Presumed apoptosis and reduced arcuate nucleus neuropeptide Y and pro-opiomelanocortin mRNA in non-coma hypoglycemia.

Abstract

Hypoglycemia reduces sympathoadrenal responses to subsequent hypoglycemic bouts by an unknown mechanism. To assess whether such hypoglycemia-associated autonomic failure is due to actual brain damage, male Sprague-Dawley rats underwent 1-h bouts of insulininduced (5 U/kg i.v.) hypoglycemia (1.6-2.8 mmol/l) 1 or 3 times on alternate days. Rats remained alert and were rescued with intravenous glucose at 60-80 min. Plasma epinephrine and corticosterone responses were significantly reduced during the second and third bouts. Brains from these rats were processed by the terminal transferase-mediated deoxyuridine triphosphatebiotin nick end-labeling ( W ith the advent of intensive insulin therapy for type 1 diabetes, there has been an increasing incidence of hypoglycemic episodes (1). Some of these are severe, resulting in coma and subsequent permanent brain damage and/or death (2-5). Other less severe hypoglycemic episodes do not impair consciousness. Regardless of degree, hypoglycemia is associated with a robust counterregulatory response (CRR) (6-9). Low levels of plasma glucose are sensed by selective neurons in the brain (10-14) as well as by glucosensors in the hepatic portal vein (15). Signals from these sensors provoke activation of the sympathoadrenal system with preferential release of epinephrine. In addition, cortisol is released from the adrenal cortex and glucagon from the pancreatic ␣-cells (6-9,16). This response maximizes the mobilization of glucose in the body. In addition to this CRR, subjects develop an awareness of their hypoglycemic state (6,9). Both the CRR and the hypoglycemia awareness can become blunted after only a single bout of hypoglycemia (9,17). Hypoglycemia awareness recovers fairly rapidly but the diminished CRR is often prolonged (6), suggesting that different mechanisms are responsible for the two phenomena. However, the mechanism underlying either of these remains unclear.Here, we present a rat model of the dampened CRR and show that a single bout of moderate hypoglycemia produces highly site-specific apparent apoptotic response in cells of the hypothalamic arcuate nucleus (ARC). This apoptosis is associated with a significant reduction in the expression of both neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA in this same nucleus. RESEARCH DESIGN AND METHODSAnimals and hypoglycemia. Male Sprague-Dawley rats were used at 300-400 g. They were housed at 22-23°C on a 12-h light-dark schedule and fed Purina rat chow (#5001) (PMI Nutrition International, Brentwood, MO) ad libitum. One group of rats (n = 12) had right atrial silastic catheters implanted via the right jugular vein under ketamine (45 mg/kg)/xylazine (9 mg/kg i.p.) anesthesia with buprenorphine (0.2 mg/kg) analgesia. Catheters were filled with a polyvinylpyrrolidone/heparin mix, which was replaced daily. The rats were allowed 4-5 days to recover and then were fasted overnight. Additional rats without venous catheters were also used (n = 34) and fasted overnight before testing. Between 0800 and 1000, a blood sample (...

Cortical Fluoro-Jade Staining and Blunted Adrenomedullary Response to Hypoglycemia after Noncoma Hypoglycemia in Rats

“…Surprisingly, we did not observe FJ staining in the arcuate nucleus of the hypothalamus, where we previously identified TUNEL staining and altered gene expression after hypoglycemic insult (Tkacs et al, 2000). The reversibility of hypoglycemic DNA damage in neurons has been shown in vitro, as striatal neurons exposed to aglycemia for 24, 48, or 72 h show increased TUNEL staining that reverses when the neurons are returned to glucosecontaining culture medium (McDermott et al, 2003).…”

“…At 12, 24, or 48 h after hypoglycemic injury, no neurons appeared to contain immunoreactivity for activated caspase-3 or for the calpain or caspase cleavage products of spectrin. Finally, at 12, 24, or 48 h after hypoglycemia in rats (n ¼ 12 HYPO, 6 CTRL) treated with a similar protocol, there were no cells positive for activated caspase-3 immunoreactivity in sections taken through the hypothalamus where we previously reported TUNEL-positive cells at 48 h (but not 24 h) after hypoglycemia (Tkacs et al, 2000). Figure 4 Total numbers of FJ þ cells over time after hypoglycemic insult.…”

“…The first experiment we report here aimed to confirm and extend our previous finding that a single episode of moderate hypoglycemia is sufficient to reduce counterregulatory hormone responses to subsequent hypoglycemia (Tkacs et al, 2000). The time points and experimental manipulations were based on our prior study with the following modifications: epinephrine, norepinephrine, glucagon, and corticosterone responses to equivalent mild hypoglycemia were compared between rats studied 48 h after a single episode of moderate hypoglycemia and control, pair-fasted rats.…”

“…We previously reported that three episodes of moderate hypoglycemia (30 to 35 mg/dL) delivered at 48 h intervals resulted in decreased epinephrine responses. Rats killed after one to three hypoglycemic episodes also had positive TUNEL staining in cells of the arcuate nucleus, indicating DNA damage, and reduced mRNA for arcuate neuropeptide Y (NPY) and proopiomelanocortin (POMC) (Tkacs et al, 2000).…”

Hypoglycaemia unawareness and the brain

“…The glucagon and catecholamine responses to localised VMH administration of 2 deoxyglucose are diminished in recurrently hypoglycaemic rats, just as the response to hypoglycaemia per se would be expected to be, suggesting an adaptation of glucose processing inside the cell membrane [96]. One concern is the possibility of apoptosis of glucose sensing neurones, in response to hypoglycaemia [97], although earlier animal studies would suggest that hypoglycaemia has to be very severe and/or prolonged to induce irreversible cellular changes. Another implicates the hypothalamic activation of the cortisol pathway during hypoglycaemia in the subsequent suppression of the responses to subsequent hypoglycaemia [98].…”