Maria M. Byrne

Glucagon-Like Peptide 1 Improves the Ability of the β-Cell to Sense and Respond to Glucose in Subjects With Impaired Glucose Tolerance

Impaired glucose tolerance (IGT) and NIDDM are both associated with an impaired ability of the β-Cell to sense and respond to small changes in plasma glucose concentrations. The aim of this study was to establish if glucagon-like peptide 1 (GLP-1), a natural enteric peptide and potent insulin secretagogue, improves this defect. Two weight-matched groups, one with eight subjects having IGT (2-h glucose, 10.1 ± 0.3 mmol/l) and another with seven subjects with diet-treated NIDDM (2-h glucose, 14.5 ± 0.9 mmol/l), were studied on two occasions during a 12-h oscillatory glucose infusion, a sensitive test of the ability of the β-Cell to sense and respond to glucose. Glucose was infused with a mean rate of 4 mg · kg−1 · min−1, amplitude 33% above and below the mean rate, and periodicity of 144 min, with infusion of saline or GLP-1 at 0.4 pmol · kg−1 · min−1 for 12 h. Mean glucose levels were significantly lower in both groups during the GLP-1 infusion compared with during saline infusion: 9.2 ± 0.4 vs. 6.4 ±0.1 mmol/l in the IGT subjects (P < 0.0004) and 14.6 ± 1.0 vs. 9.3 ± 0.7 mmol/L in NIDDM subjects (P < 0.0002). Despite this significant reduction in plasma glucose concentration, insulin secretion rates (ISRs) increased significantly in IGT subjects (513.3 ± 77.6 vs. 583.1 ± 100.7 pmol/min; P < 0.03), with a trend toward increasing in NIDDM subjects (561.7 ± 122.16 vs. 642.8 ± 128 pmol/min; P = 0.1). These results were compatible with enhanced insulin secretion in the presence of GLP-1. Spectral power was used as a measure of the ability of the β-Cell to secrete insulin in response to small changes in the plasma glucose concentration during the oscillatory infusion. Spectral power for ISR increased from 2.1 ± 0.9 during saline infusion to 7.4 ±1.3 during GLP-1 infusion in IGT subjects (P < 0.004), but was unchanged in NIDDM subjects (1.0 ± 0.4 to 1.5 ± 0.6; P = 0.3). We concluded that low dosage GLP-1 improves the ability of the β-Cell to secrete insulin in both IGT and NIDDM subjects, but that the ability to sense and respond to subtle changes in plasma glucose is improved in IGT subjects, with only a variable response in NIDDM subjects. β-Cell dysfunction was improved by GLP-1 infusion, suggesting that early GLP-1 therapy may preserve β-Cell function in subjects with IGT or mild NIDDM

Intestinal Proliferation and Delayed Intestinal Transit in a Patient with a GLP-1-, GLP-2- and PYY-Producing Neuroendocrine Carcinoma

Glucagon-like peptides (GLP) 1 and 2 are hormones derived from the post-translational processing of proglucagon in the intestinal L cells that influence intestinal motility and small bowel growth, respectively. We describe a patient with a neuroendocrine tumor of unknown primary origin with peritoneal carcinomatosis and diffuse liver metastases, who presented with constipation and nocturnal itching for over 3 years. Small bowel follow-through showed decreased small intestinal motility and marked intestinal hypertrophy. Biopsies from mesenterial lymph nodes showed, histologically, a well-differentiated neuroendocrine tumor (G1), with positive immunostaining for chromogranin A, GLP-1, GLP-2 and polypeptide YY (PYY). Jejunal biopsy demonstrated marked intestinal mucosal hypertrophy. HPLC analysis combined with RIA of tumor and serum extracts revealed that the tumor was producing and releasing fasting levels of GLP-1 of 738±20.7 pg/ml (normal levels (nl) <100 pg/ml), GLP-2 of 3,150±9 pg/ml (nl <100 pg/ml) as well as PYY 550 pg/ml (nl <100 pg/ml). Octreotide administration decreased levels of GLP-1 and GLP-2 and reduced small intestinal transit time from 150 to 50 min. However, tumor growth was not inhibited by octreotide, interferon or dacarbazine therapy and the patient died 8 months later. This is the first case report demonstrating the overproduction of GLP-1, GLP-2 and PYY from an neuroendocrine tumor, in a patient with intestinal hypertrophy and delayed intestinal transit time.

INS-1 Cells Undergoing Caspase-Dependent Apoptosis Enhance the Regenerative Capacity of Neighboring Cells

OBJECTIVE In diabetes, β-cell mass is not static but in a constant process of cell death and renewal. Inactivating mutations in transcription factor 1 (tcf-1)/hepatocyte nuclear factor1a (hnf1a) result in decreased β-cell mass and HNF1A–maturity onset diabetes of the young (HNF1A-MODY). Here, we investigated the effect of a dominant-negative HNF1A mutant (DN-HNF1A) induced apoptosis on the regenerative capacity of INS-1 cells. RESEARCH DESIGN AND METHODS DN-HNF1A was expressed in INS-1 cells using a reverse tetracycline-dependent transactivator system. Gene(s)/protein(s) involved in β-cell regeneration were investigated by real-time quantitative RT-PCR, Western blotting, and immunohistochemistry. Pancreatic stone protein/regenerating protein (PSP/reg) serum levels in human subjects were detected by enzyme-linked immunosorbent assay. RESULTS We detected a prominent induction of PSP/reg at the gene and protein level during DN-HNF1A–induced apoptosis. Elevated PSP/reg levels were also detected in islets of transgenic HNF1A-MODY mice and in the serum of HNF1A-MODY patients. The induction of PSP/reg was glucose dependent and mediated by caspase activation during apoptosis. Interestingly, the supernatant from DN-HNF1A–expressing cells, but not DN-HNF1A–expressing cells treated with zVAD.fmk, was sufficient to induce PSP/reg gene expression and increase cell proliferation in naïve, untreated INS-1 cells. Further experiments demonstrated that annexin-V–positive microparticles originating from apoptosing INS-1 cells mediated the induction of PSP/reg. Treatment with recombinant PSP/reg reversed the phenotype of DN-HNF1A–induced cells by stimulating cell proliferation and increasing insulin gene expression. CONCLUSIONS Our results suggest that apoptosing INS-1 cells shed microparticles that may stimulate PSP/reg induction in neighboring cells, a mechanism that may facilitate the recovery of β-cell mass in HNF1A-MODY.

AMP-activated Protein Kinase Mediates Apoptosis in Response to Bioenergetic Stress through Activation of the Pro-apoptotic Bcl-2 Homology Domain-3-only Protein BMF

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation.

Identification of circulating microRNAs in HNF1A-MODY carriers

Aims/hypothesisHNF1A-MODY is a monogenic form of diabetes caused by mutations in the HNF1A gene. Here we identify, for the first time, HNF1A-MODY-associated microRNAs (miRNAs) that can be detected in the serum of HNF1A-MODY carriers.MethodsAn miRNA array was carried out in rat INS-1 insulinoma cells inducibly expressing the common human Pro291fsinsC-HNF1A frame shift mutation. Differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of miRNAs in the serum of HNF1A-MODY carriers (n = 31), MODY-negative family members (n = 10) and individuals with type 2 diabetes mellitus (n = 17) was quantified by absolute real-time PCR analysis.ResultsInducible expression of Pro291fsinsC-HNF1A in INS-1 cells caused a significant upregulation of three miRNAs (miR-103, miR-224, miR-292-3p). The differential expression of two miRNAs (miR-103 and miR-224) was validated in vitro. Strongly elevated levels of miR-103 and miR-224 could be detected in the serum of HNF1A-MODY carriers compared with MODY-negative family controls. Serum levels of miR-103 distinguished HNF1A-MODY carriers from HbA1c-matched individuals with type 2 diabetes mellitus.Conclusions/interpretationOur study demonstrates that the pathophysiology of HNF1A-MODY is associated with the overexpression of miR-103 and miR-224. Furthermore, our study demonstrates that these miRNAs can be readily detected in the serum of HNF1A-MODY carriers.

“In-Situ” Teicoplanin for central venous catheter infection

SummaryInfection of central venous catheters (CVC) is a relatively common occurrence in immunocompromised patients, management of which has included I.V. antibiotics ± removal of catheter. We have previously demonstrated that intracatheter administration of Amikacin empirically, successfully eradicated all bacterial infections except those due to S. epidermidis. A study was subsequently undertaken to treat gram positive cocci infections of CVC with intracatheter Teicoplanin. Eleven patients attending a single institution with documented gram positive cocci infection of CVC over a one year period were included in the study. Teicoplanin was instilled with heparinised saline once daily into the infected lumen of the CVC and allowed to remain for 24 hours. Treatment was continued for 48 hours after negative cultures were reported. Teicoplanin was successful in eradicating infection in 100% of cases. Mean duration of treatment was six days (range 4–9 days). Four patients subsequently developed a further infection, a mean of 13 weeks from first infection, only one of which was due to the same organism and this was successfully treated by a further course of Teicoplanin. No side effects were reported and catheter life was prolonged a mean of 132 days after completion of treatment. The use of Teicoplanin in this way for treatment of gram positive cocci infection of CVC is highly effective; once daily administration of antibiotic enables treatment to be given on an outpatient basis, thereby minimising hospital admission.

Successful maintenance on sulphonylurea therapy and low diabetes complication rates in a HNF1A–MODY cohort

HNF1A gene mutations are the most common cause of maturity‐onset diabetes of the young (MODY) in the UK. Persons with HNF1A–MODY display sensitivity to sulphonylurea therapy; however, the long‐term efficacy is not established. There is limited literature as to the prevalence of micro‐ and macrovascular complications in this unique cohort. The aim of this study was to determine the natural progression and clinical management of HNF1A–MODY diabetes in a dedicated MODY clinic.

Teenage pregnancy in type 1 diabetes mellitus

Carmody D, Doyle A, Firth RGR, Byrne MM, Daly S, Mc Auliffe F, Foley M, Coulter‐Smith S, Kinsley BT. Teenage pregnancy in type 1 diabetes mellitus

The clinical management of hyperglycemia in pregnancy complicated by maturity-onset diabetes of the young

OBJECTIVE Women with maturity-onset diabetes of the young (MODY) are often first identified and diagnosed with diabetes during pregnancy. Genetics and hyperglycemia play an important role in determining fetal size in MODY pregnancies. The principal objective of the current study is to determine the outcomes and clinical management of hyperglycemia in pregnancies complicated by glucokinase gene (GCK) and hepatocyte nuclear factor (HNF)-1α MODY mutations. STUDY DESIGN A retrospective chart review of 37 women with a GCK/HNF-1α mutation was conducted. Data on variables such as birthweight, mode of delivery, and the treatment of hyperglycemia were available on 89 pregnancies. RESULTS The birthweight in unaffected GCK offspring was significantly higher than in the affected GCK offspring (4.8 [4.1-5.2] kg vs 3.2 [3.1-3.7] kg; P = .01). Seven-point home blood glucose monitoring over a 7-day period in each trimester demonstrated higher fasting and postprandial glycemic excursions in the first trimester of GCK pregnancies when compared to HNF-1α pregnancies (fasting 104 [90-115] mg/dL vs 84 [77-88] mg/dL; P = .01 and postprandial 154 [135-196] mg/dL vs 111 [100-131] mg/dL; P = .04) despite insulin treatment. There was a higher percentage of miscarriages in the GCK group when compared to the HNF-1α MODY group (33.3% vs 14%; P = .07), which was similar to the background population. Insulin initiated at an early gestation appeared to lower the incidence of macrosomia in GCK unaffected offspring. CONCLUSION Hyperglycemia in HNF-1α pregnancies is easily managed with current insulin protocols; in contrast, glycemic excursions are difficult to manage in GCK pregnancies. There was an increased percentage of miscarriages in GCK pregnancies highlighting the importance of a diagnosis of GCK-MODY in women prior to conception and the necessity for preconception care.

Serum levels of pancreatic stone protein (PSP)/reg1A as an indicator of beta-cell apoptosis suggest an increased apoptosis rate in hepatocyte nuclear factor 1 alpha (HNF1A-MODY) carriers from the third decade of life onward

BackgroundMutations in the transcription factor hepatocyte nuclear factor-1-alpha (HNF1A) result in the commonest type of maturity onset diabetes of the young (MODY). HNF1A-MODY carriers have reduced pancreatic beta cell mass, partially due to an increased rate of apoptosis. To date, it has not been possible to determine when apoptosis is occurring in HNF1A-MODY.We have recently demonstrated that beta cell apoptosis stimulates the expression of the pancreatic stone protein/regenerating (PSP/reg) gene in surviving neighbour cells, and that PSP/reg1A protein is subsequently secreted from these cells. The objective of this study was to determine whether serum levels of PSP/reg1A are elevated during disease progression in HNF1A-MODY carriers, and whether it may provide information regarding the onset of beta-cell apoptosis.MethodsWe analysed serum PSP/reg1A levels and correlated with clinical and biochemical parameters in subjects with HNF1A-MODY, glucokinase (GCK-MODY), and type 1 diabetes mellitus. A control group of normoglycaemic subjects was also analysed.ResultsPSP/reg1A serum levels were significantly elevated in HNF1A-MODY (n = 37) subjects compared to controls (n = 60) (median = 12.50 ng/ml, IQR = 10.61-17.87 ng/ml versus median = 10.72 ng/ml, IQR = 8.94-12.54 ng/ml, p = 0.0008). PSP/reg1A correlated negatively with insulin levels during OGTT, (rho = −0.40, p = 0.02). Interestingly we noted a significant positive correlation of PSP/reg1A with age of the HNF1A-MODY carriers (rho = 0.40 p = 0.02) with an age of 25 years separating carriers with low and high PSP/reg1A levels. Patients with type 1 diabetes mellitus also had elevated serum levels of PSP/reg1A compared to controls, however this was independent of the duration of diabetes.ConclusionOur data suggest that beta cell apoptosis contributes increasingly to the pathophysiology of HNF1A-MODY in patients 25 years and over. PSP/reg1A may be developed as a serum marker to detect increased beta-cell apoptosis, or its therapeutic response.