Casey M. Donovan

HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise☆

Exercise causes heat shock (muscle temperatures of up to 45 degrees C, core temperatures of up to 44 degrees C) and oxidative stress (generation of O2- and H2O2), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32P]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30-60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O2- generation with increasing temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Sweet talk in the brain: Glucosensing, neural networks, and hypoglycemic counterregulation

Glucose is the primary fuel for the vast majority of cells, and animals have evolved essential cellular, autonomic, endocrine, and behavioral measures to counteract both hypo- and hyperglycemia. A central component of these counterregulatory mechanisms is the ability of specific sensory elements to detect changes in blood glucose and then use that information to produce appropriate counterregulatory responses. Here we focus on the organization of the neural systems that are engaged by glucosensing mechanisms when blood glucose concentrations fall to levels that pose a physiological threat. We employ a classic sensory-motor integrative schema to describe the peripheral, hindbrain, and hypothalamic components that make up counterregulatory mechanisms in the brain. We propose that models previously developed to describe how the forebrain modulates autonomic reflex loops in the hindbrain offer a reasoned framework for explaining how counterregulatory neural mechanisms in the hypothalamus and hindbrain are structured.

Importance of Hepatic Glucoreceptors in Sympathoadrenal Response to Hypoglycemia

To ascertain whether hepatic glucoreceptors are important to hypoglycemic counterregulation, a localized euglycemic clamp was employed across the liver during general hypoglycemia. Dogs were infused peripherally with insulin (18–21 pmol · kg−1 · min−1) for 150 min to induce systemic hypoglycemia. During the liver-clamp (LC) protocol, glucose was infused via the portal vein to maintain euglycemia at the liver. In control experiments, i.e., matched infusion (Ml), glucose was infused peripherally at a rate determined to yield similar arterial glycemia levels in the two protocols. Arterial glucose concentrations were not different between protocols during the final hour of insulin infusion (3.26 ± 0.21 and 3.25 ± 0.21 mM during LC and MI, respectively; P = 0.91). Calculated hepatic glucose concentrations during the same period were significantly higher for LC (5.22 ± 0.23 mM) than for MI (3.25 ± 0.21 mM). During MI, both epinephrine and norepinephrine rose significantly from basal values of 562 ± 87 pM and 1.21 ± 0.19 nM to plateaus of 3691 ± 1097 pM (P = 0.0001) and 2.38 ± 0.35 nM (P = 0.0002), respectively. However, during LC, the elevation in epinephrine was suppressed by 42 ± 8% (P = 0.015) relative to MI. Six of seven animals demonstrated a suppression in the norepinephrine response, averaging 32 ± 13% (NS, P = 0.068). The glucagon response to hypoglycemia was unaffected by the level of hepatic glycemia. Hepatic hypoglycemia is essential to produce the full sympathoadrenal response to insulin-induced hypoglycemia.

The Role of Liver Glucosensors in the Integrated Sympathetic Response Induced by Deep Hypoglycemia in Dogs

The significance of the portohepatic glucosensors for counterregulation in deep hypoglycemia (i.e., glycemia < 2.8 mM) was studied in chronically cannulated male mongrel dogs in the conscious state. A total of 16 experiments were carried out on 6 dogs using the liver clamp technique under hyperinsulinemic conditions (insulin infusion, 39 pmol · min−1 · kg−1, 0–150 min). The level of glycemia presented to the liver was made to differ from the systemic arterial glucose level via portal glucose infusion. Tracer-determined rates of glucose clearance and hepatic glucose output (HGO) were assessed using D-[3-3H]glucose (0.26 μCi · min−1). Three protocols were used. In protocol I, liver clamp, systemic hypoglycemia at 2.60 ± 0.09 mM, and liver glycemia at 3.86 ± 0.05 mM were achieved with portal glucose infusion (28.2 ± 3.0 μmol · min−1 · kg−1). For protocol II, glucose was infused peripherally (18.2 ± 4.3 μmol · min−1 · kg−1), while systemic and liver glycemia were sustained at deep hypoglycemia, 2.50 ± 0.08 mM. In protocol III, via peripheral glucose infusion (62.9 ± 5.8 μmol · min−1 · kg−1), systemic and liver glycemia were maintained at a level matched to the liver glycemia during protocol I (3.98 ± 0.05 mM, P > 0.10). When compared with protocols I and III, the catecholamine response above basal was significantly greater during protocol II with liver and systemic deep hypoglycemia (7.30 ± 1.51 and 2.89 ± 0.5 nM for epinephrine and norepinephrine, respectively, P < 0.005). These values reflect net increases in the catecholamine responses of 100% and 85% for epinephrine and norepinephrine when compared with protocol I. Glucose clearance was similar among protocols (13.03 ± 1.26 ml · min−1 · kg−1 P > 0.10). HGO was essentially unchanged from basal during protocol II but suppressed during protocols I and III (P < 0.05). These results are consistent with the hypothesis that portohepatic glucosensors play a significant role in eliciting the sympathoadrenal response to deep hypoglycemia.

The Locus for Hypoglycemic Detection Shifts With the Rate of Fall in Glycemia: The Role of Portal–Superior Mesenteric Vein Glucose Sensing

OBJECTIVE—To ascertain whether portal glucose sensing extends beyond the portal vein to the superior mesenteric vein and then test whether the role of portal–superior mesenteric glucose sensors varies with the rate of fall in glycemia. RESEARCH DESIGN AND METHODS—Chronically cannulated rats underwent afferent ablation of the portal vein (PV) or portal and superior mesenteric veins (PMV) or sham operation (control). One week later, animals underwent hyperinsulinemic-hypoglycemic clamps in which the hypoglycemic nadir, 2.48 ± 0.06 mmol/l, was reached at a rate of decline in glucose of −0.09 or −0.21 mmol · l−1 · min−1 (PMV and control only). Additional PMV and control animals received an intravenous injection of the glucopenic agent 2-deoxyglucose. RESULTS—Inducing hypoglycemia slowly, at a rate of −0.09 mmol · l−1 · min−1, resulted in a 26-fold increase in epinephrine (23.39 ± 0.62 nmol/l) and 12-fold increase in norepinephrine (11.42 ± 0.92 nmol/l) for controls (P < 0.001). The epinephrine response to hypoglycemia was suppressed by 91% in PMV (2.09 ± 0.07 nmol/l) vs. 61% in PV (9.05 ± 1.59 nmol/l) (P < 0.001). The norepinephrine response to hypoglycemia was suppressed by 94 and 80% in PMV and PV, respectively, compared with that in controls. In contrast, when arterial glucose was lowered to 2.49 ± 0.06 mmol/l within 20 min, no significant differences were observed in the catecholamine responses for PMV and controls over the first 45 min of hypoglycemia (20–65 min). Only at min 105 were catecholamines significantly lower for PMV vs. controls. Injection of 2-deoxyglucose induced a very rapid sympathoadrenal response with no significant differences between PMV and controls. CONCLUSIONS—The critical locus for hypoglycemic detection shifts away from the portal-mesenteric vein to some other loci (e.g., the brain) when hypoglycemia develops rapidly.

Training enhanced hepatic gluconeogenesis: the importance for glucose homeostasis during exercise.

Endurance training has long been known to improve the individuals resistance to exercise-induced hypoglycemia. Traditionally attributed to a reduction in glucose uptake subsequent to enhanced fat oxidation, this issue has only recently been directly addressed. This paper briefly reviews the evidence for reduced glucose uptake versus enhanced glucose production in the improved hypoglycemic resistance following training. While whole body glucose removal and production may be reduced following training, this has only been demonstrated under exercising conditions in which glycemia demonstrates little deviation from rest. Under exercise conditions where untrained animals demonstrate substantial reductions in blood glucose, training enhanced hypoglycemic resistance has been shown to result entirely from enhanced glucose production via gluconeogenesis. Using the in situ perfused liver preparation, the authors have provided direct evidence for a training enhanced hepatic gluconeogenic capacity. The site of adaptation within the gluconeogenic pathway has now been constrained to below the level of the triose phosphates. Lack of evidence for suppressed skeletal muscle glucose uptake following training, a uniform observation for humans and rats, is also discussed. It is concluded that the improved hepatic gluconeogenic capacity of endurance trained individuals, at least in rats, is critical to their demonstrated resistance to exercise-induced hypoglycemia.

Continuous 24-h nicotinic acid infusion in rats causes FFA rebound and insulin resistance by altering gene expression and basal lipolysis in adipose tissue.

Nicotinic acid (NA) has been used as a lipid drug for five decades. The lipid-lowering effects of NA are attributed to its ability to suppress lipolysis in adipocytes and lower plasma FFA levels. However, plasma FFA levels often rebound during NA treatment, offsetting some of the lipid-lowering effects of NA and/or causing insulin resistance, but the underlying mechanisms are unclear. The present study was designed to determine whether a prolonged, continuous NA infusion in rats produces a FFA rebound and/or insulin resistance. NA infusion rapidly lowered plasma FFA levels (>60%, P < 0.01), and this effect was maintained for ≥5 h. However, when this infusion was extended to 24 h, plasma FFA levels rebounded to the levels of saline-infused control rats. This was not due to a downregulation of NA action, because when the NA infusion was stopped, plasma FFA levels rapidly increased more than twofold (P < 0.01), indicating that basal lipolysis was increased. Microarray analysis revealed many changes in gene expression in adipose tissue, which would contribute to the increase in basal lipolysis. In particular, phosphodiesterase-3B gene expression decreased significantly, which would increase cAMP levels and thus lipolysis. Hyperinsulinemic glucose clamps showed that insulins action on glucose metabolism was improved during 24-h NA infusion but became impaired with increased plasma FFA levels after cessation of NA infusion. In conclusion, a 24-h continuous NA infusion in rats resulted in an FFA rebound, which appeared to be due to altered gene expression and increased basal lipolysis in adipose tissue. In addition, our data support a previous suggestion that insulin resistance develops as a result of FFA rebound during NA treatment. Thus, the present study provides an animal model and potential molecular mechanisms of FFA rebound and insulin resistance, observed in clinical studies with chronic NA treatment.

Metabolic sensors mediate hypoglycemic detection at the portal vein.

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 β-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.

Peripheral and Central Glucose Sensing In Hypoglycemic Detection

Hypoglycemia poses a serious threat to the integrity of the brain, owing to its reliance on blood glucose as a fuel. Protecting against hypoglycemia is an extended network of glucose sensors located within the brain and in the periphery that serve to mediate responses restoring euglycemia, i.e., counterregulatory responses. This review examines the various glucose sensory loci involved in hypoglycemic detection, with a particular emphasis on peripheral glucose sensory loci and their contribution to hypoglycemic counterregulation.

Activation of Hindbrain Neurons Is Mediated by Portal-Mesenteric Vein Glucosensors During Slow-Onset Hypoglycemia

Hypoglycemic detection at the portal-mesenteric vein (PMV) appears mediated by spinal afferents and is critical for the counter-regulatory response (CRR) to slow-onset, but not rapid-onset, hypoglycemia. Since rapid-onset hypoglycemia induces Fos protein expression in discrete brain regions, we hypothesized that denervation of the PMV or lesioning spinal afferents would suppress Fos expression in the dorsal medulla during slow-onset hypoglycemia, revealing a central nervous system reliance on PMV glucosensors. Rats undergoing PMV deafferentation via capsaicin, celiac-superior mesenteric ganglionectomy (CSMG), or total subdiaphragmatic vagotomy (TSV) were exposed to hyperinsulinemic–hypoglycemic clamps where glycemia was lowered slowly over 60–75 min. In response to hypoglycemia, control animals demonstrated a robust CRR along with marked Fos expression in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus. Fos expression was suppressed by 65–92% in capsaicin-treated animals, as was epinephrine (74%), norepinephrine (33%), and glucagon (47%). CSMG also suppressed Fos expression and CRR during slow-onset hypoglycemia, whereas TSV failed to impact either. In contrast, CSMG failed to impact upon Fos expression or the CRR during rapid-onset hypoglycemia. Peripheral glucosensory input from the PMV is therefore required for activation of hindbrain neurons and the full CRR during slow-onset hypoglycemia.