Histological insights into the pathogenesis of post-Roux-en-Y hyperinsulinaemic hypoglycaemia.

BACKGROUND: ß-cell hyperplasia has been implicated in the aetiology of post Roux-en-Y gastric bypass hyperinsulinaemic hypoglycaemia, but the pathogenesis of this condition is still unclear. CASE REPORT: We report a case of a 52-year-old man with post-Roux-en-Y gastric bypass hyperinsulinaemic hypoglycaemia who underwent distal pancreatectomy to alleviate his symptoms. Pancreatic histopathology showed chronic pancreatitis with a corresponding loss of exocrine tissue and islet retention. Amyloid deposition was found in pancreatic islets. These features are more typically associated with Type 2 diabetes. DISCUSSION: This case highlights the potential multifactorial pathogenesis of symptomatic hypoglycaemia after Roux-en-Y gastric bypass.

Endocrinologic and metabolic complications in the intensive care unit.

Critical illness provides major stresses on all body systems, including those serving important regulatory functions. Endocrinologic and metabolic abnormalities are common on presentation and during hospitalization in the intensive care unit. Some of these abnormalities are the focus of this article. The authors review abnormalities of the adrenal and thyroid glands and in the metabolism of glucose, and include a brief review of abnormalities of sodium and calcium metabolism.

Thyroid disease mediated by molecular defects in cell surface and nuclear receptors.

The proposed mechanisms of RTH are not mutually exclusive. In fact, there is considerable experimental evidence that many if not all of these complex receptor interactions with elements of the transcriptional unit are involved in RTH. Several aspects of RTH remain unclear, in particular on a clinical level. We still do not completely understand the seeming paradox of a tight distribution of receptor mutations and wide variability in phenotypic presentation. The discovery that many of the RTH receptors have defects in corepressor interaction makes it tempting to speculate that the variability in RTH phenotype within kindreds is secondary to differences in corepressor expression. These issues may be better understood as research further proceeds into cofactors and their control of transcription. We also need better tools to determine thyroid status at a peripheral level. Basal metabolic rate, serum measurement of thyroid-responsive gene products, echocardiographic techniques, and other clinical measures have for the most part been unhelpful in determining thyroid status of specific organ systems. Consequently, therapeutic interventions for RTH are directed toward normalizing biochemical indices of thyroid homeostasis, without really knowing whether these efforts correct imbalances within crucial tissues. These studies, and the more widespread investigation of hormone receptor action in general, are moving at a breathtaking pace, and there is a keen interest in applying these principals to understanding the pathophysiologic mechanism of a variety of diseases.

Large scale synthesis of recombinant human thyrotropin using methotrexate amplification: chromatographic, immunological, and biological characterization.

Studies of human TSH (hTSH) structure and function have been limited by difficulties in producing large quantities of recombinant hormone. We describe a system for the stable expression of high levels of recombinant human TSH (rec hTSH) using a mutant form of dihydrofolate reductase (dhfr) as an amplifiable dominant selectable marker. A vector expressing both the hTSH alpha-subunit and the mutant dhfr was cotransfected with a hTSH beta-subunit expression vector into dhfr-deficient cells. Amplification of the transfected sequences by methotrexate selection, followed by cell culture in a hollow fiber perfusion system, yielded rec hTSH production as high as 100,000 microU/ mL. Immunoradiometric assays using five different antibodies revealed no differences in the immunological activities of rec hTSH and pituitary hTSH. Bioactivity was measured in a novel TSH bioassay coupling the generation of cAMP by a transfected hTSH receptor to the cAMP-dependent regulation of a luciferase reporter gene. The ED50 for bovine TSH in this bioassay was 1.4 ng/mL (3.5 x 10(-11) mol/L). The ratio of the ED50 values for rec hTSH and pituitary hTSH was 1.0:1.1 (P = NS), indicating that the two TSHs were of equivalent potency. In conclusion, we have developed techniques for the high level production of rec hTSH that is immunologically and biologically equivalent to pituitary hTSH. The ability to produce large quantities of rec hTSH using standard laboratory techniques should facilitate future studies, such as the development of clinically useful TSH analogs.

Mutations of the human thyrotropin-beta subunit glycosylation site reduce thyrotropin synthesis independent of changes in glycosylation status.

In recent studies, site-directed mutagenesis has been used to alter the tripeptide glycosylation recognition sequences of glycoprotein hormone subunits, thereby affecting their structure and function. However, it is not known whether these effects result from changes in glycosylation status, amino acid sequence, or both. We therefore studied the synthesis of wild-type and mutant recombinant human thyrotropins produced by transient transfection of a human cell line. Mutating the TSH-beta subunit glycosylation recognition sequence, Asn-Thr-Thr (codons 23-25), to either Gln-Thr-Thr or Asn-Thr-Tyr abolished subunit glycosylation, as demonstrated by the inability to incorporate 3H-carbohydrates. However, a third mutation (Asn-Thr-Ser) contained an intact glycosylation recognition sequence site, and was shown to retain glycosylation. The mutations that abolished TSH-beta subunit glycosylation resulted in greater than 90% decreases in TSH synthesis. However, the glycosylation recognition sequence mutant that retained beta subunit glycosylation exhibited a 70% decrease in TSH production. These decreases were not attributable to the intracellular accumulation of TSH or its free beta subunit. We also engineered two TSH-beta subunit mutations that did not alter the glycosylation recognition sequence. A glycine to arginine mutation adjacent to the glycosylation recognition sequence, in a region thought to be critical for heterodimer formation, abolished TSH production. In contrast, shortening the TSH-beta subunit carboxyterminus by six amino acids increased TSH synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

A new point mutation in the 3,5,3'-triiodothyronine-binding domain of the c-erbA beta thyroid hormone receptor is tightly linked to generalized thyroid hormone resistance.

Two different mutations in the c-erbA beta thyroid hormone receptor have recently been reported as genetic abnormalities responsible for the syndrome of generalized thyroid hormone resistance (GTHR). We have now found in a third kindred, D, in which GTHR is inherited as a dominant disease, a new point mutation in the T3-binding domain of c-erbA beta. A guanine to cytosine base substitution at nucleotide position 1305, which altered codon-335 from glutamine (CAG) to histidine (CAC), was found in one allele of 10 affected members and was not found in 6 unaffected members. This C-1305 sequence was not present in 106 random alleles, indicating that it was a mutation in c-erbA beta, and it was tightly linked to GTHR in kindred D, with a maximum logarithm of the odds score of 4.19 at a recombination fraction of 0. The tight linkage result confirms that GTHR maps to the c-erbA beta locus in multiple kindreds. In view of the tight linkage between the C-1305 mutation and GTHR, and that this mutation is a nonconservative alteration in a crucial region of the T3-binding domain, it is probably the genetic defect in kindred D responsible for GTHR. The kindred D receptor appears to result in a different phenotype of tissue resistance compared to the previously reported kindred. A receptor with a mutation in the carboxy-terminus of c-erbA beta.

Analysis of gonadotropin-releasing hormone gene structure in families with familial central precocious puberty and idiopathic hypogonadotropic hypogonadism.

We examined the GnRH gene structure in a family with familial central precocious puberty (eight members, four affected) and a family with idiopathic hypogonadotropic hypogonadism (eight members, three affected) using Southern blot analysis and sequencing of cloned polymerase chain reaction products. Genomic DNA samples were digested with restriction enzymes and hybridized to the human placental GnRH cDNA probe. BamHI digests revealed 6.5- and 2.7-kilobase (kb) bands; BglII, 6.0- and 4.0-kb bands; Ncol, 8.0- and 3.5-kb bands; Pstl, 4.2-kb, 2.8-kb, 1.3-kb and 950-basepair bands; XbaI, 6.5- and 5.0-kb bands. These sizes were the same as those found by this analysis in normal individuals. All family members with familial central precocious puberty or idiopathic hypogonadotropic hypogonadism showed the same size bands, except for one unaffected member of the family with idiopathic hypogonadotropic hypogonadism who had an additional band at 5.5 kb after digestion with NcoI, which is thought to be a rare polymorphism. Sequencing of exon 2 of the GnRH gene from these families, including the exon-intron borders, revealed a polymorphism in the signal sequence of GnRH that predicts an amino acid change from tryptophan (nucleotide sequence: TGG) to serine (TCG) at the -8 position of the GnRH preprohormone. Although this polymorphism did not cosegregate with the clinical disorder in either family, this novel polymorphism may prove useful in the evaluation of linkage to the GnRH gene in other families with pubertal disorders. No other nucleotide sequence abnormality was found in 1.2 kb of the 5' flanking region or the four exons and their splice sites.

A base mutation of the C-erbA beta thyroid hormone receptor in a kindred with generalized thyroid hormone resistance. Molecular heterogeneity in two other kindreds.

Generalized thyroid hormone resistance (GTHR) is a disorder of thyroid hormone action that we have previously shown to be tightly linked to one of the two thyroid hormone receptor genes, c-erbA beta, in a single kindred, A. We now show that in two other kindreds, B and D, with differing phenotypes, there is also linkage between c-erbA beta and GTHR. The combined maximum logarithm of the odds score for all three kindreds at a recombination fraction of 0 was 5.77. In vivo studies had shown a triiodothyronine (T3)-binding affinity abnormality in nuclear receptors of kindred A, and we therefore investigated the defect in c-erbA beta in this kindred by sequencing a major portion of the T3-binding domain in the 3'-region of fibroblast c-erbA beta cDNA and leukocyte c-erbA beta genomic DNA. A base substitution, cytosine to adenine, was found at cDNA position 1643 which altered the proline codon at position 448 to a histidine. By allelic-specific hybridization, this base substitution was found in only one allele of seven affected members, and not found in 10 unaffected members of kindred A, as expected for a dominant disease. Also, this altered base was not found in kindreds B or D, or in 92 random c-erbA beta alleles. These results and the fact that the mutation is predicted to alter the secondary structure of the crucial T3-binding domain of the c-erbA beta receptor suggest this mutation is an excellent candidate for the genetic cause of GTHR in kindred A. Different mutations in the c-erbA beta gene are likely responsible for the variant phenotypes of thyroid hormone resistance in kindreds B and D.

Tight linkage between the syndrome of generalized thyroid hormone resistance and the human c-erbA beta gene.

Multiple cDNAs belonging to the c-erbA gene family encode proteins that bind T3 with high affinity. However, the biological functions of these multiple thyroid hormone receptors have not yet been clarified. Generalized thyroid hormone resistance (GTHR) refers to a human syndrome characterized by tissue refractoriness to the action of thyroid hormones; several studies have suggested quantitative or qualitative defects in T3 binding to nuclear receptors in certain kindreds. To investigate the biological functions of the c-erbA genes, c-erbA alpha and c-erbA beta, we tested the hypothesis that an abnormal c-erbA gene product is present in GTHR by examining these genes in members of one kindred. Restriction enzyme analysis failed to identify an abnormal pattern in affected individuals suggesting no rearrangements or large deletions. However, we demonstrated that the gene conferring the GTHR phenotype is tightly linked to the c-erbA beta locus on chromosome 3. This linkage strongly suggests that the c-erbA beta gene is important in man as a thyroid hormone receptor and identifies a putative c-erbA beta mutant phenotype with central nervous system, pituitary, liver, metabolic, and growth abnormalities.

Animal models of alcoholic neuropathy: morphologic, electrophysiologic, and biochemical findings.

A chronic high alcohol intake was induced in rats through the use of two procedures: the schedule-induced polydipsia technique and the liquid diet technique. Rats consumed 11-12 g of ethanol per kilogram body weight per day for 16 to 18 weeks. Morphologic evidence of a mild distal axonal neuropathy in the ventral caudal nerve was proposed. The red blood cell transketolase levels were normal, indicating that the rats were not deficient in thiamine and suggesting that the axonal degeneration was due to the direct toxic effect of alcohol. Axonal transport studies demonstrated a significant increase in the amount of acetylcholinesterase transported in an orthograde direction in the sciatic nerves of alcohol-exposed rats, and indicated no change in the transport of choline acetyltransferase or in the specific binding of colchicine by neurotubulin.