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Objective: Although insulin is the most effective diabetes medication for lowering blood glucose, how insulin is used in clinical practice and how well patients respond to insulin therapy over the course of several years has not been documented. Our objective was to describe glycemic control, side-effects and dose titration over 7 years among persons starting insulin in a health plan that has long used a treatment algorithm similar to the current American Diabetes Association/European Association for the Study of Diabetes (ADA-EASD) algorithm for the management of hyperglycemia., Research Design and Methods: Patients (n = 2417) who initiated insulin therapy between 1 January 1999 and 31 December 2004 were followed for a mean of 49.5 months until 30 June 2007, death, or health plan termination. Mean hemoglobin A1C, number of units of insulin purchased and body weight were assessed on a quarterly basis. The proportion experiencing edema or hypoglycemia was assessed annually., Results: Mean population A1C declined from 9.3 to 7.8% following insulin initiation and remained at that level for 7 years. However, A1C remained above 8% for 40% of patients, half of whom remained above 9.0%. The mean individual coefficient of variation in A1C was 0.12 (inter-quartile range 0.072-0.143). Mean daily insulin dosage started at 55 units and increased to approximately 100 units. Patients gained a mean of 6 lb (2.7 kg) during the first year then gained weight more gradually thereafter. Physicians diagnosed edema in 8-9% of patients annually. Hypoglycemia occurred in fewer than 2% of patients in any given year, with no cases requiring hospitalization., Conclusions: Insulin lowered mean A1C by about 1.5 percentage points to stable levels, but this required ongoing dosage increases. Nevertheless, many patients remained in poor control. Insulin is effective when used per ADA-EASD guidelines but health plans wishing to optimize diabetes care may need to intensify insulin therapy or consider the use of adjunct therapies in the years after initiation. This study was limited by its observational descriptive design, and its reliance on insulin purchases rather than actual consumption.

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the Caenorhabditis elegans protein DAF-16 binds the IGFBP-1 small middle dotIRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1.IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1.IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.

In primary hepatocytes, overexpression of an insulin response element-A binding protein (IRE-ABP), a member of the SRY family of high-mobility group (HMG) proteins, inhibits CCAAT/enhancer-binding protein alpha (C/EBPalpha)-mediated activation of the female-specific cytochrome P450 2C12 (CYP2C12) gene, but not the male-specific cytochrome P450 2C11 (CYP2C11) gene. IRE-ABP and C/EBPalpha have overlapping specificity for the C/EBPalpha target site in the CYP2C12 promoter and compete for binding to CYP2C12 DNA in vitro. In contrast, IRE-ABP and C/EBPalpha bind distinct sequences in the CYP2C11 promoter. A single amino acid substitution in the HMG domain of IRE-ABP impairs its ability to bind DNA and to inhibit the effect of C/EBPalpha on CYP2C12 gene expression. Therefore, the ability of IRE-ABP to inhibit C/EBPalpha-stimulated CYP2C12 gene expression requires a functional DNA-binding domain. Taken together, our findings suggest that SRY-like proteins can bind to a subset of sequences recognized by the C/EBP family of DNA-binding proteins and modulate gene transcription in a context-specific manner.

Multiple elements in the upstream region of the GAPDH gene play a role in mediating the acute and chronic effect of insulin on GAPDH gene expression. The complexity of this regulation provides many layers of control. In differentiated tissues, the transcriptional response to insulin results from the additive effects of g/TRE, IRE-A and IRE-B. The gTRE may interact with newly synthesized c-fos/c-jun heterodimer to activate GAPDH gene transcription. Studies are underway to determine whether protein synthesis inhibitors affect the regulation of GAPDH. Because there are several elements that mediate the effect, it will be difficult to determine the significance of these findings until each cis-acting factor and its binding protein can be studied in isolation. IRE-A and IRE-B act together to promote a 5- to 8-fold insulin effect on HGAPDH-CAT in H35 hepatoma cells and a 3-fold effect in 3T3 adipocytes. We have succeeded in detecting an insulin-sensitive DNA-binding protein referred to as IREA-BP with an element -480 to -435. Insulin treatment of differentiated 3T3 adipocytes for 1 hr results in a 4-fold increase in the amount of this binding protein, as estimated by the amount of 32P-labelled oligonucleotide retarded on non-denaturing PAGE (11). The effect of insulin on IRP-B is comparable. Furthermore, IREA-BP is induced during the process of fasting and refeeding rats, an important in vivo correlate with our tissue culture models (11). These observations imply that the binding proteins IREA-BP and IRP-B are essential components in the signal transduction pathway of insulin action on GAPDH gene expression in metabolically active tissues such as fat and liver. Differentiation-dependence and tissue-specificity are achieved through multiple regulatory elements and involve pre- and post-translational regulation of multiple transcription factors. IREA-BP is present in preadipocytes but activity in highly induced upon differentiation of preadipocytes to adipocytes. The IRE-B (-408 to -269) DNA binding protein is not detected in 3T3 preadipocytes. A gC/EBP like-protein takes part in the formation of this complex which may explain the inductive effect of differentiation on binding. Finally, footprint and cotransfection studies indicate that the differentiation-dependent protein C/EBP also regulates GAPDH gene transcription through a motif located within one hundred nucleotides of the promoter. We have begun to clone the IRE-A and IRE-B DNA binding proteins. An IRE-A binding protein that footprints the 3' domain of the IRE-A has been cloned.(ABSTRACT TRUNCATED AT 400 WORDS)

THE upstream region of the human glyceraldehyde-3-phosphate dehydrogenase gene contains an insulin-response element (IRE-A) responsible for insulin-dependent transcription of the gene. The open reading frame of a rat complementary DNA encoding a protein (IRE-ABP) that binds to this sequence contains an HMG box motif that is 67% identical to the mouse candidate gene for the testis-determining factor SRY, and 98% identical to the mouse SRY-like gene, a4. Here we report that IRE-ABP and SRY bind to IRE-A DNA with comparable specificity in a DNase-I footprinting assay. Two females with sex reversal were found to have a single amino-acid substitution in the HMG box domain of SRY at position 3 and 7, respectively. SRY derivatives containing corresponding mutations do not make contact with IRE-A DNA. These results are direct evidence that mouse SRY-like proteins are sequence-specific DNA-binding proteins and identify two amino acids critical to this interaction. Moreover, IRE-A is a candidate SRY-response element.