David W. Cooke

Analysis of the human type I insulin-like growth factor receptor promoter region

We isolated genomic fragments containing the 5 region of the human type I insulin-like growth factor receptor gene. A unique transcription start site was identified, defining a 1038 bp 5-untranslated region. No TATA or CCAAT elements were identified in the proximal 480 nucleotides of 5-flanking region. The region surrounding the transcription start site was similar to a recently described initiator sequence. The 5-flanking and 5-untranslated regions were highly GC-rich, with numerous potential Sp1 binding sites. A potential AP-2 binding site was identified in the 5-flanking region and a potential thyroid response element was identified in the 5-untranslated region. The 5 region of the human gene was very similar to that of the rat gene, with conservation of many of the potential regulatory elements.

The transcription factor nuclear factor I mediates repression of the GLUT4 promoter by insulin.

Insulin represses GLUT4 expression in 3T3-L1 adipocytes through an insulin response element located at bases −706 to −676 in the 5′-flanking sequence. Nuclear proteins related to the nuclear factor I (NF1) family of transcription factors bind to this insulin response element. Mutations that disrupt binding of NF1 proteins to the insulin response element impair the insulin response in reporter gene assays. Insulin treatment of 3T3-L1 adipocytes induces a rapid change in the level of phosphorylation of NF1 proteins, providing a potential mechanism for insulin’s ability to regulate gene expression through NF1. Another as yet unidentified protein, not related to NF1, also binds to the GLUT4 insulin response element and is able to mediate partial repression of the GLUT4 promoter in reporter gene assays.

A Sequence Element in the GLUT4 Gene That Mediates Repression by Insulin

Prolonged treatment of 3T3-L1 adipocytes decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of promoter-reporter gene constructs that contained 2900 or 785 base pairs of 5′-flanking region of the murine GLUT4 gene was down-regulated by insulin (p < 0.0005), whereas expression of constructs that contained 641, 469, or 78 base pairs of 5′-flanking region was not. Nuclear extract from 3T3-L1 adipocytes protected the region from −707 to −681 in the GLUT4 5′-flanking region from DNase I digestion. Using an oligonucleotide probe that corresponded to this footprinted region, two major protein-DNA complexes were identified by a gel mobility shift assay. Southwestern analysis identified four protein bands with molecular masses from 38 to 46 kDa that bound to the insulin-responsive region probe. A reporter gene construct in which bases −706 to −676 were deleted was not repressed by insulin treatment, confirming that this sequence is necessary for the repression of the GLUT4 promoter by insulin in 3T3-L1 adipocytes. This sequence does not show homology to previously described insulin response elements and thus represents a distinct mechanism of gene regulation by insulin.

Repression of GLUT4 expression by the endoplasmic reticulum stress response in 3T3-L1 adipocytes.

Expression of GLUT4 is decreased in adipocytes in obesity and type 2 diabetes, contributing to the insulin resistance of these states. Recent investigations suggest a role for activation of the ER stress response in the pathophysiology of type 2 diabetes. We investigated activation of the ER stress response in 3T3-L1 adipocytes. We show that activation of the ER stress response decreased GLUT4 expression at the level of gene transcription. Activation of the ER stress response also increased the expression of CHOP10, an inhibitor of the activity and expression of C/EBPalpha. As expected, activation of the ER stress response decreased expression of C/EBPalpha, an activator of GLUT4 expression, providing a mechanism to account for the repression of GLUT4 by ER stress activation. Our studies identify repression of GLUT4 expression as another potential mechanism for obesity-induced activation of the ER stress response to contribute to the insulin resistance of obesity.

GLUT4 expression in 3T3-L1 adipocytes is repressed by proteasome inhibition, but not by inhibition of calpains

Because of recent studies showing linkage of type 2 diabetes with the calpain 10 gene, we investigated the ability of calpains to regulate GLUT4 expression in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the calpain inhibitor ALLN significantly decreased the mRNA and protein expression of GLUT4. GLUT4 expression was not affected by treatment with the more selective calpain inhibitors PD150606, calpeptin, or a calpastatin peptide. In contrast, treatment with the proteasome inhibitors lactacystin or MG132 repressed GLUT4 mRNA level to 35% (10 microM lactacystin) and 12% (10 microM MG132) of control levels. Therefore, the expression of GLUT4 in 3T3-L1 adipocytes was repressed by proteasome inhibition, but not by inhibition of calpains; the effect of ALLN was due to its ability to inhibit proteasome function, rather than its action to inhibit calpains. Concomitant with the repression of GLUT4 mRNA levels, proteasome inhibition decreased GLUT4 protein levels in 3T3-L1 adipocytes. The decrease in GLUT4 expression occurred at the transcriptional level, as treatment with proteasome inhibitors decreased GLUT4 transcription measured by a nuclear run-on assay. Thus, these data demonstrate a new pathway for the regulation of GLUT4 expression that involves proteasomal degradation of factors that regulate GLUT4 expression.

Mineralocorticoid Disorders and Endocrine Hypertension

The renin-angiotensin-aldosterone system regulates intravascular volume and the serum potassium concentration through its regulation of sodium and potassium handling in the distal nephron of the kidneys. Isolated disorders of this system in children are rare but when present can cause significant morbidity from either deficient or excess mineralocorticoid action. This chapter describes the physiology of mineralocorticoid action followed by the mechanisms responsible for disordered mineralocorticoid action. For each disorder, the appropriate diagnostic evaluation and treatment recommendations are presented.