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L S Phillips

Publications and source records attributed to L S Phillips.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of core sequences involved in metabolism-dependent nuclear protein binding to the rat insulin-like growth factor I gene.

In the liver, most insulin-like growth factor I (IGF-I) transcripts originate in exon 1, where important cis-regulatory regions are located downstream from the major transcription initiation sites. Within these regions, we have attempted to identify sequences which are involved in the decrease in IGF-I gene transcription associated with diabetes mellitus. The function of different genomic templates was assessed by in vitro transcription, which revealed a consistent 50-80% decrease in the activity of nuclear extracts from streptozotocin-diabetic as compared with normal rats. The disparity in transcriptional activity between normal and diabetic nuclear extracts was reduced with templates containing 11-bp mutations within DNase I protected regions III or V (+42 and +129 bp, respectively, from the major transcription initiation site), but a mutation between regions IV and V had little effect. Within region III, gel mobility shift analysis and methylation interference studies indicated that DNA-protein interactions involve a GCGC core sequence. In region V, gel mobility shift studies and uracil interference analysis revealed interactions involving a TTAT core. While gel mobility shift analysis and transient transfection studies indicate that the GCGC core sequence in region III recognizes C/EBP, the AT-rich sequence in region V is likely to recognize a protein with homeodomain characteristics. Identification of the nuclear factor(s) interacting with regions III and V, downstream from exon 1 initiation sites, will be important for understanding the mechanism of reduced IGF-I gene transcription due to diabetes mellitus.

Animals↗
PubMed1999

Diabetes in urban African-Americans. XV. Identification of barriers to provider adherence to management protocols.

OBJECTIVE: To determine whether health care providers appropriately identify patients with poor glycemic control and to investigate reasons why providers may fail to intensify therapy in these patients. RESEARCH DESIGN AND METHODS: Our management protocol calls for providers to advance diabetes therapy in patients with fasting plasma glucose levels > 7.8 mmol/l or random plasma glucose levels > 10.0 mmol/l. During a 3-month period, providers completed a questionnaire at the end of individual patient visits by asking whether the patient was well controlled and whether therapy was advanced. If therapy was not advanced in patients perceived to have poor control, providers were asked to provide a justification. RESULTS: Providers appropriately identified 88% of well-controlled patients and 94% of patients with poor glycemic control. Out of 1,144 patient visits, control was reported to be good in 508 and poor in 636. In these 636 visits, therapy was advanced in 490 but not in 146 visits. The dominant reasons for failure to intensify therapy were the perception by the provider that control was improving (34%) or the belief that the patient was not compliant with diet or medications (25%). Less common reasons included acute illness, patient refusal, and recurrent hypoglycemia. Based on fasting glucose levels, protocol adherence was 55% before the questionnaire, 64% during the questionnaire (P = 0.006), and 63% afterwards. CONCLUSIONS: Providers in a specialty diabetes clinic appropriately classified patients according to glycemic control and tended to intensify therapy when indicated in most poorly controlled patients. Provider self-survey of behavior and decision making may be an effective strategy to improve adherence to management protocols.

Black or African American↗
PubMed1999

What do internal medicine residents need to enhance their diabetes care?

OBJECTIVE: To identify areas that should be targeted for improvement in care, we examined internal medicine resident practice patterns and beliefs regarding diabetes in a large urban hospital outpatient clinic. RESEARCH DESIGN AND METHODS: Internal medicine residents were surveyed to assess the frequency at which they performed key diabetes quality of care indicators. Responses were compared with recorded performance derived from chart and laboratory database reviews. Resident attitudes about diabetes were determined using the Diabetes Attitude Survey for Practitioners. Finally, an eight-item scale was used to assess barriers to diabetes care. RESULTS: Both self-described and recorded performance of recommended diabetes services short of national recommendations. For yearly eye examinations and lipid screening, recorded performance levels were similar to trainees' reports. However, documented inquiries about patient self-monitoring of blood glucose, performance of foot examinations, and urine protein screening were lower than trainees' reports. Some 49% of the residents selected a target HbA1c of 6.6-7.5% as an attainable goal, yet half of the patients using oral agents or insulin had HbA1c values > 8.0%. No differences in self-described or recorded performance were found by year of training. Most residents did not perceive themselves to need additional training related to diabetes care, and residents were generally neutral about patient autonomy. Patient nonadherence and time constraints within the clinic were most often cited as barriers to care. CONCLUSIONS: The study identifies several areas that require improvement in resident care of diabetes in the ambulatory setting. Because experience during training contributes to future practice patterns, developing a program that teaches trainees how to implement diabetes practice guidelines and methods to achieve optimal glycemic control may be key to future improvements in the quality of diabetes care.

Adult↗
PubMed1999

Diabetes in urban African-Americans. XVII. Availability of rapid HbA1c measurements enhances clinical decision-making.

OBJECTIVE: To assess the impact of rapid-turnaround HbA1c results on providers' clinical decision-making and on follow-up HbA1c levels. RESEARCH DESIGN AND METHODS: The research design was a randomized clinical trial in which rapid HbA1c results were made available to providers on even days of the month (rapid, n = 575), but delayed by 24 h on odd days (conventional, n = 563). Adjustment of therapy for patients with type 2 diabetes was considered appropriate if therapy was intensified for HbA1c values >7% or not intensified for HbA1c values < or =7%. A post-hoc analysis was also performed using patients (n = 574) who returned for follow-up 2-7 months later to ascertain the effect of rapid HbA1c availability on subsequent glycemic control. RESULTS: Rapid HbA1c availability resulted in more appropriate management compared with conventional HbA1c availability (79 vs. 71%, P = 0.003). This difference was due mainly to less frequent intensification when HbA1c levels were < or =7% (10 vs. 22%, P < 0.0001) and slightly to more frequent intensification for patients with HbA1c values >7% (67 vs. 63%, P = 0.33). For both groups, intensification was greatest for patients on insulin (51%) compared with patients on oral agents (35%) and diet alone (14%) (P < 0.0001). Regression analysis confirmed that providers receiving conventional HbA1c results were more likely to intensify therapy in patients who already had HbA1c levels < or =7%. Over 2-7 months of follow-up, HbA1c rose more in patients with conventional HbA1c results compared with rapid results (0.8 vs. 0.4%, P = 0.02). In patients with initial HbA1c >7%, rapid HbA1c results had a favorable impact on follow-up HbA1c independent of the decision to intensify therapy (P = 0.03). CONCLUSIONS: Availability of rapid HbA1c determinations appears to facilitate diabetes management. The more favorable follow-up HbA1c profile in the rapid HbA1c group occurs independently of the decision to intensify therapy, suggesting the involvement of other factors such as enhanced provider and/or patient motivation.

Black People↗
PubMed1999

Diabetes in urban African-Americans. XVI. Overcoming clinical inertia improves glycemic control in patients with type 2 diabetes.

OBJECTIVE: Diabetes care can be limited by clinical inertia-failure of the provider to intensify therapy when glucose levels are high. Although disease management programs have been proposed as a means to improve diabetes care, there are few studies examining their effectiveness in patient populations that have traditionally been underserved. We examined the impact of our management program in the Grady Diabetes Unit, which provides care primarily to urban African-American patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: We assessed glycemic outcomes in patients with type 2 diabetes who had an intake evaluation between 1992 and 1996 and who were identified on the basis of compliance with keeping the recommended number of return visits. For 698 patients, we analyzed changes in HbA1c values between baseline and follow-up visits at 6 and 12 months, and the proportion of patients achieving a target value of < or =7.0% at 12 months. Since a greater emphasis on therapeutic intensification began in 1995, we also compared HbA1c values and clinical management in 1995-1996 with that of 1992-1994. RESULTS: HbA1c averaged 9.3% on presentation. After 12 months of care, HbA1c values averaged 8.2, 8.4, 8.5, 7.7, and 7.3% for the 1992-1996 cohorts, respectively, and were significantly lower compared with values on presentation (P < 0.0025); the average fall in HbA1c was 1.4%. The percentage of patients achieving a target HbA1c < or =7.0% improved progressively from 1993 to 1996, with 57% of the patients attaining this goal in 1996. Mean HbA1c after 12 months was 7.6% in 1995-1996, significantly improved over the level of 8.4% in 1992-1994 (P < 0.0001). HbA1c levels after 12 months of care were lower in 1995-1996 versus 1992-1994, whether patients were managed with diet alone, oral agents, or insulin (P < 0.02). Improved HbA1c in 1995-1996 versus 1992-1994 was associated with increased use of pharmacologic therapy CONCLUSIONS: Structured programs can improve glycemic control in urban African-Americans with diabetes. Self-examination of performance focused on overcoming clinical inertia is essential to progressive upgrading of care.

Black People↗
PubMed1999

Insulin replacement therapy for the rat model of streptozotocin-induced diabetes mellitus.

OBJECTIVE: This study was conducted to compare various strategies for insulin replacement therapy in the streptozotocin-induced diabetic rat model. METHODS: Control and diabetic Sprague Dawley rats were fed ad libitum, blood glucose concentration was measured twice daily, and outcome was assessed over the final 5 days of a 10-day treatment period, with adjustment of insulin dosage toward the goal of normal glucose values. RESULTS: All insulin regimens induced weight gain at least comparable to that of controls, but glucose regulation differed. It was not possible to normalize glucose values by use of protamine zinc insulin (PZI) or Ultralente insulin given once daily. In contrast, PZI and neutral protamine Hagedorn (NPH) insulin given twice daily provided glucose values comparable to those in controls, whereas glucose values were modestly higher in response to a 70% human insulin isophane suspension and 30% soluble human insulin solution (70/ 30 insulin) given twice daily. Attempted normalization of glucose values was limited by hypoglycemia, which was most common after administration of PZI once daily, and least common after 70/30 insulin given twice daily. Dosage requirements for Ultralente insulin were four- to fivefold higher than those for all other insulins. CONCLUSION: In streptozotocin-diabetic rats, normal weight gain can be achieved by treatment with PZI insulin once daily, but attainment of near-normal glucose values requires administration of PZI, NPH, or 70/ 30 insulin twice daily. Ultralente insulin may have reduced bioeffectiveness in this animal model.

Animals↗
PubMed1998

Diabetes in urban African Americans. XII. Anthropometry for assessing municipal hospital outpatients recently diagnosed with type 2 diabetes.

OBJECTIVE: Abdominal obesity is associated with insulin resistance and cardiovascular risk factors, but there has been little information published to advance the use of abdominal anthropometry in the care of diabetic patients. RESEARCH METHODS AND PROCEDURES: A cross-sectional survey of municipal hospital outpatients recently diagnosed with type 2 diabetes (73 men and 142 women of whom 89% were African Americans). Age-adjusted linear regression was used to compare the supine sagittal abdominal diameter (SAD), supine waist circumference, four anthropometric ratios, and the body mass index (kg/m2) for their ability to predict serum fasting C-peptide and lipid levels. RESULTS: The best predictor of log-transformed C-peptide was SAD/height (p<0.0001 for men; p=0.0003 for women). SAD/thigh circumference was the best predictor of log-transformed triglycerides for men (p=0.002) and of total cholesterol/HDL cholesterol for women (p=0.043). The body mass index was less able to predict C-peptide, HDL cholesterol and total cholesterol/HDL cholesterol than was SAD/height or SAD/thigh circumference or waist circumference/height. DISCUSSION: Anthropometric indices of abdominal obesity appear to be correlated with insulin production and lipid risk factors among municipal-hospital, type 2 diabetic patients much as they are in other studied populations. Since anthropometric data are inexpensively obtained and immediately available to the practitioner, their utility for preliminary clinical assessment deserves to be tested in prospective outcome studies.

Abdomen↗
PubMed1998

Molecular regulation of insulin-like growth factor-I and its principal binding protein, IGFBP-3.

The insulin-like growth factors (IGFs) have diverse anabolic cellular functions, and structure similar to that of proinsulin. The distribution of IGFs and their receptors in a wide variety of organs and tissues enables the IGFs to exert endocrine, paracrine, and autocrine effects on cell proliferation and differentiation, caloric storage, and skeletal elongation. IGF-I exhibits particular metabolic responsiveness, and circulating IGF-I originates predominantly in the liver. Hepatic IGF-I production is controlled at the level of gene transcription, and transcripts are initiated largely in exon 1. Hepatic IGF-I gene transcription is reduced in conditions of protein malnutrition and diabetes mellitus, and our laboratory has used in vitro transcription to study mechanisms related to diabetes. We find that the presence of sequences downstream from the major transcription initiation sites in exon 1 is necessary for the diabetes-induced decrease in IGF-I transcription. Six nuclear factor binding sites have been identified within the exon 1 downstream region, and footprint sites III and V appear to be necessary for metabolic regulation; region V probes exhibit a decrease in nuclear factor binding with hepatic nuclear extracts from diabetic animals. IGFs in biological fluids are associated with IGF binding proteins, and IGFs circulate as a 150-kDa complex that consists of an IGF, an IGFBP-3, and an acid-labile subunit. Circulating IGFBP-3 originates mainly in hepatic nonparenchymal cells, where IGF-I increases IGFBP-3 mRNA stability, but insulin increases IGFBP-3 gene transcription. Regulation of IGFBP-3 gene transcription by insulin appears to be mediated by an insulin-responsive element, which recognizes insulin-responsive nuclear factors in both gel mobility shift assays and southwestern blots. Studies of mechanisms underlying the modulation of IGF-I and IGFBP-3 gene transcription, and identification of critical nuclear proteins involved, should lead to new understanding of the role and regulation of these important growth factors in diabetes mellitus and other metabolic disorders.

Animals↗
PubMed1998

Diabetes in urban African-Americans. VI. Utility of fasting or random glucose in identifying poor glycemic control.

OBJECTIVE: African-Americans have an increased prevalence of both diabetes and diabetes complications, creating an imperative for improved metabolic control. Because American Diabetes Association guidelines recommend that action be taken when HbA1c is > 8.0%, but access to rapid-turnaround HbA1c assays remains limited, we tested the utility of fasting and random plasma glucose cutoffs as indicators of HbA1c > 8.0%. RESEARCH DESIGN AND METHODS: Using receiver operating characteristics (ROC) analysis, we evaluated the sensitivity, specificity, and predictive value of fasting and random plasma glucose measurements in identifying an HbA1c > 8.0% (fasting n = 974, random n = 552). The population studied was predominantly African-American, middle-aged, and non-insulin-dependent. RESULTS: Fasting plasma glucose was a significant indicator of HbA1c > 8.0%, both in the whole group and in subgroups for diet, sulfonylureas, and insulin; the corresponding areas under the ROC curve were 0.87, 0.90, 0.87, and 0.84, respectively (all P < 0.0001). A fasting plasma glucose cutoff of > 9.2 mmol/l (165 mg/dl) provided a sensitivity of 80% and a specificity of 83% for the whole group and a 77% positive predictive value. Random plasma glucose was also a good indicator of HbA1c > 8.0%, both in the whole group and in subgroups for diet, sulfonylureas, and insulin; the corresponding areas under the ROC curve were 0.85, 0.91, 0.85, and 0.77, respectively (all P < 0.0001). A cutoff > 9.8 mmol/l (177 mg/dl) provided a sensitivity of 78% and a specificity of 77% for the whole group and a 78% positive predictive value. Overall, a plasma glucose > 11.1 mmol/l (200 mg/dl) identified an HbA1c > 8.0% with a predictive value of approximately 90% if done while fasting and a predictive value of approximately 80-85% if random. The utility of both fasting and random plasma glucose cutoffs was subsequently confirmed in a prospective study of another 2,309 and 1,396 patients, respectively. CONCLUSIONS: Although glucose levels cannot replace HbA1c determinations, measurement of fasting or random plasma glucose may be used during a clinic visit to identify poorly controlled type 2 patients with reasonable certainty and allow timely patient education and therapeutic intervention.

Black or African American↗
PubMed1997

Identification of an insulin-responsive element in the rat insulin-like growth factor-binding protein-3 gene.

The hepatic expression and serum levels of insulin-like growth factor-binding protein-3 (IGFBP-3) are decreased in insulin-dependent and insulin-resistant diabetes. Insulin increases hepatic IGFBP-3 expression by enhancing gene transcription. This report identifies sequences within the IGFBP-3 promoter that are necessary and sufficient for the response to insulin in hepatic nonparenchymal cells. By transient transfection, we mapped the insulin response element to the -1150 to -1124 base pair (bp) region of the rat IGFBP-3 promoter. Three tandem repeats of the -1150 to -1117 bp region conferred insulin responses in a heterologous promoter. Gel shift analyses revealed a 3-fold increase in DNA-protein complex formation with nuclear extracts obtained from insulin-stimulated nonparenchymal cells compared with cells incubated without insulin and revealed 3-4-fold decrease in complex formation with nuclear extracts obtained from the livers of streptozotocin-diabetic rats compared with control rats. Mutational analysis of this 34-bp region showed a core sequence of 10 bp (-1148 to -1139) that is critical for interaction with insulin-induced trans-acting factors. Southwestern blotting revealed a approximately 90-kDa protein that was increased 2-3-fold by the addition of insulin. Thus, we have identified cis-acting DNA sequences that are responsible for regulation of IGFBP-3 transcription by insulin and essential for binding of insulin-responsive nuclear factors.

Animals↗
PubMed1997

Diabetes in urban African-Americans. IX. Provider adherence to management protocols.

OBJECTIVE: Staged diabetes management should permit glycemic goals to be attained in a timely manner, but the success of such an approach requires conformity by health care providers. To test performance, we analyzed the adherence of practitioners to a protocol for staged management of NIDDM patients. RESEARCH DESIGN AND METHODS: Records of patients treated at the Grady Memorial Hospital Diabetes Clinic were reviewed retrospectively over a 3-year period. For each patient, intensification of therapy was indicated if fasting plasma glucose was > 7.8 mmol/l and a prior HbA1c was > 7.0%. Protocols dictated a progression from dietary therapy alone to increasing dosages of sulfonylureas to increasing dosages of insulin. Patients were seen at bimonthly intervals. RESULTS: During the 3-year period, 1,051 patient visits met protocol criteria for intensification. Adherence to the protocol improved significantly in the 3rd year compared with the first 2 years (30, 31, and 47% adherence in the 1st, 2nd, and 3rd years, respectively). Patients treated with diet alone were significantly less likely to have their therapy intensified than patients on sulfonylureas or insulin (intensification rates 25, 41, and 47%, respectively). In the management of patients treated with diet alone, practitioners were reluctant to intensify therapy at early visits, but were more likely to do so later, 19% of patients beyond goal range at the 2-month visit were started on pharmacological therapy vs. 28% at the 4-month visit, and 39% at the 6-month visit (P < 0.01). In contrast, there was no significant difference in the frequency of therapy intensification between early and late visits for patients on sulfonylureas or insulin. Practitioners appeared to base the decision to intensify on the fasting plasma glucose level more than on the most recent HbA1c. Age did not appear to be a significant factor in the decision to intensify. CONCLUSIONS: Although staged management protocols constitute critical tools to achieve glycemic goals, the adherence of health care providers may be suboptimal. Special efforts may be needed to assure compliance.

Black or African American↗
PubMed1996

Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription.

In normal subjects and diabetic patients, insulin suppresses whole body proteolysis suggesting that the loss of lean body mass and muscle wasting in insulinopenia is related to increased muscle protein degradation. To document how insulinopenia affects organ weights and to identify the pathway for accelerated proteolysis in muscle, streptozotocin-treated and vehicle-injected, pair-fed control rats were studied. The weights of liver, adipose tissue, and muscle were decreased while muscle protein degradation was increased 75% by insulinopenia. This proteolytic response was not eliminated by blocking lysosomal function and calcium-dependent proteases at 7 or 3 d after streptozotocin. When ATP synthesis in muscle was inhibited, the rates of proteolysis were reduced to the same level in insulinopenic and control rats suggesting that the ATP-dependent, ubiquitin-proteasome pathway is activated. Additional evidence for activation of this pathway in muscle includes: (a) an inhibitor of proteasome activity eliminated the increased protein degradation; (b) mRNAs encoding ubiquitin and proteasome subunits were increased two- to threefold; and (c) there was increased transcription of the ubiquitin gene. We conclude that the mechanism for muscle protein wasting in insulinopenia includes activation of the ubiquitin-proteasome pathway with increased expression of the ubiquitin gene.

Adenosine Triphosphate↗
PubMed1996

In vitro transcription of the rat insulin-like growth factor-I gene.

Although the liver is the major source of circulating insulin-like growth factor-I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are initiated largely in exon 1, we established an in vitro transcription system to evaluate activation of transcription via the major exon 1 initiation site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter was used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared with liver. 54 base pairs (bp) of upstream sequence were sufficient to direct IGF-I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between -300 and -60 bp; binding was confirmed by gel shift analysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to such sites was established by competition analysis, which revealed a specific decrease in IGF-I transcription in the presence of a competing fragment. Use of this in vitro transcription system should permit analysis of the function of individual transcription factors involved in regulation of IGF-I gene expression.

Animals↗
PubMed1996

Diabetes in urban African Americans. III. Management of type II diabetes in a municipal hospital setting.

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.

Black or African American↗
PubMed1996

Insulin and insulin-like growth factor-I regulate hepatic insulin-like growth factor binding protein-3 by different mechanisms.

UNLABELLED: Insulin-dependent diabetes mellitus is associated with decreased levels of circulating insulin-like growth factor binding protein-3 (IGFBP-3), which are restored toward normal by treatment with insulin and/or infusion of insulin-like growth factor-I (IGF-I). To understand underlying mechanisms, we studied IGFBP-3 production in cocultures of parenchymal and nonparenchymal cells from the livers of normal rats. Release of IGFBP-3 was measured by ligand blotting and was increased 1.9- and 15-fold by 10(-8) and 10(-8) M Insulin compared with 10(-10) M (P < 0.05 for 10(-6) vs. 10(-10) M). Expression of IGFBP-3 mRNA was increased concomitantly by 23 and 226% (P < 0.05 for 10(-6) M vs. 10(-10) M), consistent with regulation in part at pretranslational levels. To evaluate mRNA stability, transcription was inhibited with 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB): IGFBP-3 mRNA t1/2 was estimated at 13 h and 17 h with addition of 10(-5) M and 10(-10) M insulin, respectively, ruling out regulation at the level of mRNA turnover. IGFBP-3 gene transcription rates were evaluated by nuclear run-on assays and were increased 2,9-fold with the addition of 10(-6) M insulin, as compared with 10(-10) M insulin, comparable to stimulation of expression. Addition of IGF-I at 2.6 x 10(-8) M and 5.3 x 10(-8) M increased IGFBP-3 release by 5.2- and 8.2-fold (both P < 0.05 vs. no IGF-I), with concomitant increase in IGFBP-3 mRNA expression by 14- and 29-fold (both P < 0.05 vs. no IGF-I), suggesting regulation at a pretranslational level. Further studies showed that IGF-I did not have a significant effect on transcription initiation rates but prolonged the apparent half-life of IGFBP-3 mRNA about 2-fold. Stimulation of IGFBP-3 via type 1 IGF-I receptors was evaluated by studies with [QAYL] IGF-I; the analog increased IGFBP-3 mRNA expression 220 +/- 27% above the level obtained without IGF-I (vs. 133 +/- 9% with wild type IGF-I, P < 0.05), suggesting involvement of receptor-mediated synthesis. CONCLUSION: Insulin stimulates IGFBP-3 gene transcription but provides proportionally greater increases in IGFBP-3 release, consistent with regulation at both transcriptional and posttranslational levels; in contrast, IGF-I alters IGFBP-3 expression by decreasing IGFBP-3 mRNA degradation, consistent with regulation at pretranslational and posttranscriptional levels. Decreased IGFBP-3 levels in conditions of diabetes mellitus may be due to decreased hepatic IGFBP-3 release, and secondary both to decreased gene transcription (caused by insulin deficiency), as well as to decreased IGFBP-3 mRNA half-life (caused by low levels of IGF-I).

Animals↗
PubMed1996

Transcription initiation of the rat insulin-like growth factor-I gene in hepatocyte primary culture.

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.

Animals↗
PubMed1995

Transcriptional regulation of the rat insulin-like growth factor-I gene involves metabolism-dependent binding of nuclear proteins to a downstream region.

Insulin-like growth factor-I (IGF-I) gene transcription is mediated largely via exon 1. In an initial search for regulatory regions, rat hepatocytes were transfected with IGF-I constructs. Since omission of downstream sequences led to reduced expression, we then used in vitro transcription to evaluate potential metabolic regulation via downstream regions. With templates including 219 base pairs of downstream sequence, transcriptional activity was reduced 70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates lacking downstream sequences. The downstream region contained six DNase I footprints, and templates with deletion of either region III or V no longer provided reduced transcriptional activity with nuclear extracts from diabetic rats. Nuclear protein binding to regions III and V appeared to be metabolically regulated, as shown by reduced DNase I protection and activity in gel mobility shift assays with nuclear extracts from diabetic rats. Southwestern blotting probes corresponding to regions III and V recognized a approximately 65-kDa nuclear factor present at reduced levels in diabetic rats. These findings indicate that a downstream region in exon 1 may be important for both IGF-I expression and metabolic regulation. Altered concentration or activity of a transcription factor(s) binding to this region may contribute to reduced IGF-I gene transcription associated with diabetes mellitus.

Animals↗