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Effective and safe insulin gene therapy will require regulation of transgenic insulin secretion. We have created a liver-targeted insulin transgene by engineering glucose responsive elements into a hepatic promoter containing an inhibitory insulin response sequence. In this work, we demonstrate application of this transgene for the treatment of diabetes mellitus in vivo, by administering a recombinant adenovirus vector, Ad/(GIRE)3BP-1 2xfur, to rats made diabetic with streptozotocin. We verified hepatic expression of transgenic insulin by RT-PCR, and confirmed glucose responsive stimulation of transgenic insulin secretion in vivo by serum RIA. Following a portal system injection of either Ad/(GIRE)3BP-1 2xfur, or an empty adenoviral vector, animals made diabetic with either low (120 mg/kg), or high (290 mg/kg) dose streptozotocin (STZ) were monitored for changes in body weight, and blood glucose. Without subcutaneous insulin injections, blood glucose values of sham-treated animals (n = 8) remained elevated, and animals failed to gain weight (n = 4), or died (n = 4). In contrast, body weight of Ad/(GIRE)3BP-1 2xfur-treated animals (n = 13) increased, and blood glucose remained at near normal levels from one to 12 weeks. Glucose values <50 mg/dl were infrequently observed, and no Ad/(GIRE)3BP-1 2xfur-treated animal succumbed to hypoglycemia. Treatment with the insulin transgene enabled diabetic animals to reduce blood sugars following a glucose load, and to maintain blood sugar levels during a 10-h fast. Hepatic production of human insulin produced near normal glycemia, and weight gain, without exogenous insulin, and without lethal hypoglycemia. In conclusion, we have demonstrated the feasibility of utilizing transcription to control transgenic insulin production in a rodent model of diabetes mellitus.

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Insulin gene therapy requires that insulin secretion be coupled to metabolic requirements. To this end, we have developed an insulin transgene whose transcription is stimulated by glucose and inhibited by insulin. Glucose- and insulin-sensitive promoters were constructed by inserting glucose-responsive elements (GlREs) from the rat L-pyruvate kinase (L-PK) gene into the insulin-sensitive, liver-specific, rat insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Glucose (5 to 25 mM) stimulated, and insulin (10-10 to 10-7 M) inhibited, luciferase expression driven by these promoters in primary cultured rat hepatocytes. The capacity of transfected hepatocytes to secrete mature, biologically active insulin was demonstrated using a human proinsulin cDNA (2xfur), modified to allow protein processing by endogenous endopeptidase activity. Medium conditioned by insulin-producing hepatocytes contained greater than 300 microU/ml immunoreactive insulin, while denaturing SDS-PAGE of an anti-insulin immunoprecipitate revealed bands with the mobilities of insulin A, and B chains. Biological activity of hepatocyte-produced insulin was demonstrated in a transfection assay, in which medium conditioned by insulin-producing hepatocytes exerted an effect similar to 10-7 M insulin. We then combined the glucose- and insulin-sensitive promoter with the modified human proinsulin cDNA to create a metabolically sensitive insulin transgene ((GlRE)3BP-1 2xfur). In both H4IIE hepatoma cells stably transfected with this construct, and normal rat hepatocytes (GlRE)3BP-1 2xfur-mediated insulin secretion increased in response to stimulation by glucose. Moreover, a capacity to decrease insulin production in response to diminishing glucose exposure was also demonstrated. We conclude that the transcriptional regulation of insulin production using these glucose- and insulin-sensitive constructs meets the requirements for application in a rodent model of insulin gene therapy. Gene Therapy (2000) 7, 205-214.

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Transcription initiation in the insulin-like growth factor-I (IGF-I) gene is complex, involving multiple sites in two exons. While most transcripts are initiated in exon 1 in vivo, critical regulatory mechanisms are difficult to assess in intact animals. To examine the impact of insulin and growth hormone (GH) under more controlled conditions, we have studied the utilization of different exon 1 and exon 2 transcription-initiation sites in normal rat hepatocytes in primary culture. Normal rat hepatocytes were cultured for 48 h in serum-free medium, with insulin at 10(-6) or 10(-11) M, and with or without human GH 200 ng/ml. Relative abundance of IGF-I transcripts was evaluated by the RNase-protection assay, using a probe which permitted identification of initiation in exon 1 (site 1 (-380 bp from the 3' end of exon 1), site 2 (-343 bp), site 3 (-242 bp), sites 1 and 2 spliced, and site 4 (-32 bp)), as well as in exon 2. After normalization of signal intensity to adjust for differences in length of protected probe, the utilization of initiation sites in vitro was remarkably similar to that in vivo: 1, 14, 6, 23, 19 and 37% for sites 1, 2, 3, 1 and 2 spliced, 4 and exon 2 respectively in the cultured hepatocytes, compared with 1, 12, 18, 21, 18 and 40% for these sites in normal liver. Insulin alone increased transcripts initiated from exon 1, site 2 by over 3 times, and both sites 1 and 2 spliced and exon 2 transcripts by about 5 times. GH alone had similar effects, producing a 4-5 times increase in transcripts from these initiation sites. Addition of both insulin and GH had additive effects, increasing transcripts from exon 1, sites 2, 3 and 4 by 4-6 times, and from exon 1, sites 1 and 2 spliced, and exon 2 by over 8 times. Of the total IGF-I mRNA transcripts, 37% were initiated from sites 2 and/or sites 1 and 2 spliced, and 37% from exon 2. Analysis of the relative contribution of individual initiation sites revealed hormone-induced increases which were statistically significant only for exon 2, in the presence of insulin alone and in combination with GH. In conclusion, in cultured hepatocytes, insulin or GH alone produced a coordinated increase in all exon 1 transcripts, and the effect of the combination of insulin and GH was additive for these transcripts. Exon 2 appeared to be more sensitive to insulin alone, and to GH in the presence of insulin, than exon 1. Since utilization of initiation sites in hepatocytes mimics that found in liver, this in vitro system should be useful for examining underlying transcriptional regulatory mechanisms.

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OBJECTIVE: Management of type II diabetes is difficult, particularly in urban populations with limited resources and access to care. To evaluate the effectiveness of structured care delivered by non-physician providers, patients were studied prospectively for 6 months in a municipal hospital diabetes clinic. DESIGN AND METHODS: The population was approximately 90% African American and had median known diabetes duration of approximately 1 year, 54% had incomes below the Federal Poverty Guideline. Primary management was provided by nurse-practitioners and dietitians, and primary outcome measures were hemoglobin A1c (HbA1c), fasting plasma glucose, and changes in body weight. RESULTS: Responses were analyzed in 325 new patients returning for visits at 2, 4, 6, and 12 months; metabolic profiles at presentation were similar to those of subjects who missed intervening visits. Lean patients largely continued on pharmacologic therapy and improved HbA1c from 9.4% to 7.4% at 2 months (P < 0.001), remained stable through 6 months, then rose to 7.9% at 1 year. Obese patients (71%) received dietary instruction. Weaning of pharmacologic therapy was attempted for the first 2 months, resulting in a decline of HbA1c from 9.6% to 8.0% (P < 0.001), with 70% treated with diet alone. In the obese, HbA1c continued to decrease through 6 months (7.7%). Thereafter, providers saw patients at their own discretion and intensified therapy as needed. Although by 1 year, HbA1c had risen to only 8.2%, some patients required reinstitution of pharmacologic therapy; 59% were on diet alone. While 52% lost 4 lb or more (mean 9.3) by 2 months, little additional weight was lost. Interestingly, glycemic control was improved both in those who lost > or = 8.5 lb in the first 2 months (HbA1c 9.6% to 8.1% at 12 months), and in those who gained weight (HbA1c 10.2% to 8.2%). In the obese patients using pharmacologic agents at presentation, 35% were able to discontinue oral agents or insulin by 1 year, with good glycemic control (HbA1c < 8%). For patients who were initially on diet alone, a fasting plasma glucose > 177 mg/dL predicted the need for pharmacologic therapy with 97% certainty. CONCLUSIONS: In urban African American patients, nonpharmacologic management of type II diabetes substantially improves metabolic control; decreases in HbA1c are comparable in those who do and do not lose weight. Therapy managed by nonphysician providers can be an effective cornerstone of diabetes care in this socioeconomically disadvantaged population.

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Although the major endocrine source of insulin-like growth factor-I (IGF-I) is the liver, little is known about IGF-I transcriptional mechanisms in this tissue. To evaluate the role of cis-regulatory elements, rat hepatocytes in primary culture were transfected with DNA constructs containing IGF-I 5'flanking sequences, fused to a luciferase reporter. We demonstrate the presence of a novel promoter approximately 0.5 kb upstream from exon 1 transcription initiation sites, together with a repressor element in this region, and a downstream repressor element which can modulate the activity of both endogenous and heterologous promoters.

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Hepatic expression of insulin-like growth factor binding protein-1 is regulated by insulin and glucocorticoids. To study underlying mechanisms, rat hepatocytes in primary culture were transfected with deletion mutants and heterologous promoter constructs, identifying a 41 bp region of the rat insulin-like growth factor binding protein-1 promoter which is sufficient to mediate regulation by both insulin and glucocorticoids. Half maximal suppression of promoter activity by insulin occurred at a physiologic concentration, 5 x 10(-10) M, and regulation by insulin was dominant in that insulin suppressed promoter activity at all dexamethasone concentrations. Transfection of rat hepatocytes in primary culture should be a useful approach for exploring the regulation of gene expression by insulin.

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Three hundred six patients with adenocarcinoma of the prostate underwent pelvic lymphadenectomy and had Stage D1 (T0-3,N1-2,M0) disease; 171 patients underwent radical retropubic prostatectomy with or without immediate adjuvant therapy (hormonal or radiation or both) or conservative (hormonal or radiation or both) treatment alone (n = 135). Follow-up was one-half to eighteen and one-half years (mean, 5 yrs). Immediate adjuvant orchiectomy significantly (P = 0.01) improved survival (87.4% at 10 years) and nonprogression rates for patients who underwent radical prostatectomy, but not for those who had lymphadenectomy. Overall patient survival was significantly better (P = 0.005) after prostatectomy than lymphadenectomy. Residual disease (n = 43) in patients who underwent prostatectomy and received adjuvant treatment (orchiectomy or radiation or both) did not affect disease outcome. Bilateral pelvic lymphadenectomy and radical prostatectomy with immediate adjuvant orchiectomy provided survival comparable to the expected survival; conservative treatment alone was associated with rapid disease progression and poor survival and significantly (P = 0.02) higher local morbidity.

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