory response to systemic hypoglycemia, it is not clear that the central nervous system (CNS) is the locus for those metabolic sensors responding to plasma lactate elevations in vivo as previously reported (16,17). The current findings clearly demonstrate that the selective elevation of portal vein plasma lactate levels, similar to that used in studies of whole-body plasma lactate elevations, substantially suppresses the sympathoadrenal response to systemic hypoglycemia. The suppression in the epinephrine response to hypoglycemia with local portal vein lactate elevation in the current study, a 64% suppression, is comparable with that reported for whole-body lactate elevation during hypoglycemia, i.e., 60eC70% (16,17). As noted above, normalizing glycemia or elevating lactate in the portal vein results in a comparable suppression in the sympathoadrenal response to hypoglycemia, which is similar in magnitude to that reported for whole-body lactate infusions. In contrast, elevating brain glucose levels during systemic hypoglycemia is purported to result in a substantially greater suppression of the epinephrine response to hypoglycemia (90%) (7). Thus, the response to elevating whole-body plasma lactate levels during hypoglycemia in quantitative terms appears more consistent with the blunting of portal vein metabolic sensors, as opposed to CNS metabolic sensors.
Nutrient sensing by hypothalamic glucose-responsive neurons has been attributed to intracellular changes in the ATP-to-ADP ratio and the subsequent binding of ATP to KATP, leading to changes in cell potential (19,24). Exposure of hypothalamic glucose-responsive neurons to glucose or lactate results in closure of KATP channels, which can be reversed by the application of diazoxide (19). The intracerebroventricular application of the KATP channel closer, glibenclamide, blunts the sympathoadrenal response to hypoglycemia, suggestive of an ATP-dependent mechanism (36). The current observation that elevating portal vein pyruvate concentration during hypoglycemia results in a suppression of the sympathoadrenal response comparable with that of lactate or glucose is consistent with such an ATP-dependent mechanism. However, Yang et al. (20,21) reported that glucose-responsive and glucose-inhibited neurons in the hypothalamus do not respond to elevated pyruvate despite the fact that pyruvate is apparently metabolized by such neurons. These same investigators observed that early glycolytic intermediates, e.g., glyceraldehyde and lactate, were able to substitute for glucose and alter the firing rate in both glucose-responsive and glucose-inhibited hypothalamic neurons. Based on the inability of pyruvate to stimulate hypothalamic glucose sensors, Yang et al. concluded that ATP production was not the primary metabolic event responsible for "glycemic detection" in these cells. They postulated that hypothalamic glucose sensing must be mediated by products of glucose metabolism proximal to pyruvate oxidation, e.g., cytosolic NADH. This hypothesis is supported by a recent report demonstrating a glucose-induced increase in cytosolic NADH, but not ATP levels, in hypothalamic glucose-responsive neurons (37).
Although it remains unclear as to whether glucose-sensing neurons in the hypothalamus and portal vein use a common metabolic event(s), our findings are consistent with a postulated mechanism for pancreatic -cell nutrient sensing (38). It is proposed that the intracellular rise in malonyl CoA signals nutrient abundance in the -cell, and thus only those compounds capable of both substrate oxidation and citric acid cycle anaplerosis should be effective (39,40). Elevating portal vein pyruvate, lactate, or glucose but not BHB proved comparable in their efficacy for suppressing the sympathoadrenal response to hypoglycemia (Fig. 5). Of these substrates, only BHB is incapable of supplying citric acid cycle intermediates via anaplerosis. That the metabolic sensing mechanisms for the -cell and portal vein glucose sensors might be similar is supported by reports that GLUT2 (41) and the pancreatic form of glucokinase (42), critical components of the -cell glucose-sensing "machinery," are both expressed in the portal region.
As noted above, elevating portal vein BHB levels fails to impact either epinephrine or norepinephrine secretion during hypoglycemia (Fig. 4). This is in contrast to whole-body ketone infusions, which have previously been shown to suppress the counterregulatory responses to insulin-induced hypoglycemia (16). Clearly the impact of whole-body hyperketonemia on the sympathoadrenal response to hypoglycemia must be manifest at some locus distal from the portal vein, most likely the CNS. Iontophoretic application of BHB has been observed to modify neuronal discharge of hypothalamic glucose-responsive and glucose-inhibited neurons in vitro in a dose dependent manner (18). Although it is possible that the portal vein BHB level achieved in our current study (2.02 ± 0.03 mmol/l) was insufficient to mask portal vein metabolic sensing or that BHB is not transported into portal glucose sensors, neither proposal appears plausible. Previous studies showed that elevating ketone levels 10-fold above basal to arterial concentrations of 1eC2 mmol/l significantly diminishes the sympathoadrenal response to hypoglycemia in humans (16), dogs (43), and rats (44). Additionally, transport of lactate, pyruvate, and BHB across cellular plasma membranes uses the same family of monocarboxylate transporter proteins (45). Therefore, transport across plasma membrane would not account for the differences observed between lactate or pyruvate and BHB. Assuming that the impact of BHB is effected at the level of the CNS, the inability of portal vein metabolic sensors to respond to ketones may be further evidence distinguishing that metabolic sensing at the portal vein is distinct from that which governs sensing at the brain.
Metabolic sensors have long been known to reside in the hepatoportal area and are understood to be involved in the regulation of feeding (46). Early studies by Niijima (47,48), using an in situ guinea pig liver perfusion preparation, demonstrated that the rates of hepatic vagal afferent discharge are inversely proportional to glucose availability in the perfusion medium. Subsequent studies showed that afferent discharge was also augmented by intraportal administrations of 2-deoxy-D-glucose (glucose oxidation inhibitor) (47) or mercaptoacetate (fatty acid oxidation inhibitor) (49), suggesting that the glucose sensory mechanism is dependent on the intracellular fuel oxidation and the subsequent ATP production. In support of this, intraperitoneal administration of lactate, pyruvate, or BHB has been shown to suppress feeding; however, no change in food intake in response to these metabolites was observed after hepatic branch vagatomy (50). It was therefore proposed that glucose-sensitive hepatic vagal afferents regulate feeding as a result of intracellular fuel oxidation and subsequent changes in mitochondrial ATP production (50).
Despite the fact that some investigators have suggested that glucose-sensitive vagal afferents are also involved in the regulation of the sympathoadrenal response (47), studies have failed to observe this relationship in vivo (15,51). On the other hand, we have shown that sectioning the celiac ganglion, through which spinal afferents originating from the portohepatis ascend, substantially impairs the ability of the animal to mount a full sympathoadrenal response to hypoglycemia (15). Thus, it appears that feeding in the periphery is mediated by glucose-sensitive hepatic vagal afferents, where hypoglycemic detection is mediated by spinal afferents transcending the celiac ganglion. Furthermore, this study points out yet another revealing distinction between the two sets of hepatoportal sensors, at least in respect to their responsiveness to ketone bodies. Clearly, further studies are warranted to elucidate exact molecular and cellular mechanisms of portal vein metabolic sensors.
In summary, our current findings indicate that the portal vein glucose sensor responds to alternative metabolic substrates such as lactate and pyruvate and therefore behaves as a metabolic sensor rather than a glucose-specific receptor. Portal vein elevations of glucose, lactate, and pyruvate but not BHB equally suppressed (60eC80%) the sympathoadrenal response to hypoglycemia. These findings suggest a mechanism of cellular glycemic detection mediated by events distal to pyruvate oxidation, requiring anaplerotic input into the citric acid cycle. In this regard, the mechanism for glycemic detection at the portal vein appears similar to that of the -cell. That pyruvate and BHB demonstrate differential effects at the portal vein and CNS indicates distinct mechanisms for hypoglycemic detection in these two loci. Elucidation of the cellular mechanism(s) involved in portal vein metabolic sensing and how that information is integrated with glucose sensors in the CNS remains a significant challenge to our understanding of hypoglycemic counterregulation in diabetes.
ACKNOWLEDGMENTS
C.M.D. has received National Institutes of Health Grant DK-55257.
We thank Satoshi Fujita, Maziyar Saberi, and MaryAnn Bohland for extensive contributions to the study.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Cryer PE: Hypoglycemia-associated autonomic failure in diabetes. Am J Physiol Endocrinol Metab 281:E1115eCE1121, 2001
The Diabetes Control and Complications Trial Research Group: Hypoglycemia in the Diabetes Control and Complications Trial. Diabetes 46:271eC286, 1997
Bolli G, de Feo P, Compagnucci P, Cartechini MG, Angeletti G, Santeusanio F, Brunetti P, Gerich JE: Abnormal glucose counterregulation in insulin-dependent diabetes mellitus: interaction of anti-insulin antibodies and impaired glucagon and epinephrine secretion. Diabetes 32:134eC141, 1983
Kleinbaum J, Shamoon H: Impaired counterregulation of hypoglycemia in insulin-dependent diabetes mellitus. Diabetes 32:493eC498, 1983
White NH, Skor DA, Cryer PE, Levandoski LA, Bier DM, Santiago JV: Identification of type I diabetic patients at increased risk for hypoglycemia during intensive therapy. N Engl J Med 308:485eC491, 1983
Rattarasarn C, Dagogo-Jack S, Zachwieja JJ, Cryer PE: Hypoglycemia-induced autonomic failure in IDDM is specific for stimulus of hypoglycemia and is not attributable to prior autonomic activation. Diabetes 43:809eC818, 1994
Biggers DW, Myers SR, Neal D, Stinson R, Cooper NB, Jaspan JB, Williams PE, Cherrington AD, Frizzell RT: Role of brain in counterregulation of insulin-induced hypoglycemia in dogs. Diabetes 38:7eC16, 1989
Borg WP, During MJ, Sherwin RS, Borg MA, Brines ML, Shulman GI: Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to hypoglycemia. J Clin Invest 93:1677eC1682, 1994
Hevener AL, Bergman RN, Donovan CM: Novel glucosensor for hypoglycemic detection localized to the portal vein. Diabetes 46:1521eC1525, 1997
Hevener AL, Bergman RN, Donovan CM: Portal vein afferents are critical for the sympathoadrenal response to hypoglycemia. Diabetes 49:8eC12, 2000
Lamarche L, Yamaguchi N, Peronnet F: Hepatic denervation reduces adrenal catecholamine secretion during insulin-induced hypoglycemia. Am J Physiol 268:R50eCR57, 1995
Donovan CM, Halter JB, Bergman RN: Importance of hepatic glucoreceptors in sympathoadrenal response to hypoglycemia. Diabetes 40:155eC158, 1991
Donovan CM, Hamilton-Wessler M, Halter JB, Bergman RN: Primacy of liver glucosensors in the sympathetic response to progressive hypoglycemia. Proc Natl Acad Sci U S A 91:2863eC2867, 1994
Smith D, Pernet A, Reid H, Bingham E, Rosenthal JM, Macdonald IA, Umpleby AM, Amiel SA: The role of hepatic portal glucose sensing in modulating responses to hypoglycaemia in man. Diabetologia 45:1416eC1424, 2002
Fujita S, Donovan CM: Celiac-superior mesenteric ganglionectomy, but not vagotomy, suppresses the sympathoadrenal response to insulin-induced hypoglycemia. Diabetes 54:3258eC3264, 2005
Veneman T, Mitrakou A, Mokan M, Cryer P, Gerich J: Effect of hyperketonemia and hyperlacticacidemia on symptoms, cognitive dysfunction, and counterregulatory hormone responses during hypoglycemia in normal humans. Diabetes 43:1311eC1317, 1994
Maran A, Cranston I, Lomas J, Macdonald I, Amiel SA: Protection by lactate of cerebral function during hypoglycaemia. Lancet 343:16eC20, 1994
Minami T, Shimizu N, Duan S, Oomura Y: Hypothalamic neuronal activity responses to 3-hydroxybutyric acid, an endogenous organic acid. Brain Res 509:351eC354, 1990
Song Z, Routh VH: Differential effects of glucose and lactate on glucosensing neurons in the ventromedial hypothalamic nucleus. Diabetes 54:15eC22, 2005
Yang XJ, Kow LM, Funabashi T, Mobbs CV: Hypothalamic glucose sensor: similarities to and differences from pancreatic -cell mechanisms. Diabetes 48:1763eC1772, 1999
Yang XJ, Kow LM, Pfaff DW, Mobbs CV: Metabolic pathways that mediate inhibition of hypothalamic neurons by glucose. Diabetes 53:67eC73, 2004
Leloup C, Arluison M, Lepetit N, Cartier N, Marfaing-Jallat P, Ferre P, Penicaud L: Glucose transporter 2 (GLUT 2): expression in specific brain nuclei. Brain Res 638:221eC226, 1994
Lynch RM, Tompkins LS, Brooks HL, Dunn-Meynell AA, Levin BE: Localization of glucokinase gene expression in the rat brain. Diabetes 49:693eC700, 2000
Ashford ML, Boden PR, Treherne JM: Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive K+ channels. Pflugers Arch 415:479eC483, 1990
Donovan CM, Pagliassotti MJ: Endurance training enhances lactate clearance during hyperlactatemia. Am J Physiol 257:E782eCE789, 1989
Stacpoole PW, Greene YJ: Dichloroacetate. Diabetes Care 15:785eC791, 1992
Gao J, Islam MA, Brennan CM, Dunning BE, Foley JE: Lactate clamp: a method to measure lactate utilization in vivo. Am J Physiol 275:E729eCE733, 1998
Koch DD, Feldbruegge DH: Optimized kinetic method for automated determination of beta-hydroxybutyrate. Clin Chem 33:1761eC1766, 1987
Passonnea J LO: Enzymatic Analysis: A Practical Guide. Totowa, NJ, Humana Press, 1993
Peuler JD, Johnson GA: Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci 21:625eC636, 1977
Ishise S, Pegram BL, Yamamoto J, Kitamura Y, Frohlich ED: Reference sample microsphere method: cardiac output and blood flows in conscious rat. Am J Physiol 239:H443eCH449, 1980
Tsacopoulos M, Magistretti PJ: Metabolic coupling between glia and neurons. J Neurosci 16:877eC885, 1996
Borg MA, Tamborlane WV, Shulman GI, Sherwin RS: Local lactate perfusion of the ventromedial hypothalamus suppresses hypoglycemic counterregulation. Diabetes 52:663eC666, 2003
Patil GD, Briski KP: Lactate is a critical "sensed" variable in caudal hindbrain monitoring of CNS metabolic stasis. Am J Physiol Regul Integr Comp Physiol 289:R1777eCR1786, 2005
Himmi T, Perrin J, Dallaporta M, Orsini JC: Effects of lactate on glucose-sensing neurons in the solitary tract nucleus. Physiol Behav 74:391eC397, 2001
Evans ML, McCrimmon RJ, Flanagan DE, Keshavarz T, Fan X, McNay EC, Jacob RJ, Sherwin RS: Hypothalamic ATP-sensitive K+ channels play a key role in sensing hypoglycemia and triggering counterregulatory epinephrine and glucagon responses. Diabetes 53:2542eC2551, 2004
Ainscow EK, Mirshamsi S, Tang T, Ashford ML, Rutter GA: Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels. J Physiol 544:429eC445, 2002
Prentki M, Tornheim K, Corkey BE: Signal transduction mechanisms in nutrient-induced insulin secretion. Diabetologia 40 (Suppl. 2):S32eCS41, 1997
Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802eC5810, 1992
Schuit F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T, Prentki M: Metabolic fate of glucose in purified islet cells: glucose-regulated anaplerosis in beta cells. J Biol Chem 272:18572eC18579, 1997
Burcelin R, Dolci W, Thorens B: Glucose sensing by the hepatoportal sensor is GLUT2-dependent: in vivo analysis in GLUT2-null mice. Diabetes 49:1643eC1648, 2000
Jetton TL, Liang Y, Pettepher CC, Zimmerman EC, Cox FG, Horvath K, Matschinsky FM, Magnuson MA: Analysis of upstream glucokinase promoter activity in transgenic mice and identification of glucokinase in rare neuroendocrine cells in the brain and gut. J Biol Chem 269:3641eC3654, 1994
Flatt JP, Blackburn GL, Randers G, Stanbury JB: Effects of ketone body infusion on hypoglycemic reaction in postabsorptive dogs. Metabolism 23:151eC158, 1974
Stricker EM, Rowland N, Saller CF, Friedman MI: Homeostasis during hypoglycemia: central control of adrenal secretion and peripheral control of feeding. Science 196:79eC81, 1977
Poole RC, Halestrap AP: Transport of lactate and other monocarboxylates across mammalian plasma membranes. Am J Physiol 264:C761eCC782, 1993
Novin D, VanderWeele DA, Rezek M: Infusion of 2-deoxy-D-glucose into the hepatic-portal system causes eating: evidence for peripheral glucoreceptors. Science 181:858eC860, 1973
Niijima A: Glucose sensitive afferent nerve fibers in the liver and regulation of blood glucose. Brain Res Bull 5:175eC179, 1980
Niijima A: Afferent impulse discharges from glucoreceptors in the liver of the guinea pig. Ann N Y Acad Sci 157:690eC700, 1969
Ritter S, Taylor JS: Vagal sensory neurons are required for lipoprivic but not glucoprivic feeding in rats. Am J Physiol 258:R1395eCR1401, 1990
Langhans W, Egli G, Scharrer E: Regulation of food intake by hepatic oxidative metabolism. Brain Res Bull 15:425eC428, 1985
Jackson PA, Pagliassotti MJ, Shiota M, Neal DW, Cardin S, Cherrington AD: Effects of vagal blockade on the counterregulatory response to insulin-induced hypoglycemia in the dog. Am J Physiol 273:E1178eCE1188, 1997(Aleksey V. Matveyenko, an)
BHB, -hydroxybutyrate; CNS, central nervous system; DCA, dichloroacetic acid
ABSTRACT
The current study sought to ascertain whether portal vein glucose sensing is mediated by a metabolic fuel sensor analogous to other metabolic sensors presumed to mediate hypoglycemic detection (e.g., hypothalamic metabosensors). We examined the impact of selectively elevating portal vein concentrations of lactate, pyruvate, or -hydroxybutyrate (BHB) on the sympathoadrenal response to insulin-induced hypoglycemia. Male Wistar rats (n = 36), chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein (infusion), underwent hyperinsulinemic-hypoglycemic (2.5 mmol/l) clamps with either portal or jugular vein infusions of lactate, pyruvate, or BHB. By design, arterial concentrations of glucose and the selected metabolite were matched between portal and jugular (NS). Portal vein concentrations were significantly elevated in portal versus jugular (P < 0.0001) for lactate (5.03 ± 0.2 vs. 0.84 ± 0.08 mmol/l), pyruvate (1.81 ± 0.21 vs. 0.42 ± 0.03 mmol/l), or BHB (2.02 ± 0.1 vs. 0.16 ± 0.03 mmol/l). Elevating portal lactate or pyruvate suppressed both the epinephrine (64% decrease; P < 0.01) and norepinephrine (75% decrease; P < 0.05) responses to hypoglycemia. In contrast, elevating portal BHB levels failed to impact epinephrine (P = 0.51) or norepinephrine (P = 0.47) levels during hypoglycemia. These findings indicate that hypoglycemic detection at the portal vein is mediated by a sensor responding to some metabolic event(s) subsequent to the uptake and oxidation of glucose.
Hypoglycemia presents a major obstacle in the treatment of type 1 diabetes and late stage type 2 diabetes (1). Attributed primarily to imperfect insulin replacement therapy (2) and compromised glucagon secretion (3), hypoglycemia is further exacerbated in diabetes by the failure to mount an appropriate sympathoadrenal response, i.e., epinephrine and norepinephrine secretion (3,4). Patients demonstrating both suppressed glucagon and epinephrine secretion have been shown to be severalfold more likely to suffer a severe hypoglycemic episode than individuals with an uncompromised sympathoadrenal response (5). Because the defect in the sympathoadrenal response for type 1 diabetic patients is specific to hypoglycemia (6) and is often associated with hypoglycemic unawareness (1), impaired glucose sensing has been implicated as a cause. However, the mechanism(s) underlying hypoglycemic detection in vivo and the integration of that afferent input toward appropriate counterregulation remain largely unknown.
Hypoglycemic detection has often been attributed exclusively to the brain (7), more specifically the glucose-sensitive areas of the hypothalamus (8). However, our laboratory (9,10) and others (11) have provided evidence supporting the importance of portal vein glucosensing in mediating the full sympathoadrenal response to hypoglycemia. Normalizing portal vein glycemia has been shown to suppress the sympathoadrenal response under a variety of hypoglycemic conditions (12,13) for rats (9,10), dogs (13), and humans (14). Furthermore, ablation of portal vein afferents via total surgical hepatic denervation (11) or by the topical application of phenol (10) resulted in a blunting of the sympathoadrenal response to hypoglycemia comparable with that achieved via portal vein glucose normalization. More recently, we reported that sectioning the celiac ganglion (15), through which spinal afferents innervating the portohepatis pass, results in a similar suppression of the sympathoadrenal response to hypoglycemia.
Despite our improved insight into the neuronal axis of portal vein glucose sensing, the cellular mechanism(s) by which glycemic detection occurs in the portal vein remains obscure. Whole-body infusions of lactate and -hydroxybutyrate (BHB) have been shown to suppress the counterregulatory response to hypoglycemia (16,17). These findings suggest that those cells primarily responsible for hypoglycemic detection in vivo respond to a cellular metabolic event(s) subsequent to glucose uptake, i.e., metabolic sensors as opposed to specific glucose receptors. In vitro studies have demonstrated that glucose-sensing neurons of the hypothalamus respond to a variety of metabolic substrates, including lactate and BHB (18,19,20,21). Additionally, these neurons express characteristic components of -cell glucose-sensing "machinery," such as GLUT2 glucose transporter protein (22), the pancreatic form of glucokinase (23), and ATP-sensitive K+ channels (24). In the current study, we sought to ascertain whether hypoglycemic detection at the portal vein is also metabolic in nature using fuel-sensing mechanisms similar to those of the hypothalamus. This was achieved by examining the impact of selective portal vein elevations of substrates known to impact brain metabolic sensors, i.e., lactate and BHB, as well as pyruvate, a glycolytic intermediate that apparently fails stimulate glucose-sensitive neurons in the hypothalamus (20,21).
RESEARCH DESIGN AND METHODS
Male Wistar rats (241 ± 7 g, n = 36) in the conscious relaxed state were used in all experimental procedures. All animals were housed in individual cages, fed ad libitum, and subjected to standard 12-h light-dark cycle. The University of Southern California Institutional Animal Care and Use Committee approved all surgical and experimental procedures. One week before the study, rats were anesthetized (3:3:1 ketamine HCl:xylazine:acepromizine malate; 0.1 ml/0.1 kg body wt i.m.), and indwelling catheters were inserted into the portal vein (silastic tubing, 0.051 cm internal diameter [ID] with 0.03-cm-ID tip) for infusion of studied metabolites, into the carotid artery (polyethylene tubing, 0.058 cm ID) for arterial blood sampling, and into the jugular vein (dual silastic cannula, 0.03 cm ID) for peripheral insulin, glucose, and metabolite infusions. All cannulas were tunneled subcutaneously, exteriorized on the back of the neck, and incased in an infusion harness (Instech Laboratories, Plymouth Meeting, PA). All animals were allowed at least 6 days to recover from surgery and to regain their original body weight. All animals were fasted overnight before initiation of the experiment.
Hyperinsulinemic-hypoglycemic clamp.
All rats were exposed to a general protocol for the induction of hypoglycemia. On the day of the experiment, animals were placed in a metabolic chamber and allowed to acclimatize to the new environment for at least 60 min (eC90 to eC30 min). At min 0, whole-body hypoglycemia was induced via jugular vein insulin infusion (25 mU · kgeC1 · mineC1) and variable jugular glucose infusions (20% dextrose). Basal glucose concentrations were measured at eC30 and 0 min, and subsequent serial sampling for glucose was performed at 10-min intervals throughout the duration of the clamp (0eC105 min). Plasma concentrations of epinephrine, norepinephrine, and infused metabolites (lactate, pyruvate, or BHB) were determined at basal (eC30 and 0 min) and during sustained hypoglycemia (60, 75, 90, and 105 min). Plasma samples for the insulin assay were drawn during the basal period (eC30 min) and at the end of hypoglycemia (105 min).
Study design.
Experiments were subdivided according to the infused metabolite, i.e., portal lactate clamp (n = 13), portal pyruvate clamp (n = 11), and portal BHB clamp (n = 12). Animals were randomly selected to take part in one of six separate protocols distinguished by the site of infusion, portal vein versus jugular vein, and metabolite infused, lactate, pyruvate, and BHB.
Portal lactate clamp.
For portal-lactate animals (n = 7), whole-body hypoglycemia was induced as described above with a lactate (2.5 mol/l lactic acid in phosphate buffer, pH 4.5; Sigma, St. Louis, MO) infused via the portal vein at variable rates starting at 40 min to attain local portal vein hyperlactatemia (5 mmol/l) in the midst of systemic hypoglycemia. This lactate infusate has previously been shown to elevate lactate levels without impacting blood pH (25). Jugular-lactate control animals (n = 6) were exposed to an identical hyperinsulinemic-hypoglycemic clamp with lactate infused (variable rate) via the jugular vein beginning at 40 min to match arterial lactate concentrations in the portal lactate group. To isolate the increase in lactate levels to the portal vein, dichloroacetic acid (DCA; 300 mg/kg) was infused in both groups via the jugular vein to prevent any elevation in arterial lactate concentration above basal. Dichloroacetate selectively inhibits tissue lactate release via activation of pyruvate dehydrogenase and has been used in a number of animal and human studies without any adverse affects (26,27).
Portal pyruvate clamp.
Portal-pyruvate (n = 6) and jugular-pyruvate (n = 5) rats were exposed to a protocol identical to that above for the lactate clamp with the exception that animals received pyruvate infusions (1 mol/l sodium pyruvate in sterile 0.9 mol/l NaCl, pH 7.4; Sigma), without DCA.
Portal BHB clamp.
Portal-BHB (n = 5) and jugular-BHB (n = 7) animals were exposed to a protocol identical to that above for the lactate clamp with the exception that animals received BHB infusions (1 mol/l sodium BHB in sterile 0.9 mol/l NaCl, pH 7.4; Sigma), without DCA.
Analytical procedures.
Glucose and lactate were assayed by the glucose oxidase method (YSI, Yellow Springs, OH). Arterial BHB (28) and pyruvate (29) concentrations were assayed spectrophotometrically. Catecholamines were analyzed using a single-isotope radioenzymatic method (30). Insulin samples were assayed using a radioimmunoassay kit (Linco Research, St. Charles, MO).
Calculations and data analysis.
The estimated local portal vein metabolite concentrations (MPV) were calculated as MPV = MA+ (MINFPOR/PVPF), where MA is arterial metabolite concentration (in micromoles per milliliter), MINFPOR is the portal vein metabolite infusion rate (in micromoles per minute), and PVPF is portal vein plasma flow rate in (milliliters per minute). The portal vein plasma flow rate was assumed to be 80% of hepatic plasma flow rate, where hepatic plasma flow rate was estimated to be 1.3 ml · g liver wteC1 · mineC1 (31). Mean steady-state metabolite concentrations were determined as the average of at least four serial samples that differ nondirectionally by <0.5% per minute. Experimental results were expressed as means ± SE. A one-way ANOVA was used to compare characteristics between independent groups. Repeated measures ANOVAs were used to determine group differences over time with Tukey’s test for post hoc analysis. Significance was set at P < 0.05.
RESULTS
Portal lactate clamp.
Arterial plasma insulin increased from an average basal value of 98 ± 10.41 pmol/l (P = 0.76, between groups) to a hyperinsulinemic value of 5,246.34 ± 310.59 pmol/l (P = 0.52, between groups). Basal glucose concentrations for portal-lactate versus jugular-lactate (6.76 ± 0.18 vs. 7.10 ± 0.16 mmol/l, P = 0.12) and glucose concentrations at the hypoglycemic nadir (2.54 ± 0.14 vs. 2.51 ± 0.14 mmol/l, P = 0.81) were not significantly different between the groups (Fig. 1A). Furthermore, no significant differences were found between portal-lactate versus jugular-lactate protocols with respect to basal arterial lactate (0.88 ± 0.08 vs. 0.9 ± 0.09 mmol/l; P = 0.81; Fig. 1B) and arterial lactate during sustained (60eC105 min) hypoglycemia (1.01 ± 0.1 vs. 0.91 ± 0.09 mmol/l; P = 0.36; Fig. 1B). As expected, portal vein lactate infusions significantly elevated mean portal vein plasma lactate concentrations in the portal-lactate group, whereas portal vein lactate concentrations in the jugular-lactate group remained at basal levels (5.03 ± 0.20 vs. 0.91 ± 0.09 mmol/l, P < 0.0001; Fig. 1C)
In response to insulin-induced hypoglycemia, jugular-lactate animals demonstrated a robust sympathoadrenal response where plasma epinephrine concentrations increased from 1.68 ± 0.51 nmol/l at basal to 14.44 ± 2.70 nmol/l by 105 min (Fig. 2A). In contrast, selective elevation of portal vein lactate resulted in a 64% suppression of the epinephrine response (14.44 ± 2.70 vs. 5.26 ± 0.77 nmol/l, P < 0.01; Fig. 2A) at 105 min of hypoglycemia. Additionally, statistically significant suppressions in the epinephrine response to hypoglycemia were observed in the portal-lactate group at min 60, 75, and 90 of hypoglycemia. Correspondingly, the norepinephrine response above basal was also suppressed by 67% in the portal-lactate versus the jugular-lactate group (1.66 ± 0.57 vs. 5.05 ± 1.21 nmol/l, P < 0.01; Fig. 2B).
Portal pyruvate clamp.
Mean arterial glucose concentrations were not significantly different between the portal-pyruvate versus jugular-pyruvate groups at basal (5.77 ± 0.2 mmol/l, P = 0.69) nor were they statistically different during the hypoglycemic plateau (2.55 ± 0.12 mmol/l, P = 0.84; Fig. 3A). Furthermore, at basal (P = 0.76) and during sustained hypoglycemia (P = 0.17), no significant differences between the two groups were observed with respect to mean arterial pyruvate concentrations (Fig. 3B). On the other hand, as a result of the selective portal vein pyruvate infusion, mean portal vein pyruvate levels were significantly elevated (1.81 ± 0.21 vs. 0.42 ± 0.05 mmol/l, P < 0.0001; Fig. 3B) in portal-pyruvate versus jugular-pyruvate animals, respectively. In jugular-pyruvate animals, hypoglycemia elicited 16-fold increase in the epinephrine response from basal 1.00 ± 0.19 to 16.15 ± 3.21 nmol/l at min 105 of the hyperinsulinemic-hypoglycemic clamp. On the other hand, as a result of selective elevation of portal vein pyruvate concentrations, epinephrine response to whole-body hypoglycemia in the portal-pyruvate group was suppressed by 64% at min 105 compared with the jugular-pyruvate group (5.84 ± 0.71 vs. 16.15 ± 3.21 nmol/l, P < 0.01; Fig. 3C). In jugular-pyruvate animals, norepinephrine concentrations increased approximately threefold from the basal value of 2.25 ± 0.38 to 6.13 ± 0.77 nmol/l at the end point of hypoglycemia. Similar to the epinephrine response, elevation of portal vein pyruvate resulted in a 80% suppression of the norepinephrine response above basal at min 105 of hypoglycemia (0.78 ± 0.41 vs. 3.88 ± 0.69 nmol/l, P < 0.01; Fig. 3D).
Portal BHB clamp.
By design, there were no significant differences between the portal-BHB and jugular-BHB groups with respect to arterial glucose concentrations at basal (5.86 ± 0.17 vs. 5.61 ± 0.16 mmol/l; P = 0.45; Fig. 4A), during the fall in glycemia, and during sustained hypoglycemia (2.53 ± 0.14 vs. 2.52 ± 0.14 mmol/l; P = 0.91; Fig. 4A). Also, there were no significant differences between portal-BHB and jugular-BHB treatments with respect to mean arterial BHB levels at basal (0.55 ± 0.23 vs. 0.67 ± 0.10 mmol/l; P = 0.59, data not shown) and during hypoglycemia (0.12 ± 0.03 vs. 0.16 ± 0.03 mmol/l; P = 0.32; Fig. 4B). Portal vein BHB infusions elevated BHB concentrations in the portal vein 12-fold in the portal-BHB versus jugular-BHB groups (2.02 ± 0.03 vs. 0.163 ± 0.03 mmol/l; P < 0.0001; Fig. 4B). In contrast to selective elevation of portal vein lactate or pyruvate, no significant differences were observed between the portal-BHB versus jugular-BHB groups with to respect epinephrine (5.42 ± 0.92 vs. 6.84 ± 1.59 nmol/l; P = 0.51 at min 105; Fig. 4C) and norepinephrine (2.67 ± 0.49 vs. 3.45 ± 0.81 nmol/l; P = 0.47 at min 105; Fig. 4D) responses to hypoglycemia.
DISCUSSION
In the current study, we investigated the effects of selective portal vein elevation of lactate, pyruvate, or BHB on the sympathoadrenal response to insulin-induced systemic hypoglycemia. Clamping portal vein lactate or pyruvate concentrations at approximately fivefold above basal, while maintaining arterial concentrations during systemic hypoglycemia, resulted in a substantial suppression of the epinephrine and norepinephrine responses, 64 and 75%, respectively. In contrast, elevating portal vein BHB to levels 12-fold above basal failed to impact the sympathoadrenal response to hypoglycemia. The ability to detect alternative metabolic substrates known to enter the glycolytic pathway distal to the uptake of glucose is indicative of a portal vein metabolic sensor rather than a glucoreceptor specific for the glycosyl moiety. The metabolic nature of hypoglycemic detection at the portal vein is further underscored by the fact that portal elevation of either lactate or pyruvate led to a quantitative blunting of the sympathoadrenal response comparable with that achieved via portal glucose normalization (Fig. 5).
Whole-body infusions of lactate have been shown to blunt the sympathoadrenal response to insulin-induced hypoglycemia in humans (16,17). It is generally assumed that such lactate-induced suppression of the sympathoadrenal response is mediated via its action on brain metabolic sensors. Lactate can be readily used by nervous tissue (32) and has been shown, both in vitro and in vivo, to activate glucose-sensitive neurons in the brain (19eC21,33,34). The ability of lactate to alter the firing rate of glucose-responsive neurons has been demonstrated in the hypothalamus (19,20) and in the hindbrain (35). However, in vitro studies have not been uniform in their observations of the effect of lactate on glucose-inhibited hypothalamic neurons. Yang et al. (21), reported that the addition of lactate, similar to glucose, decreased the firing rate of hypothalamic glucose-inhibited neurons, whereas Song and Routh (19) observed an increased discharge from such neurons. Local microdialysis of the ventromedial hypothalamus with 100 mmol/l lactate has been shown to substantially suppress the sympathoadrenal response to systemic hypoglycemia in rats in vivo (33). Exogenous lactate delivery (50 e蘭ol/h) into the caudal fourth ventricle has also been observed to prolong recovery from insulin-induced hypoglycemia (34).
Although the abovementioned findings clearly demonstrate that elevated brain lactate levels can impact the counterregulatory response to systemic hypoglycemia, it is not clear that the central nervous system (CNS) is the locus for those metabolic sensors responding to plasma lactate elevations in vivo as previously reported (16,17). The current findings clearly demonstrate that the selective elevation of portal vein plasma lactate levels, similar to that used in studies of whole-body plasma lactate elevations, substantially suppresses the sympathoadrenal response to systemic hypoglycemia. The suppression in the epinephrine response to hypoglycemia with local portal vein lactate elevation in the current study, a 64% suppression, is comparable with that reported for whole-body lactate elevation during hypoglycemia, i.e., 60eC70% (16,17). As noted above, normalizing glycemia or elevating lactate in the portal vein results in a comparable suppression in the sympathoadrenal response to hypoglycemia, which is similar in magnitude to that reported for whole-body lactate infusions. In contrast, elevating brain glucose levels during systemic hypoglycemia is purported to result in a substantially greater suppression of the epinephrine response to hypoglycemia (90%) (7). Thus, the response to elevating whole-body plasma lactate levels during hypoglycemia in quantitative terms appears more consistent with the blunting of portal vein metabolic sensors, as opposed to CNS metabolic sensors.
Nutrient sensing by hypothalamic glucose-responsive neurons has been attributed to intracellular changes in the ATP-to-ADP ratio and the subsequent binding of ATP to KATP, leading to changes in cell potential (19,24). Exposure of hypothalamic glucose-responsive neurons to glucose or lactate results in closure of KATP channels, which can be reversed by the application of diazoxide (19). The intracerebroventricular application of the KATP channel closer, glibenclamide, blunts the sympathoadrenal response to hypoglycemia, suggestive of an ATP-dependent mechanism (36). The current observation that elevating portal vein pyruvate concentration during hypoglycemia results in a suppression of the sympathoadrenal response comparable with that of lactate or glucose is consistent with such an ATP-dependent mechanism. However, Yang et al. (20,21) reported that glucose-responsive and glucose-inhibited neurons in the hypothalamus do not respond to elevated pyruvate despite the fact that pyruvate is apparently metabolized by such neurons. These same investigators observed that early glycolytic intermediates, e.g., glyceraldehyde and lactate, were able to substitute for glucose and alter the firing rate in both glucose-responsive and glucose-inhibited hypothalamic neurons. Based on the inability of pyruvate to stimulate hypothalamic glucose sensors, Yang et al. concluded that ATP production was not the primary metabolic event responsible for "glycemic detection" in these cells. They postulated that hypothalamic glucose sensing must be mediated by products of glucose metabolism proximal to pyruvate oxidation, e.g., cytosolic NADH. This hypothesis is supported by a recent report demonstrating a glucose-induced increase in cytosolic NADH, but not ATP levels, in hypothalamic glucose-responsive neurons (37).
Although it remains unclear as to whether glucose-sensing neurons in the hypothalamus and portal vein use a common metabolic event(s), our findings are consistent with a postulated mechanism for pancreatic -cell nutrient sensing (38). It is proposed that the intracellular rise in malonyl CoA signals nutrient abundance in the -cell, and thus only those compounds capable of both substrate oxidation and citric acid cycle anaplerosis should be effective (39,40). Elevating portal vein pyruvate, lactate, or glucose but not BHB proved comparable in their efficacy for suppressing the sympathoadrenal response to hypoglycemia (Fig. 5). Of these substrates, only BHB is incapable of supplying citric acid cycle intermediates via anaplerosis. That the metabolic sensing mechanisms for the -cell and portal vein glucose sensors might be similar is supported by reports that GLUT2 (41) and the pancreatic form of glucokinase (42), critical components of the -cell glucose-sensing "machinery," are both expressed in the portal region.
As noted above, elevating portal vein BHB levels fails to impact either epinephrine or norepinephrine secretion during hypoglycemia (Fig. 4). This is in contrast to whole-body ketone infusions, which have previously been shown to suppress the counterregulatory responses to insulin-induced hypoglycemia (16). Clearly the impact of whole-body hyperketonemia on the sympathoadrenal response to hypoglycemia must be manifest at some locus distal from the portal vein, most likely the CNS. Iontophoretic application of BHB has been observed to modify neuronal discharge of hypothalamic glucose-responsive and glucose-inhibited neurons in vitro in a dose dependent manner (18). Although it is possible that the portal vein BHB level achieved in our current study (2.02 ± 0.03 mmol/l) was insufficient to mask portal vein metabolic sensing or that BHB is not transported into portal glucose sensors, neither proposal appears plausible. Previous studies showed that elevating ketone levels 10-fold above basal to arterial concentrations of 1eC2 mmol/l significantly diminishes the sympathoadrenal response to hypoglycemia in humans (16), dogs (43), and rats (44). Additionally, transport of lactate, pyruvate, and BHB across cellular plasma membranes uses the same family of monocarboxylate transporter proteins (45). Therefore, transport across plasma membrane would not account for the differences observed between lactate or pyruvate and BHB. Assuming that the impact of BHB is effected at the level of the CNS, the inability of portal vein metabolic sensors to respond to ketones may be further evidence distinguishing that metabolic sensing at the portal vein is distinct from that which governs sensing at the brain.
Metabolic sensors have long been known to reside in the hepatoportal area and are understood to be involved in the regulation of feeding (46). Early studies by Niijima (47,48), using an in situ guinea pig liver perfusion preparation, demonstrated that the rates of hepatic vagal afferent discharge are inversely proportional to glucose availability in the perfusion medium. Subsequent studies showed that afferent discharge was also augmented by intraportal administrations of 2-deoxy-D-glucose (glucose oxidation inhibitor) (47) or mercaptoacetate (fatty acid oxidation inhibitor) (49), suggesting that the glucose sensory mechanism is dependent on the intracellular fuel oxidation and the subsequent ATP production. In support of this, intraperitoneal administration of lactate, pyruvate, or BHB has been shown to suppress feeding; however, no change in food intake in response to these metabolites was observed after hepatic branch vagatomy (50). It was therefore proposed that glucose-sensitive hepatic vagal afferents regulate feeding as a result of intracellular fuel oxidation and subsequent changes in mitochondrial ATP production (50).
Despite the fact that some investigators have suggested that glucose-sensitive vagal afferents are also involved in the regulation of the sympathoadrenal response (47), studies have failed to observe this relationship in vivo (15,51). On the other hand, we have shown that sectioning the celiac ganglion, through which spinal afferents originating from the portohepatis ascend, substantially impairs the ability of the animal to mount a full sympathoadrenal response to hypoglycemia (15). Thus, it appears that feeding in the periphery is mediated by glucose-sensitive hepatic vagal afferents, where hypoglycemic detection is mediated by spinal afferents transcending the celiac ganglion. Furthermore, this study points out yet another revealing distinction between the two sets of hepatoportal sensors, at least in respect to their responsiveness to ketone bodies. Clearly, further studies are warranted to elucidate exact molecular and cellular mechanisms of portal vein metabolic sensors.
In summary, our current findings indicate that the portal vein glucose sensor responds to alternative metabolic substrates such as lactate and pyruvate and therefore behaves as a metabolic sensor rather than a glucose-specific receptor. Portal vein elevations of glucose, lactate, and pyruvate but not BHB equally suppressed (60eC80%) the sympathoadrenal response to hypoglycemia. These findings suggest a mechanism of cellular glycemic detection mediated by events distal to pyruvate oxidation, requiring anaplerotic input into the citric acid cycle. In this regard, the mechanism for glycemic detection at the portal vein appears similar to that of the -cell. That pyruvate and BHB demonstrate differential effects at the portal vein and CNS indicates distinct mechanisms for hypoglycemic detection in these two loci. Elucidation of the cellular mechanism(s) involved in portal vein metabolic sensing and how that information is integrated with glucose sensors in the CNS remains a significant challenge to our understanding of hypoglycemic counterregulation in diabetes.
ACKNOWLEDGMENTS
C.M.D. has received National Institutes of Health Grant DK-55257.
We thank Satoshi Fujita, Maziyar Saberi, and MaryAnn Bohland for extensive contributions to the study.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Cryer PE: Hypoglycemia-associated autonomic failure in diabetes. Am J Physiol Endocrinol Metab 281:E1115eCE1121, 2001
The Diabetes Control and Complications Trial Research Group: Hypoglycemia in the Diabetes Control and Complications Trial. Diabetes 46:271eC286, 1997
Bolli G, de Feo P, Compagnucci P, Cartechini MG, Angeletti G, Santeusanio F, Brunetti P, Gerich JE: Abnormal glucose counterregulation in insulin-dependent diabetes mellitus: interaction of anti-insulin antibodies and impaired glucagon and epinephrine secretion. Diabetes 32:134eC141, 1983
Kleinbaum J, Shamoon H: Impaired counterregulation of hypoglycemia in insulin-dependent diabetes mellitus. Diabetes 32:493eC498, 1983
White NH, Skor DA, Cryer PE, Levandoski LA, Bier DM, Santiago JV: Identification of type I diabetic patients at increased risk for hypoglycemia during intensive therapy. N Engl J Med 308:485eC491, 1983
Rattarasarn C, Dagogo-Jack S, Zachwieja JJ, Cryer PE: Hypoglycemia-induced autonomic failure in IDDM is specific for stimulus of hypoglycemia and is not attributable to prior autonomic activation. Diabetes 43:809eC818, 1994
Biggers DW, Myers SR, Neal D, Stinson R, Cooper NB, Jaspan JB, Williams PE, Cherrington AD, Frizzell RT: Role of brain in counterregulation of insulin-induced hypoglycemia in dogs. Diabetes 38:7eC16, 1989
Borg WP, During MJ, Sherwin RS, Borg MA, Brines ML, Shulman GI: Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to hypoglycemia. J Clin Invest 93:1677eC1682, 1994
Hevener AL, Bergman RN, Donovan CM: Novel glucosensor for hypoglycemic detection localized to the portal vein. Diabetes 46:1521eC1525, 1997
Hevener AL, Bergman RN, Donovan CM: Portal vein afferents are critical for the sympathoadrenal response to hypoglycemia. Diabetes 49:8eC12, 2000
Lamarche L, Yamaguchi N, Peronnet F: Hepatic denervation reduces adrenal catecholamine secretion during insulin-induced hypoglycemia. Am J Physiol 268:R50eCR57, 1995
Donovan CM, Halter JB, Bergman RN: Importance of hepatic glucoreceptors in sympathoadrenal response to hypoglycemia. Diabetes 40:155eC158, 1991
Donovan CM, Hamilton-Wessler M, Halter JB, Bergman RN: Primacy of liver glucosensors in the sympathetic response to progressive hypoglycemia. Proc Natl Acad Sci U S A 91:2863eC2867, 1994
Smith D, Pernet A, Reid H, Bingham E, Rosenthal JM, Macdonald IA, Umpleby AM, Amiel SA: The role of hepatic portal glucose sensing in modulating responses to hypoglycaemia in man. Diabetologia 45:1416eC1424, 2002
Fujita S, Donovan CM: Celiac-superior mesenteric ganglionectomy, but not vagotomy, suppresses the sympathoadrenal response to insulin-induced hypoglycemia. Diabetes 54:3258eC3264, 2005
Veneman T, Mitrakou A, Mokan M, Cryer P, Gerich J: Effect of hyperketonemia and hyperlacticacidemia on symptoms, cognitive dysfunction, and counterregulatory hormone responses during hypoglycemia in normal humans. Diabetes 43:1311eC1317, 1994
Maran A, Cranston I, Lomas J, Macdonald I, Amiel SA: Protection by lactate of cerebral function during hypoglycaemia. Lancet 343:16eC20, 1994
Minami T, Shimizu N, Duan S, Oomura Y: Hypothalamic neuronal activity responses to 3-hydroxybutyric acid, an endogenous organic acid. Brain Res 509:351eC354, 1990
Song Z, Routh VH: Differential effects of glucose and lactate on glucosensing neurons in the ventromedial hypothalamic nucleus. Diabetes 54:15eC22, 2005
Yang XJ, Kow LM, Funabashi T, Mobbs CV: Hypothalamic glucose sensor: similarities to and differences from pancreatic -cell mechanisms. Diabetes 48:1763eC1772, 1999
Yang XJ, Kow LM, Pfaff DW, Mobbs CV: Metabolic pathways that mediate inhibition of hypothalamic neurons by glucose. Diabetes 53:67eC73, 2004
Leloup C, Arluison M, Lepetit N, Cartier N, Marfaing-Jallat P, Ferre P, Penicaud L: Glucose transporter 2 (GLUT 2): expression in specific brain nuclei. Brain Res 638:221eC226, 1994
Lynch RM, Tompkins LS, Brooks HL, Dunn-Meynell AA, Levin BE: Localization of glucokinase gene expression in the rat brain. Diabetes 49:693eC700, 2000
Ashford ML, Boden PR, Treherne JM: Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive K+ channels. Pflugers Arch 415:479eC483, 1990
Donovan CM, Pagliassotti MJ: Endurance training enhances lactate clearance during hyperlactatemia. Am J Physiol 257:E782eCE789, 1989
Stacpoole PW, Greene YJ: Dichloroacetate. Diabetes Care 15:785eC791, 1992
Gao J, Islam MA, Brennan CM, Dunning BE, Foley JE: Lactate clamp: a method to measure lactate utilization in vivo. Am J Physiol 275:E729eCE733, 1998
Koch DD, Feldbruegge DH: Optimized kinetic method for automated determination of beta-hydroxybutyrate. Clin Chem 33:1761eC1766, 1987
Passonnea J LO: Enzymatic Analysis: A Practical Guide. Totowa, NJ, Humana Press, 1993
Peuler JD, Johnson GA: Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci 21:625eC636, 1977
Ishise S, Pegram BL, Yamamoto J, Kitamura Y, Frohlich ED: Reference sample microsphere method: cardiac output and blood flows in conscious rat. Am J Physiol 239:H443eCH449, 1980
Tsacopoulos M, Magistretti PJ: Metabolic coupling between glia and neurons. J Neurosci 16:877eC885, 1996
Borg MA, Tamborlane WV, Shulman GI, Sherwin RS: Local lactate perfusion of the ventromedial hypothalamus suppresses hypoglycemic counterregulation. Diabetes 52:663eC666, 2003
Patil GD, Briski KP: Lactate is a critical "sensed" variable in caudal hindbrain monitoring of CNS metabolic stasis. Am J Physiol Regul Integr Comp Physiol 289:R1777eCR1786, 2005
Himmi T, Perrin J, Dallaporta M, Orsini JC: Effects of lactate on glucose-sensing neurons in the solitary tract nucleus. Physiol Behav 74:391eC397, 2001
Evans ML, McCrimmon RJ, Flanagan DE, Keshavarz T, Fan X, McNay EC, Jacob RJ, Sherwin RS: Hypothalamic ATP-sensitive K+ channels play a key role in sensing hypoglycemia and triggering counterregulatory epinephrine and glucagon responses. Diabetes 53:2542eC2551, 2004
Ainscow EK, Mirshamsi S, Tang T, Ashford ML, Rutter GA: Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels. J Physiol 544:429eC445, 2002
Prentki M, Tornheim K, Corkey BE: Signal transduction mechanisms in nutrient-induced insulin secretion. Diabetologia 40 (Suppl. 2):S32eCS41, 1997
Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802eC5810, 1992
Schuit F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T, Prentki M: Metabolic fate of glucose in purified islet cells: glucose-regulated anaplerosis in beta cells. J Biol Chem 272:18572eC18579, 1997
Burcelin R, Dolci W, Thorens B: Glucose sensing by the hepatoportal sensor is GLUT2-dependent: in vivo analysis in GLUT2-null mice. Diabetes 49:1643eC1648, 2000
Jetton TL, Liang Y, Pettepher CC, Zimmerman EC, Cox FG, Horvath K, Matschinsky FM, Magnuson MA: Analysis of upstream glucokinase promoter activity in transgenic mice and identification of glucokinase in rare neuroendocrine cells in the brain and gut. J Biol Chem 269:3641eC3654, 1994
Flatt JP, Blackburn GL, Randers G, Stanbury JB: Effects of ketone body infusion on hypoglycemic reaction in postabsorptive dogs. Metabolism 23:151eC158, 1974
Stricker EM, Rowland N, Saller CF, Friedman MI: Homeostasis during hypoglycemia: central control of adrenal secretion and peripheral control of feeding. Science 196:79eC81, 1977
Poole RC, Halestrap AP: Transport of lactate and other monocarboxylates across mammalian plasma membranes. Am J Physiol 264:C761eCC782, 1993
Novin D, VanderWeele DA, Rezek M: Infusion of 2-deoxy-D-glucose into the hepatic-portal system causes eating: evidence for peripheral glucoreceptors. Science 181:858eC860, 1973
Niijima A: Glucose sensitive afferent nerve fibers in the liver and regulation of blood glucose. Brain Res Bull 5:175eC179, 1980
Niijima A: Afferent impulse discharges from glucoreceptors in the liver of the guinea pig. Ann N Y Acad Sci 157:690eC700, 1969
Ritter S, Taylor JS: Vagal sensory neurons are required for lipoprivic but not glucoprivic feeding in rats. Am J Physiol 258:R1395eCR1401, 1990
Langhans W, Egli G, Scharrer E: Regulation of food intake by hepatic oxidative metabolism. Brain Res Bull 15:425eC428, 1985
Jackson PA, Pagliassotti MJ, Shiota M, Neal DW, Cardin S, Cherrington AD: Effects of vagal blockade on the counterregulatory response to insulin-induced hypoglycemia in the dog. Am J Physiol 273:E1178eCE1188, 1997(Aleksey V. Matveyenko, an)