Glucagon-Like Immunoreactants in Extracts of the Rat Hypothalamus*

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Abstract
Immunohistochemical studies have revealed that materials with glucagon-like immunoreactivity but not genuine glucagon are present in rat hypothalamus (RH). In contrast, both glucagon-like immunoreactants and, presumably, genuine glucagon have been detected in canine brain extracts. We report here the extraction and partial characterization of glucagon-like immunoreactants from RH. Initial extractions of whole brain were made in acid-saline (pH 2.8) in the presence of 1,000 kallikrein international units (KIU)/ml Trasylol (T) or 2 mg/ml benzamidine. Both 30K (C-terminally directed) and K4023 (centrally directed) glucagon antisera showed considerable apparent immunoreactivity, which diluted in parallel with standard porcine glucagon. Gel filtration on Sephadex G-150 demonstrated a peak of 90,000 molecular weight, which, surprisingly, reacted identically with both 30K and K4023. However, these extracts caused marked degradation of [125I]iodoglucagon despite the presence of up to 2,000 KlU/ml T or 10 mg/ml benzamidine, 5 μM pepstatin, 10 mM EDTA, or 1 mM phenylmethylsulfonyl fluoride, regardless of whether extracts were neutralized. The apparent high molecular weight glucagon immunoreactivity in acid-saline extracts of brain is therefore an artifact due to a tracer-degrading activity; this finding brings into question the validity of previous reports on the existence of high molecular weight forms of glucagon. When RH were extracted in acidethanol in the presence of 1,000 KlU/ml T, evaporated, and reconstituted in 2 M acetic acid, no degrading activity or any nonspecific interference was detected. Gel filtration revealed a predominant 12,000 molecular weight peak reactive only with antiserum K4023, small amounts of a 9,000 molecular weight K4023-reactive material, and only traces of a 3,500 molecular weight material reactive with both 30K and K4023 antisera. The same K4023-detectable immunoreactive pattern was also observed in ileal extracts. K4023-detectable glucagon immunoreactivity in RH was unaltered in rats fasted for 7 days and in streptozotocin-diabetic rats, despite markedly reduced plasma K4023 immunoreactivity in the former and increased plasma 30K and K4023 immunoreactivity in the latter. Other brain regions showed negligible glucagon immunoreactivity. In summary, due to tracer-degrading activity in acid-saline extracts, the presence of glucagon-like immunoreactants could only be demonstrated in acid-ethanol extracts of RH. The glucagon-like immunoreactants in RH resemble those found in the gut; their contents in the brain did not change despite induced alterations in their plasma levels.