Insulin Production In Response Research Articles

FRI664 The Impact Of Prediabetes Diagnosis On The Prevalence Of Postbariatric Hypoglycemia

Abstract Disclosure: J.R. Shariff: None. L. Funk: None. A. Zaveri: None. D.B. Davis: None. Bariatric surgery is not only a mainstay of obesity treatment, but it can result in remission of metabolic conditions, such as Type 2 diabetes. Post-operatively however, patients can develop postbariatric hypoglycemia (PBH), which is thought to be due to changes in insulin sensitivity and hypersecretion after eating. It has been suggested that prior type 2 diabetes can protect against PBH as decreased β-cell function prevents hyperinsulinemia. Patients with prediabetes may not have deterioration of β-cell function resulting in increased insulin production in response to glycemic excursion and subsequent hypoglycemia. The study aimed to assess the prevalence and severity of PBH in patients with prediabetes compared to those without prediabetes prior to bariatric surgery. We conducted a retrospective cohort study of patients 18 years and older who underwent either Roux-en-Y gastric bypass or sleeve gastrectomy between 2014 and 2021 and did not have a history of type 1 or type 2 diabetes mellitus at a single academic medical center. A history of PBH (hypoglycemia reported or documented after eating, symptoms of hypoglycemia, and resolution of symptoms after eating) and prediabetes (defined as A1c 5.7-6.4%, fasting glucose of 100-125mg/dL, or oral glucose tolerance testing with 2-hour glucose 140-199mg/dL) was determined based on medical record review. 14 patients developed PBH out of 539 (2.6%). 110 patients had prediabetes (20.4%). Of these 110 patients, 4 had PBH (3.6%). Of the 429 patients without prediabetes, 10 patients had PBH (2.3%). There was a greater post-operative weight loss, on average, in patients with PBH (19.5%) than in those without PBH (15.9%), although it should be noted that all PBH patients had Roux en Y gastric bypass. Interestingly, only 15% of the PBH population appeared to be on pharmacologic treatment, despite 21.5% being hospitalized or in the emergency room for hypoglycemic events. Most PBH patients (64.3%) developed symptoms between 1 to 3 years post-operatively, which is consistent with other studies on this topic. There was no association between prediabetes and PBH in our dataset (p=0.46), however our data set was quite small and it may be difficult to draw significant conclusions. A larger scale study done in a prospective manner would be useful for further research. Presentation: Friday, June 16, 2023

Regulation and mechanism of action of miRNAs on insulin resistance in skeletal muscles.

The term "insulin resistance" is commonly understood as a decrease in the response of insulin-sensitive tissues to insulin at its sufficient concentration, leading to chronic compensatory hyperinsulinemia. Type 2 diabetes mellitus is based on mechanisms consisting in the development of resistance to insulin in target cells (hepatocytes, adipocytes, skeletal muscle cells), resulting in the termination of an adequate response of these tissues to interaction with insulin. Since in healthy people 75-80% of glucose is utilized by skeletal muscle, it is more likely that the main cause of insulin resistance is impaired insulin-stimulated glucose utilization by skeletal muscle. With insulin resistance, skeletal muscles do not respond to insulin at its normal concentration, thereby determining an increase in glucose levels and a compensatory increase in insulin production in response to this. Despite many years of studying diabetes mellitus (DM) and insulin resistance, the molecular genetic basis for the development of these pathological conditions is still the subject of numerous studies. Recent studies point to the involvement of microRNAs (miRNAs) as dynamic modifiers in the pathogenesis of various diseases. MiRNAs are a separate class of RNA molecules that play a key role in the post-transcriptional regulation of gene expression. Recent studies have shown that miRNAs dysregulation in DM is closely related to miRNAs regulatory abilities in skeletal muscle insulin resistance. This gave grounds to consider an increase or decrease in the expression of individual microRNAs in muscle tissue and consider them as new biomarkers for diagnosing and monitoring insulin resistance and promising directions for targeted therapy. This review presents the results of scientific studies examining the role of miRNAs in skeletal muscle insulin resistance.

A glucagon-like peptide-1 receptor antagonist reduces the insulin response to a glycemic meal in ponies

Abstract High plasma concentrations of insulin can cause acute laminitis. Ponies and horses with insulin dysregulation (ID) exhibit marked hyperinsulinemia in response to dietary hydrolyzable carbohydrates. Glucagon-like peptide-1 (GLP-1), an incretin hormone released from the gastrointestinal tract, enhances insulin release, and is increased postprandially in ponies with ID. The aim of this study was to determine whether blocking the GLP-1 receptor reduces the insulin response to a high glycemic meal. Five adult ponies were adapted to a cereal meal and then given two feed challenges 24 h apart of a meal containing 3 g/kg BW micronized maize. Using a randomized cross-over design all ponies received both treatments, where one of the feeds was preceded by the IV administration of a GLP-1 receptor blocking peptide, Exendin-3 (9-39) amide (80 µg/kg), and the other feed by a sham treatment of peptide diluent only. Blood samples were taken before feeding and peptide administration, and then at 30-min intervals via a jugular catheter for 6 h for the measurement of insulin, glucose, and active GLP-1. The peptide and meal challenge caused no adverse effects, and the change in plasma glucose in response to the meal was not affected (P = 0.36) by treatment: peak concentration 9.24 ± 1.22 and 9.14 ± 1.08 mmol/L without and with the antagonist, respectively. Similarly, there was no effect (P = 0.35) on plasma active GLP-1 concentrations: peak concentration 14.3 ± 1.36 pM and 13.7 ± 1.97 pM without and with the antagonist, respectively. However, the antagonist caused a significant decrease in the area under the curve for insulin (P = 0.04), and weak evidence (P = 0.06) of a reduction in peak insulin concentration (456 ± 147 μIU/mL and 370 ± 146 μIU/mL without and with the antagonist, respectively). The lower overall insulin response to the maize meal after treatment with the antagonist demonstrates that blocking the GLP-1 receptor partially reduced insulin production in response to a high starch, high glycemic index, diet. Using a different methodological approach to published studies, this study also confirmed that GLP-1 does contribute to the excessive insulin production in ponies with ID.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel –facilitated platform ideal for islet organoid engineering.

Environmental Factors Contribute to β Cell Endoplasmic Reticulum Stress and Neo-Antigen Formation in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which immune-mediated targeting and destruction of insulin-producing pancreatic islet β cells leads to chronic hyperglycemia. There are many β cell proteins that are targeted by autoreactive T cells in their native state. However, recent studies have demonstrated that many β cell proteins are recognized as neo-antigens following posttranslational modification (PTM). Although modified neo-antigens are well-established targets of pathology in other autoimmune diseases, the effects of neo-antigens in T1D progression and the mechanisms by which they are generated are not well understood. We have demonstrated that PTM occurs during endoplasmic reticulum (ER) stress, a process to which β cells are uniquely susceptible due to the high rate of insulin production in response to dynamic glucose sensing. In the context of genetic susceptibility to autoimmunity, presentation of these modified neo-antigens may activate autoreactive T cells and cause pathology. However, inherent β cell ER stress and protein PTM do not cause T1D in every genetically susceptible individual, suggesting the contribution of additional factors. Indeed, many environmental factors, such as viral infection, chemicals, or inflammatory cytokines, are associated with T1D onset, but the mechanisms by which these factors lead to disease onset remain unknown. Since these environmental factors also cause ER stress, exposure to these factors may enhance production of neo-antigens, therefore boosting β cell recognition by autoreactive T cells and exacerbating T1D pathogenesis. Therefore, the combined effects of physiological ER stress and the stress that is induced by environmental factors may lead to breaks in peripheral tolerance, contribute to antigen spread, and hasten disease onset. This Hypothesis and Theory article summarizes what is currently known about ER stress and protein PTM in autoimmune diseases including T1D and proposes a role for environmental factors in breaking immune tolerance to β cell antigens through neo-antigen formation.

Endocrine system on chip for a diabetes treatment model

The endocrine system is a collection of glands producing hormones which, among others, regulates metabolism, growth and development. One important group of endocrine diseases is diabetes, which is caused by a deficiency or diminished effectiveness of endogenous insulin. By using a microfluidic perfused 3D cell-culture chip, we developed an ‘endocrine system on chip’ to potentially be able to screen drugs for the treatment of diabetes by measuring insulin release over time. Insulin-secreting β-cells are located in the pancreas, while L-cells, located in the small intestines, stimulate insulin secretion. Thus, we constructed a co-culture of intestinal-pancreatic cells to measure the effect of glucose on the production of glucagon-like peptide-1 (GLP-1) from the L-cell line (GLUTag) and insulin from the pancreatic β-cell line (INS-1). After three days of culture, both cell lines formed aggregates, exhibited 3D cell morphology, and showed good viability (>95%). We separately measured the dynamic profile of GLP-1 and insulin release at glucose concentrations of 0.5 and 20 mM, as well as the combined effect of GLP-1 on insulin production at these glucose concentrations. In response to glucose stimuli, GLUTag and INS-1 cells produced higher amounts of GLP-1 and insulin, respectively, compared to a static 2D cell culture. INS-1 combined with GLUTag cells exhibited an even higher insulin production in response to glucose stimulation. At higher glucose concentrations, the diabetes model on chip showed faster saturation of the insulin level. Our results suggest that the endocrine system developed in this study is a useful tool for observing dynamical changes in endocrine hormones (GLP-1 and insulin) in a glucose-dependent environment. Moreover, it can potentially be used to screen GLP-1 analogues and natural insulin and GLP-1 stimulants for diabetes treatment.

Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose.

Type 1 diabetes occurs due to the autoimmune destruction of pancreatic β-cells in islets. Transplantation of islets is a promising option for the treatment of patients with type 1 diabetes that experience hypoglycemic unawareness despite maximal care, but the present shortage of donor islets hampers such transplantation. Transplantation of insulin-producing cells derived from the patients themselves would be one of the most promising approaches to cure type 1 diabetes. Previously, we demonstrated that insulin-producing cells could be produced by transfecting murine pancreatic cells with Yamanaka’s reprogramming factors. Non-obese diabetic (NOD) mice are naturally occurring mutant mice defective in insulin production due to autoimmune ablation of pancreatic β-cells. In this study, we showed that glucose-sensitive insulin-producing cells are successfully generated by transfecting primary pancreatic cells from NOD mice (aged 6 months old) with a plasmid harboring the cDNAs for Oct-3/4, Sox2, Klf4, and c-Myc. Transfection was repeated 4 times in a 2 day-interval. Sixty-five days after final transfection, cobblestone-like colonies appeared. They proliferated in vitro and expressed pluripotency-related genes as well as Pdx1, a transcription factor specific to tissue-specific stem cells for the β-cell lineage. Transplantation of these cells into nude mice failed to produce teratoma unlike induced pluripotent stem cells (iPSCs). Induction of these cells to the pancreatic β-cell lineage demonstrated their capability to produce insulin in response to glucose. These findings suggest that functional pancreatic β-cells can be produced from patients with type 1 diabetes. We call these resultant cells as “induced tissue-specific stem cells from the pancreas” (iTS-P) that could be valuable sources of safe and effective materials for cell-based therapy in type 1 diabetes.

Polycaprolactone Thin-Film Micro- and Nanoporous Cell-Encapsulation Devices.

Cell-encapsulating devices can play an important role in advancing the types of tissue available for transplantation and further improving transplant success rates. To have an effective device, encapsulated cells must remain viable, respond to external stimulus, and be protected from immune responses, and the device itself must elicit a minimal foreign body response. To address these challenges, we developed a micro- and a nanoporous thin-film cell encapsulation device from polycaprolactone (PCL), a material previously used in FDA-approved biomedical devices. The thin-film device construct allows long-term bioluminescent transfer imaging, which can be used for monitoring cell viability and device tracking. The ability to tune the microporous and nanoporous membrane allows selective protection from immune cell invasion and cytokine-mediated cell death in vitro, all while maintaining typical cell function, as demonstrated by encapsulated cells' insulin production in response to glucose stimulation. To demonstrate the ability to track, visualize, and monitor the viability of cells encapsulated in implanted thin-film devices, we encapsulated and implanted luciferase-positive MIN6 cells in allogeneic mouse models for up to 90 days. Lack of foreign body response in combination with rapid neovascularization around the device shows promise in using this technology for cell encapsulation. These devices can help elucidate the metrics required for cell encapsulation success and direct future immune-isolation therapies.

Weaning means more than no more milk

Cell Regeneration![Figure][1] Weaning enhances the regenerative potential of pancreatic beta cells PHOTO: TOM MCHUGH/SCIENCE SOURCE Nursing mothers provide much needed nutrition to offspring, but the full effects of weaning on offspring's physiology is unknown. Stolovich-Rain et al. now show that in mice, weaning affects the function of insulin-producing beta cells in the pancreas. The ability of beta cells to regenerate after injury or to modulate their insulin secretion decreases with age. However, beta cells also regenerated poorly in response to injury in very young mice and only gained this function upon weaning. These results suggest that at least for mouse beta cells, weaning jump-starts the cell cycle and modulates insulin production in response to glucose. Curr. Biol. 24 , 2733 (2014). [1]: pending:yes

Vitamin D deficiency is associated with insulin resistance in nondiabetics and reduced insulin production in type 2 diabetics.

It is not known whether the association of serum 25-hydroxyvitamin D [25(OH)D] with glycemic measurements of individuals without diabetes is similar to those with diabetes or not. This study is aimed to investigate the association of serum 25(OH)D with glycemic markers of diabetics, nondiabetics, and prediabetics. A case-control study was conducted on age and sex matched 1,195 patients with type 2 DM, 121 prediabetics, and 209 healthy controls. Anthropometric variables, lipid profile, glycemic measurements, and serum 25(OH)D levels were recorded. Serum insulin and C-peptide levels were also measured. All glycemic measurements were compared between diabetics and nondiabetics and prediabetics at different vitamin D status. Patients with DM had lower serum 25(OH)D compared to prediabetics and healthy controls. Endogenous insulin production in response to food intake and in fasting was significantly lower in vitamin D deficient patients with DM compared to those with serum 25(OH)D>40 ng/ml. Diabetic women with serum 25(OH)D<20 ng/ml had lower beta cell function as estimated by lower HOMA-B compared to their counterparts with serum 25(OH)D>40 ng/ml. Healthy individuals with serum 25(OH)D<20 ng/ml had signs of insulin resistance as estimated by significant increase of HOMA-IR, HbA1c, and fasting plasma glucose (FPG). In addition, we found that serum 25(OH)D was inversely associated with insulin resistance. Vitamin D deficiency is associated with insulin resistance in nondiabetics, which is independent of obesity. Furthermore, vitamin D deficiency is associated with reduced insulin production in type 2 diabetics, which was mainly observed in men. Accordingly, a gender disparity also exists in association of serum 25(OH)D with glycemic measurements.

Induction of the 72 kDa heat shock protein by glucose ingestion in black pregnant women

Obese Black women are at increased risk for development of gestational diabetes mellitus and have worse perinatal outcomes than do obese women of other ethnicities. Since hsp72 has been associated with the regulation of obesity-induced insulin resistance, we evaluated associations between glucose ingestion, hsp72 release and insulin production in Black pregnant women. Specifically, the effect of a 50-g glucose challenge test (GCT) on heat shock protein and insulin levels in the circulation 1h later was evaluated. Hsp27 and hsp60 levels remained unchanged. In contrast, serum levels of hsp72 markedly increased after glucose ingestion (p = 0.0054). Further analysis revealed that this increase was limited to women who were not obese (body mass index <30). Insulin levels pre-GCT were positively correlated with body mass index (p = 0.0189). Median insulin concentrations also increased post GCT in non-obese women but remained almost unchanged in obese women. Post-GCT serum hsp72 concentrations were inversely correlated with post GCT insulin concentrations (p = 0.0111). These observations suggest that glucose intake during gestation in Black women rapidly leads to an elevation in circulating hsp72 only in non-obese Black women. The release of hsp72 may regulate the extent of insulin production in response to a glucose challenge and, thereby, protect the mother and/or fetus from development of hyperglycemia, hyperinsulinemia, and/or immune system alterations.

Index of Suspicion in the Nursery

A 5-week-old previously well female born at 41 weeks gestational age presents to the pediatric emergency department in the winter with a 2-day history of poor feeding. Her mother is concerned that the baby is more tired than usual, “is not getting enough to eat,” and is having difficulty latching on to the breast. The mother explains that 2 weeks ago, the infant developed a dry cough and began having emesis after each feeding. She describes the emesis as nonbilious and nonbloody and approximately 1 ounce. An outside pediatrician prescribed a cough and cold medication and told the mother that the emesis was “normal baby spit-up.” Three days ago, the infant developed projectile vomiting immediately following each feeding. Today, the mother reports that the infant is breathing much harder and deeper than usual, with continued and more frequent coughs. She describes the cough as paroxysmal, although she experiences no color changes. The mother is concerned that her baby does not look like herself and is difficult to arouse. A review of systems reveals that the baby has not taken any human milk today, although her urine output has remained the same, with heavy, wet diapers. Her 2-year-old sister, who attends child care, has a cough and upper respiratory tract infection. The infant has no diarrhea, and her bowel movements are normal. There were no complications during the pregnancy. Her birthweight was 2,722 g, and she has been gaining weight appropriately, although the mother describes her as “scrawny” at birth. Physical examination of the infant in the emergency department shows a temperature …

The Use of Pancreas Biopsy Scoring Provides Reliable Porcine Islet Yields While Encapsulation Permits the Determination of Microbiological Safety

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of -1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26-56-month-old sows, we report here that the presence of large (>100 microm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.

416. Glucose-Dependent Insulin Production by Liver-Specific, Glucose-Regulatable Synthetic Promoters Results in the Cure of Diabetes

Top of pageAbstract Type 1 diabetes is caused by insulin deficiency. Insulin gene therapy has been attempted to cure diabetes; however, there is no satisfactory system that results in the tight regulation of insulin production in response to physiological changes in glucose levels. To develop a liver-specific, glucose-responsive synthetic promoter that can tightly regulate insulin production in response to blood glucose levels, we employed 3 kinds of transcription factor-binding cis-elements: hepatocyte nuclear factor-1, CAAT/enhancer binding protein response element, and glucose-response element. We generated a library of synthetic promoters that contained 9 of these cis-elements in random combinations and screened each promoter for transcriptional activity. Among 821 promoters tested, we selected 3 with the highest transcriptional activity for further testing. To determine the glucose responsiveness of these promoters in vivo, we generated recombinant adenoviruses expressing furin-cleavable rat insulin under the control of selected synthetic promoters (rAd-SP-rINSfur) and the cytomegalovirus promoter (rAd-CMV-rINSfur) as a control for ubiquitous strong activity. rAd-SP-rINSfur (1010 viral particles) or rAd-CMV-rINSfur (109 viral particles) was administered into streptozotocin-induced diabetic NOD.scid mice (n=6/group) via the tail vein, and blood glucose levels were examined. We found that diabetic mice treated with rAd-SP-rINSfur became normoglycemic a few days after treatment, and normoglycemia was maintained for more than 1 month, whereas mice treated with rAd-CMV-rINSfur were dead within 10 days due to uncontrolled constitutive expression of the insulin gene. Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice produced insulin in response to glucose and cleared exogenous glucose from the blood in a manner similar to non-diabetic control mice. Insulin expression in rAd-SP-rINSfur-treated mice was observed only in the liver, not in other tissues or organs, whereas insulin expression in rAd-CMV-rINSfur-treated mice was present in all tissues tested, indicating that the synthetic promoters were liver-specific. On the basis of these observations, we conclude that our newly constructed, liver-specific synthetic promoters can tightly regulate insulin production in response to glucose and may have therapeutic potential for the cure of diabetes in humans by liver-directed insulin gene therapy.

Insulin does not mediate glucose stimulation of proinsulin biosynthesis.

It has recently been suggested that insulin augments its own production by a physiologically important feed-forward autocrine loop. We studied the kinetics of glucose-regulated proinsulin gene expression and proinsulin biosynthesis in normal rat islets with emphasis on the potential role of insulin as a mediator of the glucose effect. There was a time-dependent increase in steady-state proinsulin mRNA in islets cultured at 16.7 mmol/l compared with 3.3 mmol/l glucose; no early (1-3 h) increase in proinsulin gene expression was observed. In contrast, there was a threefold increase in proinsulin biosynthesis within 1 h of glucose stimulation that was not affected by inhibition of glucose-stimulated proinsulin gene transcription with actinomycin D. In addition, inhibition of glucose-stimulated insulin secretion with diazoxide had no effect on glucose-stimulated proinsulin mRNA or biosynthesis. Furthermore, addition of different concentrations of insulin to islets cultured in low glucose failed to affect proinsulin biosynthesis. Taken together, our data suggest that the early glucose-dependent increase in proinsulin biosynthesis is mainly regulated at the translational level, rather than by changes in proinsulin gene expression. Moreover, we could not demonstrate any effect of insulin on islet proinsulin mRNA level or rate of proinsulin biosynthesis. Thus, if insulin has any effect on the proinsulin biosynthetic apparatus, it is a minor one. We conclude that the secreted insulin is not an important mediator of insulin production in response to glucose.

Glucose regulated production of human insulin in rat hepatocytes.

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.

Diabetes after removal of insulin tumours of pancreas: a long-term follow-up survey of 11 patients.

Eleven patients who had islet cell tumours of the pancreas removed over a 20-year period were reviewed. Two had died, one in the postoperative period and one 10 years post-operatively. The remaining nine were traced and eight of them were personally interviewed and tested. All nine were well and free from recurrent symptoms. Five of the eight had evidence of subnormal insulin production in response to oral glucose. Glucose tolerance was impaired in three. Minor anomalies were found in the electroencephalograms of all these patients and major abnormalities in two.The results of this survey suggest that other patients who have had islet cell tumours removed may now have unsuspected diabetes.

The role of human chorionic somatomammotropin (HCS) and gonadal steroids in gestational diabetes.

Maternal carbohydrate tolerance during gestation appears to be altered by at least two mechanisms: 1) decreased sensitivity of insulin-sensitive tissues to the hypoglycemic action of insulin, and 2) alterations in the sensitivity of the islets of Langerhans to the insulinogenic stimulus of glucose. The effect of physiologic amounts of unconjugated estriol and estradiol on these parameters is as yet unknown. Decreased sensitivity to insulin can be produced experimentally in female Rhesus monkeys by human chorionic somatomammotropin (HCS) and by progesterone administration. Progesterone administration enhances glucose-stimulated insulin production in both monkeys and man. By contrast, HCS administration reverses the progesterone-enhanced serum insulin response to glucose in monkeys and actually retards insulin production in response to glucose in subclinical diabetic women. The latter pattern of insulin response is similar to, but not identical with, that seen in women with gestational diabetes. Whether progesterone and/or the estrogens elaborated by the conceptus alter the effect of HCS on insulin production or sensitivity to insulin in normal or subclinical diabetic women remains to be determined.