Hyperglycemia does not increase basal hypothalamo-pituitary-adrenal activity in diabetes but it does impair the HPA response to insulin-induced hypoglycemia

Recently, we established that hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia were impaired in uncontrolled streptozotocin (STZ)-diabetic (65 mg/kg) rats and insulin treatment restored most of these responses. In the current study, we used phloridzin to determine whether the restoration of blood glucose alone was sufficient to normalize HPA function in diabetes. Normal, diabetic, insulin-treated, and phloridzin-treated diabetic rats were either killed after 8 days or subjected to a hypoglycemic (40 mg/dl) glucose clamp. Basal: Elevated basal ACTH and corticosterone in STZ rats were normalized with insulin but not phloridzin. Increases in hypothalamic corticotrophin-releasing hormone (CRH) and inhibitory hippocampal mineralocorticoid receptor (MR) mRNA with STZ diabetes were not restored with either insulin or phloridzin treatments. Hypoglycemia: In response to hypoglycemia, rises in plasma ACTH and corticosterone were significantly lower in diabetic rats compared with controls. Insulin and phloridzin restored both ACTH and corticosterone responses in diabetic animals. Hypothalamic CRH mRNA and pituitary pro-opiomelanocortin mRNA expression increased following 2 h of hypoglycemia in normal, insulin-treated, and phloridzin-treated diabetic rats but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal MR mRNA in control, insulin-, and phloridzin-treated diabetic rats but not uncontrolled diabetic rats, whereas glucocorticoid receptor mRNA was not altered by hypoglycemia. In conclusion, despite elevated basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that defects in the CRH response may be related to a defective MR response. It is intriguing that phloridzin did not restore basal HPA activity but it restored the HPA response to hypoglycemia, suggesting that defects in basal HPA function in diabetes are due to insulin deficiency, but impaired responsiveness to hypoglycemia appears to stem from chronic hyperglycemia.     

The selective serotonin reuptake inhibitor sertraline enhances counterregulatory responses to hypoglycemia

Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for patients with comorbid diabetes and depression. Clinical case studies in diabetic patients, however, suggest that SSRI therapy may exacerbate hypoglycemia. We hypothesized that SSRIs might increase the risk of hypoglycemia by impairing hormonal counterregulatory responses (CRR). We evaluated the effect of the SSRI sertraline on hormonal CRR to single or recurrent hypoglycemia in nondiabetic rats. Since there are time-dependent effects of SSRIs on serotonin neurotransmission that correspond with therapeutic action, we evaluated the effect of 6- or 20-day sertraline treatment on hypoglycemia CRR. We found that 6-day sertraline (SERT) treatment specifically enhanced the epinephrine response to a single bout of hypoglycemia vs. vehicle (VEH)-treated rats (t = 120: VEH, 2,573 +/- 448 vs. SERT, 4,202 +/- 545 pg/ml, P < 0.05). In response to recurrent hypoglycemia, VEH-treated rats exhibited the expected impairment in epinephrine secretion (t = 60: 678 +/- 73 pg/ml) vs. VEH-treated rats experiencing first-time hypoglycemia (t = 60: 2,081 +/- 436 pg/ml, P < 0.01). SERT treatment prevented the impaired epinephrine response in recurrent hypoglycemic rats (t = 60: 1,794 +/- 276 pgl/ml). In 20-day SERT-treated rats, epinephrine, norepinephrine, and glucagon CRR were all significantly elevated above VEH-treated controls in response to hypoglycemia. Similarly to 6-day SERT treatment, 20-day SERT treatment rescued the impaired epinephrine response in recurrent hypoglycemic rats. Our data demonstrate that neither 6- nor 20-day sertraline treatment impaired hormonal CRR to hypoglycemia in nondiabetic rats. Instead, sertraline treatment resulted in an enhancement of hypoglycemia CRR and prevented the impaired adrenomedullary response normally observed in recurrent hypoglycemic rats.     

Increases of neuropeptide Y-like immunoreactivity in plasma during insulin-induced hypoglycemia in man

Neuropeptide Y-like immunoreactivity (NPY-LI) in plasma during insulin-induced hypoglycemia was measured in 4 healthy male volunteers. Plasma NPY-LI increased from 167 +/- 11 pg/ml to 247 +/- 25 pg/ml 30 min after the administration of insulin (0.1 U/kg body weight IV), reached the maximum (296 +/- 6 pg/ml) 45 min after the insulin, and then decreased. These results suggest that NPY is released into the systemic circulation during insulin-induced hypoglycemia in man.     

Decreased response of epinephrine and norepinephrine to insulin-induced hypoglycemia in diabetic autonomic neuropathy

The responses of epinephrine, norepinephrine and other counter-regulatory hormones to insulin-induced hypoglycemia were investigated in 5 diabetics who showed signs of autonomic neuropathy, in 7 age-matched diabetics without autonomic neuropathy and in 7 healthy subjects. The presence of autonomic neuropathy was evaluated by decreased beat-to-beat variation in heat rates during hyperventilation or orthostatic hypotension. Catecholamines were determined by a totally automated plasma catecholamine analyzing system using a two-column system of high performance liquid chromatography. Plasma epinephrine and norepinephrine responses to hypoglycemia in diabetics with autonomic neuropathy were significantly lower than those in diabetics without autonomic neuropathy. Plasma glucagon response in diabetics was apparently attenuated compared to normal controls and there was no significant difference in glucagon response between the two patient groups. Other counter-regulatory hormone responses did not differ among the three groups. The data demonstrate that the responses of plasma epinephrine and norepinephrine to insulin-induced hypoglycemia are impaired in diabetics with autonomic neuropathy.     

Elevation of plasma corticosterone by swim stress and insulin-induced hypoglycemia in control and fluoxetine-pretreated rats.

Fluoxetine, a drug that inhibits serotonin inactivation by reuptake from the synaptic cleft and thereby enhances serotonin nerve function, was used to study the possible role of serotonin neurons in the activation of the pituitary-adrenal system of rats by swim stress or insulin-induced hypoglycemia. Fluoxetine pretreatment enhanced the elevation of plasma corticosterone produced by injection of L-5-hydroxytryptophan but did not significantly alter the elevation of plasma corticosterone by swim stress or by insulin-induced hypoglycemia, even when the stimulus was shown to be submaximal. The results suggest that serotonin neural pathways postulated to stimulate ACTH secretion are not involved in the activation of adrenocortical function by these stimuli.     

Modulation of insulin and glucagon secretion from the perfused rat pancreas by the neurohypophysial hormones and by desamino-D-arginine vasopressin (DDAVP)

Marked stimulation of glucagon release and modest stimulation of insulin release were observed during in situ perfusion of the rat pancreas with AVP or OT. Glucagon release in response to AVP or OT (200 pg/ml) gradually increased over a 45 min perfusion period reaching maxima of 500% and 300% of the pre-stimulatory levels, respectively. Insulin release transiently increased by 100%. In each case release rates returned to control values immediately after withdrawal of the peptides. Total glucagon release was concentration dependent and linear from 20 pg to 20 ng AVP or OT/ml (r greater than .97). Pancreatic response to DDAVP perfused at 20 ng/ml was virtually indistinguishable from that induced by AVP at 200 pg/ml. This demonstration of a glucagonotrophic action of the neurohypophysial hormones in the in situ perfused rat pancreas confirms earlier studies using isolated islets and bolus IV injection.     

Experimental dissociation of neural circuits underlying conditioned avoidance and hypophagic responses to lithium chloride

We previously reported that noradrenergic (NA) neurons in the nucleus of the solitary tract (NST) are necessary for exogenous CCK octapeptide to inhibit food intake in rats. To determine whether NST NA neurons also are necessary for lithium chloride (LiCl) to inhibit food intake and/or to support conditioned avoidance behavior, saporin toxin conjugated to an antibody against dopamine beta hydroxylase (DSAP) was microinjected bilaterally into the NST to ablate resident NA neurons. DSAP and sham control rats subsequently were tested for the ability of LiCl (0.15M, 2% body wt) to inhibit food intake and to support conditioned flavor avoidance (CFA). LiCl-induced hypophagia was significantly blunted in DSAP rats, and those with the most extensive loss of NST NA neurons demonstrated the most attenuated LiCl-induced hypophagia. Conversely, LiCl supported a robust CFA that was of similar magnitude in sham control and DSAP rats, including rats with the most extensive NA lesions. A terminal c-Fos study revealed intact LiCl-induced c-Fos expression in the lateral parabrachial nucleus and central amygdala in DSAP rats, despite significant loss of NST NA neurons and attenuated c-Fos activation of corticotropin-releasing hormone-positive neurons in the paraventricular nucleus of the hypothalamus (PVN). Thus, NST NA neurons contribute significantly to LiCl-induced hypophagia and recruitment of stress-responsive PVN neurons but appear to be unnecessary for CFA learning and expression. These findings support the view that distinct central nervous system circuits underlie LiCl-induced inhibition of food intake and conditioned avoidance behavior in rats.     

Immunotoxic catecholamine lesions attenuate 2DG-induced increase of AGRP mRNA

Injections of the immunotoxin, saporin conjugated to anti-dopamine-beta-hydroxylase (DSAP), into the hypothalamic paraventricular nucleus (PVH) selectively destroy norepinephrine (NE) and epinephrine (E) terminals in the medial hypothalamus and abolish glucoprivic feeding. We utilized PVH DSAP injections to examine the role of NE/E neurons in the previously reported 2-deoxy-D-glucose (2DG)-induced increases in mRNA levels for the orexigenic peptides, AGRP and NPY. Northern blot analysis revealed that DSAP lesions elevated basal but blocked 2DG-induced increases in AGRP mRNA levels. Changes in NPY mRNA were not detectable. AGRP neurons may contribute to circuitry activated by NE/E neurons for elicitation of glucoregulatory responses.     

Hormonal responses to hypoglycemia in orthostatic hypotension patients with adrenergic insufficiency

Glucagon, growth hormone and cortisol responses to insulin-induced hypoglycemia have been studied in nine normal subjects and four patients with orthostatic hypotension who also had markedly deficient sympathoadrenal medullary responses. Absence of catecholamine responses to hypoglycemia does not prevent the other hormonal responses. Glucagon, growth hormone and cortisol secretion appear to be evoked independently from the catecholamine response during hypoglycemia. Elevated basal cortisol levels are the probable cause of a delay in the nadir of hypoglycemia observed in patients with adrenergic insufficiency. The sympathetic nervous system dysfunction in patients with neurogenic orthostatic hypotension may include deficient adrenal medullary responses although other counterregulatory responses remain functional.     

Inactivation of the PVN during hypoglycemia partially simulates hypoglycemia-associated autonomic failure

The anatomic connections of the paraventricular nucleus of the hypothalamus (PVN) are such that it is ideally situated to modulate and/or control autonomic responses to a variety of stressors, including hypoglycemia. In our experimental model of hypoglycemia-associated autonomic failure (HAAF), a syndrome in which the counterregulatory response to hypoglycemia is partially compromised via unknown mechanisms, activation of the PVN is blunted (15). We hypothesized that this blunted PVN activation during HAAF may be sufficient to cause the impaired counterregulatory response. To test this hypothesis, we anesthetized the PVN with lidocaine during insulin-induced hypoglycemia in rats and measured counterregulatory hormone levels. PVN inactivation decreased indexes of the sympathoadrenal response (plasma epinephrine and norepinephrine) and the hypothalamic-pituitary axis response (ACTH). Inactivation decreased the peak epinephrine response to hypoglycemia by almost half (-42 +/- 6% from control; P = 0.04) and the peak norepinephrine response by 34 +/- 5% (P = 0.01). The peak plasma ACTH levels attained were suppressed by 35 +/- 6% (P = 0.02). Adrenal corticosterone and pancreatic glucagon responses were not impaired. This pattern of neuroendocrine response is unlike that previously seen with our HAAF model. Control infusions of lidocaine >or=1 mm anterior or posterior to the PVN did not simulate this neuroendocrine pattern. Thus it appears that decreased PVN activation, as occurs with HAAF, may be involved in specific components of HAAF (i.e., blunting the sympathoadrenal and hypothalamic-pituitary-adrenocortical axis response), but not in others (i.e., blunting the glucagon response).     

Vasopressin and A1 noradrenaline turnover during food or water deprivation in the rat

In the present study, we have examined in Wistar rats the effects of food or water deprivation of 3 days on the hypophyso-adrenal axis, vasopressinergic system and activity of A1 noradrenergic brain stem cell group, which is involved in the control of the hypothalamic neuro-endocrine activity. Levels of adrenocorticotropic hormone (ACTH) and vasopressin (AVP) were determined by radio-immunoassay, and corticosterone level was determined by fluorimetric method. Plasma levels of ACTH and corticosterone were greatly increased in both groups of rats. In water-deprived rats, plasma AVP (13.83 +/- 1.63 vs. 3.03 +/- 0.23 pg/ml) and osmolality levels were significantly elevated with a marked decrease of AVP hypophysis content (272 +/- 65 vs. 1098 +/- 75 ng/mg protein), but not in food-deprived rats in which osmolality did not change and AVP remained stocked (2082 +/- 216 ng/mg protein) in the hypophysis without release in the plasma (1.11 +/- 0.23 pg/ml). These observations indicated that both food-deprivation and water-deprivation stimulated the pituitary adrenal axis thereby suggesting a stress state. AVP production is stimulated both by fluid and food restriction but is secreted with differential effects: during food restriction AVP secretion is limited to supporting the hypothalamic pituitary-adrenal system.     

Effects of antecedent hypoglycemia, hyperinsulinemia, and excess corticosterone on hypoglycemic counterregulation

This study aimed to differentiate the effects of repeated antecedent hypoglycemia, antecedent marked hyperinsulinemia, and antecedent increases in corticosterone on counterregulation to subsequent hypoglycemia in normal rats. Specifically, we examined whether exposure to hyperinsulinemia or elevated corticosterone per se could impair subsequent counterregulation. Four groups of male Sprague-Dawley rats were used: 1) normal controls (N) had 4 days of sham antecedent treatment; 2) an antecedent hypoglycemia group (AH) had 7 episodes of hyperinsulinemic hypoglycemia over 4 days; 3) an antecedent hyperinsulinemia group (AE) had 7 episodes of hyperinsulinemic euglycemia; and 4) an antecedent corticosterone group (AC) had 7 episodes of intravenous corticosterone to simulate the hypoglycemic corticosterone levels in AH rats. On day 5, hyperinsulinemic euglycemic-hypoglycemic clamps were performed. Epinephrine responses to hypoglycemia were impaired (P < 0.05 vs. N) after antecedent hypoglycemia and hyperinsulinemia. This correlated with diminished (P < 0.05 vs. N) absolute glucose production responses in AH rats and diminished incremental glucose production responses in AE rats. Paradoxically, norepinephrine responses were increased (P < 0.05 vs. N) after antecedent hypoglycemia. Glucagon and corticosterone responses were unaffected by antecedent hypoglycemia and hyperinsulinemia. In AC rats, incremental but not absolute glucose production responses were decreased (P < 0.05 vs. N). However, neuroendocrine counterregulation was unaltered. We conclude that both antecedent hypoglycemia and hyperinsulinemia impair epinephrine and glucose production responses to subsequent hypoglycemia, suggesting that severe recurrent hyperinsulinemia may contribute to the development of hypoglycemia-associated autonomic failure.     

The Effect of the Removal of the Area Postrema on Insulin and IGF-1-Induced Cardiovascular and Sympathetic Nervous Responses

Previous studies have demonstrated that insulin and IGF-1 both increase lumbar sympathetic nerve activity (LSNA) and decrease mean arterial pressure (MAP). We hypothesized that the peripheral responses to insulin and IGF-1 are mediated, at least in part, via the central nervous system. In this study we determined the effects of the peripheral administration of both insulin and IGF-1 on cardiovascular dynamics and LSNA following removal of the area postrema (APX), a major site of blood-brain communication. Insulin infusion in normal rats decreased MAP but increased HR and LSNA. When insulin was infused in APX rats it also decreased the MAP but the MAP recovered rapidly and plateaued at a level equivalent to normals after 40 min. Insulin significantly increased the HR and LSNA in the APX rats compared to normals. However, when hypoglycemia was prevented by glucose infusion, the HR and LSNA responses to insulin in the APX rats were similar to normals. IGF-1 also decreased MAP and to a greater extent in the APX rats compared to normals but the increased LSNA in APX rats was equivalent to normals. The APX rats when compared to normals had a greater sensitivity to insulin-induced hypoglycemia while IGF-1 decreased the plasma glucose to a lesser degree in APX rats. We conclude that insulin and IGF-1 entry into the CNS at least via the area postrema does not contribute significantly to the hypotensive response and that the greater depressor response to IGF-1 is likely due to enhanced vascular sensitivity in APX rats. The increased HR and LSNA following insulin were likely mediated by an increased reflexive response to hypoglycemia.     

Methysergide delays the decompensatory responses to severe hemorrhage by activating 5-HT(1A) receptors

Central administration of the serotonin receptor ligand methysergide delays the decompensatory response to hypotensive hemorrhage. This study was performed to determine the receptor subtype that mediates this effect. Lateral ventricular (LV) injection of methysergide (40 microg) delayed the hypotensive, bradycardic, and sympathoinhibitory responses to blood withdrawal (1.26 ml/min) in conscious rats. The response was quantified, in part, as the blood volume withdrawal that produced a 40-mmHg fall in blood pressure. The delayed hypotensive response produced by methysergide (8.2 +/- 0.2 vs. 5.6 +/- 0.2 ml, P < 0.01) was reversed by the 5-hydroxytryptamine (HT)(1A) antagonist WAY-100635 (30 microg iv: 6.7 +/- 0.4 ml, P < 0. 01; 100 microg iv: 5.6 +/- 0.1 ml, P < 0.01). LV injection of the 5-HT(1A) agonist (+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) also delayed the hypotensive (10 microg: 8.6 +/- 0.3, P < 0.01; 20 microg: 9.2 +/- 0.3 ml, P < 0.01), bradycardic, and sympathoinhibitory responses to hemorrhage. WAY-100635 (10 microg iv) completely reversed the effects of 8-OH-DPAT (20 microg: 5.4 +/- 0.3 ml). Neither selective blockade of 5-HT(2) receptors nor stimulation of 5-HT(1B/1D) receptors had any effect on hemorrhage responses. These data indicate that methysergide stimulates 5-HT(1A) receptors to delay the decompensatory responses to hemorrhage.     

Dissociation of hypothalamic noradrenergic activity and sympathoadrenal responses to recurrent hypoglycemia

This study evaluated whether attenuation of sympathoadrenal responses to recurrent hypoglycemia is mediated by diminished noradrenergic activity in the hypothalamus. Male Sprague-Dawley rats received either once daily insulin (1.0 units/kg) injections or an equal administration of saline for 3 days. Both groups received an administration of insulin on the fourth day, during which blood glucose and plasma catecholamines were determined, and extracellular norepinephrine (NE) in the ventromedial hypothalamus (VMH) or paraventricular hypothalamic nucleus (PVN) was monitored with microdialysis. The peak response of plasma epinephrine to insulin-induced hypoglycemia (nadir approximately 3.2 mmol/l) was significantly reduced during the fourth hypoglycemic episode (774 +/- 134 pg/ml) compared with the first episode (2,561 +/- 410 pg/ml, P < 0.001). Baseline levels of extracellular NE were elevated approximately 25% (P = 0.07) in the VMH and approximately 46% (P = 0.03) in the PVN after multiple hypoglycemic episodes. There was no difference in noradrenergic activity during the first or fourth hypoglycemic episode in either brain area. The reduced sympathoadrenal output after recurrent hypoglycemia is likely postsynaptic from hypothalamic NE release or is mediated via a collateral pathway.     

Opposite effect of adrenalectomy on rat brain prostaglandin synthesis in basal conditions and in response to insulin or 2-deoxy-glucose

In the present study we evaluated the role of endogenous glucocorticoids in the biosynthesis of prostaglandins (PG) in cortical brain tissue of the rat. Experiments were carried out under basal conditions and in response to insulin-induced hypoglycemia and 2-deoxyglucose (2-DG) induced-cytoglucopenia. In intact rats, following hypoglycemia and cytoglucopenia, the production of brain PG was decreased. These two stress stimuli also activated adrenocortical secretory responses, as manifested by an increase in circulating ACTH and corticosterone. Bilateral adrenalectomy did not modify the brain production of PG under basal conditions. In contrast, in adrenal-ectomized rats, the biosynthesis of brain cortical PG was markedly increased in response to insulin and 2-DG. These results suggest that adrenal hormones may be involved in the modulation of cortical PG production under stressful conditions.     

Portal vein hypoglycemia is essential for full induction of hypoglycemia-associated autonomic failure with slow-onset hypoglycemia

Antecedent hypoglycemia leads to impaired counterregulation and hypoglycemic unawareness. To ascertain whether antecedent portal vein hypoglycemia impairs portal vein glucose sensing, thereby inducing counterregulatory failure, we compared the effects of antecedent hypoglycemia, with and without normalization of portal vein glycemia, upon the counterregulatory response to subsequent hypoglycemia. Male Wistar rats were chronically cannulated in the carotid artery (sampling), jugular vein (glucose and insulin infusion), and mesenteric vein (glucose infusion). On day 1, the following three distinct antecedent protocols were employed: 1) HYPO-HYPO: systemic hypoglycemia (2.52 +/- 0.11 mM); 2) HYPO-EUG: systemic hypoglycemia (2.70 +/- 0.03 mM) with normalization of portal vein glycemia (portal vein glucose = 5.86 +/- 0.10 mM); and 3) EUG-EUG: systemic euglycemia (6.33 +/- 0.31 mM). On day 2, all groups underwent a hyperinsulinemic-hypoglycemic clamp in which the fall in glycemia was controlled so as to reach the nadir (2.34 +/- 0.04 mM) by minute 75. Counterregulatory hormone responses were measured at basal (-30 and 0) and during hypoglycemia (60-105 min). Compared with EUG-EUG, antecedent hypoglycemia (HYPO-HYPO) significantly blunted the peak epinephrine (10.44 +/- 1.35 vs. 15.75 +/- 1.33 nM: P = 0.01) and glucagon (341 +/- 16 vs. 597 +/- 82 pg/ml: P = 0.03) responses to next-day hypoglycemia. Normalization of portal glycemia during systemic hypoglycemia on day 1 (HYPO-EUG) prevented blunting of the peak epinephrine (15.59 +/- 1.43 vs. 15.75 +/- 1.33 nM: P = 0.94) and glucagon (523 +/- 169 vs. 597 +/- 82 pg/ml: P = 0.66) responses to day 2 hypoglycemia. Consistent with hormonal responses, the glucose infusion rate during day 2 hypoglycemia was substantially elevated in HYPO-HYPO (74 +/- 12 vs. 49 +/- 4 micromol x kg(-1) x min(-1); P = 0.03) but not HYPO-EUG (39 +/- 7 vs. 49 +/- 4 micromol x kg(-1) x min(-1): P = 0.36). Antecedent hypoglycemia local to the portal vein is required for the full induction of hypoglycemia-associated counterregulatory failure with slow-onset hypoglycemia.