Laurence A. Gavin

The Mechanism of Impaired T3 Production from T4 in Diabetes

Uncontrolled diabetes in man is associated with low serum T3 values and impaired production of T3 from T4. Because the thiol-dependent enzyme T4-5′-deiodinase catalyzes T4-deiodination to T3, the present study was conducted to determine whether these alterations in iodothyronine metabolism consequent to diabetes were due to a reduction in the tissue content of active enzyme or thiol cofactor availability. T4-5′- deiodinase activity and nonprotein sulfhydryl (NP-SH) groups were examined in liver homogenate preparations from groups of rats (T4-treated) diabetic (streptozotocin, 100 mg/kg i.p.) for 48-96 h and compared with controls. The mean hepatic T4-5′-deiodinase activity in the control group was 4.8 ± 0.4 pmol/min/100 mg protein (mean ± SEM). In each of the diabetic groups (48, 72, and 96 h), the mean enzyme activity was significantly less (P < 0.005) than the control mean. Homogenate enrichment with the thiol reagent dithiothreitol (DTT) (5 mM) failed to reverse this diabetic effect. Moreover, although the hepatic content of NP-SH groups was significantly less than the controls after 48 and 72 h of hyperglycemia, it had spontaneously reverted to normal by 96 h. Thus, the impaired enzyme activity could not be attributed to a deficiency of thiol cofactors. The dissociation between enzyme activity and tissue sulfhydryl groups was further illustrated by the response to insulin treatment. Insulin was given by continuous s.c. infusion to 48-h diabetic rats for 48-96 h. Although insulin therapy normalized both enzyme activity and the hepatic thiol content, the temporal profile of each response was different. Insulin therapy rapidly (48 h) corrected the hepatic content of NP-SH groups, whereas the normalization of T4-5′-deiodinase activity required 72-96 h of insulin treatment. There was a positive correlation (r = + 0.69, P < 0.025) between the serum T3 values and hepatic T4-5′-deiodinase activity. The mean serum T3 concentration was significantly reduced (P < 0.001) by the diabetes after 48 h and remained low at 72 and 96 h. Insulin treatment reversed this defect but, as in the case of enzyme activity, the normalization was delayed until 72-96 h of insulin therapy. These results indicate that (1) diabetes reduces serum T3 levels and hepatic T4-5′-deiodinase activity in T4-treated rats, (2) T4-5′-deiodinase is probably the major regulator of serum T3 levels under these conditions, and (3) the reduced T4 -5′-deiodinase activity is independent of changes in the hepatic content of NPSH groups and probably reflects a reduction in the hepatic content of active enzyme.

Conversion of thyroxine to 3,3′,5′-triiodothyronine (reverse-T3) by a soluble enzyme system of rat liver

Abstract The reaction by which thyroxine (T 4 ) undergoes monodeiodination in the nonphenolic ring to form 3,3′,5′-triiodothyronine (reverse-T 3 ) has been studied in rat liver. In whole homogenate the conversion of T 4 to rT 3 is obscured by rapid degradation of the product, whereas in cytosol accumulation of rT 3 is linear for 60 min of incubation. In the cytosol system, the rate of rT 3 formation is maximal at pH 8.2, is thiol-dependent, is inactivated by heat, but is not inhibited by anaerobiosis or absence of light. Propylthiouracil is a potent inhibitor of the reaction. The higher pH-optimum of the rT 3 -forming system compared to that of T 4 -to-T 3 conversion indicates that the former reaction is mediated by an enzyme which is distinct from that controlling 3,5,3′-triiodothyronine (T 3 ) formation.

Modulation of Adipose Lipoprotein Lipase by Thyroid Hormone and Diabetes: The Significance of the Low T3 State

The present study was performed to assess the potential relationship between the low T3 syndrome and hypothyroidism. Comparative studies were performed on the relative effects of diabetes and insulin on heparinreleasable adipose lipoprotein lipase (LPL) in the intact and hypothyroid rat. Hypothyroidism for 10 days (Tx) significantly increased adipose LPL activity (5.8 ± 0.2 μeq/g/h) compared with the activity (3.6 ± 0.4 μeq/g/h) in the normal group. Diabetes for 72 h (streptozocin, STZ, 10 mg/100 g body wt, i.p.) significantly reduced (P < 0.005) adipose LPL activity in the Tx model. However, despite the suppressant effect of diabetes (43 ± 11%), the enzyme activity remained equivalent to the normal group. Insulin stimulated adipose LPL in the Tx-diabetic group. The enzyme demonstrated a synergistic response to insulin and hypothyroidism. Subsequent studies were performed in the intact diabetic rat, a low T3 state. Adipose LPL activity was reduced to a similar degree by diabetes (79 ± 2%) irrespective of the serum T3 concentration. Furthermore, the magnitude of the adipose LPL stimulation by insulin was not modulated by the endogenous serum T3. However, co-treatment of the diabetic group with T3 and insulin blunted the adipose LPL response to insulin. These various modulations in adipose LPL activity were associated with significant but opposite changes in serum triglyceride levels in both the hypothyroid and intact rat. These studies demonstrate that hypothyroidism counteracts the suppressant effect of diabetes on heparin-releasable rat adipose LPL activity and magnifies the enzyme response to insulin. The synergistic effect of hypothyroidism and insulin on adipose LPL activity suggests that the enzyme responds through different mechanisms. In contrast, the low T3 state associated with diabetes did not influence the adipose LPL response to diabetes or insulin therapy. Thus, the low T3 state in the rat does not reflect hypothyroidism. The low T3 state may, however, have a permissive role as it facilitated the adipose LPL response to insulin in the diabetic rats. Therefore, T3 therapy is contraindicated under these condition.

Molecular Cloning and Characterization of c‐erb‐A mRNA Species in Mouse Neuroblastoma Cells

The mouse neuroblastoma cell line is an excellent model in which to study thyroid hormone action and metabolism, particularly in neural tissue. We therefore undertook the molecular cloning and characterization in these cells of putative thyroid hormone receptors related to c‐erb‐A. Since rat brain tissue contains multiple cell types, and because of possible subtle differences between species (mouse and rat), we therefore screened a new cDNA library constructed from mouse neuroblastoma cell mRNA with synthetic oligonucleotide probes based on the published nucleotide sequence of the c‐erb‐A gene in whole rat brain. Despite the fact that this rat brain cDNA sequence is now recognized to represent a c‐erb‐A α 1 form, the cDNA clones that we isolated were all members of the newly‐recognized c‐erb‐A α 2 form. This identification was made on the basis of nucleotide sequence divergence downstream of the nucleotide corresponding to amino‐acid residue 370 in the predicted coding region. The two longest mouse neuroblastoma cDNA clones, clone 29 (1796 bp) and clone 32 (1410 bp), were 93% to 94% homologous with the c‐erb‐A α 2 and c‐erb‐A α 1 forms in their DNA binding and thyroid hormone binding domains (up to amino‐acid residue 370 in the latter). Both clones 29 and 32 were incomplete in that they terminated at their 3’ends at an internal Eco R1 site. Fortunately this 120 bp (40 derived amino‐acid) truncation was downstream of the reported thyroid hormone binding domain. The 5’untranslated end of clone 29 (446 bp) was of interest because a region of its nucleotide sequence (279 bp) revealed a high degree of homology (87%) with rat brain c‐erb‐A α 1. This highly conserved region in clone 29 appeared to be important in the regulation of translation because only clone 32, in which this region was truncated, efficiently translated protein in a cell‐free system. The protein product of clone 32 did not bind thyroid hormone. Northern blot analysis of mouse neuroblastoma mRNA with site‐specific synthetic oligonucleotides revealed that the c‐erb‐A α 2 species was dominant (major band of 2.4 kb), with a lesser amount of the c‐erb‐A α1 species (major band of 1.8 kb).