The effect of serum glucose alterations on selected verbal skills was examined in a group of diabetic men between 18 and 35 yr of age. An artificial insulin infusion system was used to set and maintain glucose concentrations during testing at each of three levels: hypoglycemia (55 mg/dl), euglycemia (110 mg/dl), and hyperglycemia (300 mg/dl). Subjects were used as their own controls, with performance at euglycemia serving as the comparison standard. A double-blind crossover design was employed as described in Holmes et al. (see ref. 14). Results showed significantly disrupted naming or labeling skills at hypoglycemia, with a trend toward poorer performance at hyperglycemia. During hypoglycemia, rate of responding was slowed from 6% to 18%, compared with euglycemic performance, but accuracy was not impaired. In contrast, word recognition skills were not affected by deviations in glucose. These performance effects were not correlated with duration of disease except for one of the five tests administered. This one exception, on the most difficult task, was less notable than the general finding of no relation between disease duration (from 6 mo to 17.5 yr) and test performance. Thus, in addition to considering long-term consequences of blood glucose alterations, clinicians and diabetic patients may wish to consider acute neuropsychological consequences of disrupted euglycemia.

Thyroid-stimulating hormone (TSH) is a glycoprotein comprised of two noncovalently linked subunits, or and p. The structure of TSH from a variety of species has been elucidated, including the amino acid sequence and carbohydrate composition.'Y2 Bovine TSH has been particularly well characterized and its a subunit has a molecular weight of 14,000, of which 11,000 is composed of a protein core of 96 amino acids and 3,000 represents two oligosaccharide units linked to asparagine residues. Bovine TSHP has a molecular weight of 15,000 of which 13,000 is comprised of a protein core of 113 amino acids and 2,000 represents one asparagine-linked oligosaccharide unit. TSH is structurally related to the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well to the placental hormone chorionic gonadotropin (CG). Within a single species the a subunits from each of these glycoprotein hormones are virtually identical, whereas the p subunits are unique and confer immunologic, receptorbinding, and biologic specificity. Attainment of the conformation necessary for hormonal activity is dependent on proper assembly and carbohydrate processing of the TSH subunits, whereas the free subunits are essentially devoid of receptor binding and biologic activity.2 TSH BIOSYNTHESIS AND PROCESSINGThe a and / 3 subunits of TSH are synthesized from separate messenger RNAs encoded by DNA from genes located on separate chromosomes that may differ betweenThe separate chromosomal location of two TSH subunit genes raises interesting questions about how their synthesis is coordinated in various physiological states. Preliminary studies suggest that there are 5' regulatory elements upstream of the DNA coding regions for both of these genes, and these common regulatory elements may ultimately prove to be responsible for coordinate r e g~l a t i o n .~,~ The nucleotide sequence for TSHa and TSHP has confirmed for each subunit the presence of an amino-terminal signal peptide, a sequence of 24 (for or) or 20 (for p) amino acids that is important for the binding of ribosomes containing incomplete polypeptide chains to the rough endoplasmic reticulum. Moreover, the hydrophobic nature of the signal peptide permits insertion of the chains through the lipid bilayer of the membrane and into the lumen of the endoplasmic reticulum. Each signal peptide is cleaved from the growing polypeptide before completion of messenger RNA translation, and these signal peptides are not found in subunits in intact 205

The effects of hypothalamic hypothyroidism vs. primary hypothyroidism on TSH carbohydrate structure were studied in the rat. Adult male rats with bilateral paraventricular nuclear lesions (n = 10), sham lesions (n = 10), and thyroidectomies (n = 6) were studied 2 weeks postoperatively and compared to normal animals without surgery (n = 6). Pituitaries were incubated in medium containing [3H]glucosamine for 24 h. TSH was immunoprecipitated from medium and pituitary sonicates using anti-TSH beta serum, digested with pronase to obtain TSH glycopeptides, desalted, then analyzed by Concanavalin-A (Con-A) chromatography. Compared to sham controls, hypothalamus-lesioned animals contained a greater proportion of secreted TSH glycopeptides that bound weakly to Con-A, indicating a shift from bisecting and/or multiantennary structures in control animals to biantennary and/or truncated hybrid forms in hypothalamus-lesioned animals. In contrast, thyroidectomized animals, compared to normal and lesioned animals, contained a greater proportion of secreted TSH glycopeptides that did not bind to Con-A, indicating a shift from biantennary and/or truncated hybrid forms to bisecting and/or multiantennary forms. The characteristics of the carbohydrate chains on secreted TSH differed markedly in hypothalamic vs. primary hypothyroidism despite equally low thyroid hormone levels in vivo. Thus, in addition to regulating TSH secretion, hypothalamic hormones alter TSH carbohydrate structure, which may affect its bioactivity and MCR.

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.