Occurrence of giant focal forms of congenital hyperinsulinism with incorrect visualization by (18) F DOPA-PET/CT scanning.

CONTEXT: Congenital hyperinsulinism (CHI) is a rare disease characterized by severe hypoglycaemic episodes due to pathologically increased insulin secretion from the pancreatic beta cells. When untreated, CHI might result in irreversible brain damage and death. Currently, two major subtypes of CHI are known: a focal form, associated with local distribution of affected beta cells and a nonfocal form, affecting every single beta cell. The identification of focal forms is important, as the patients can be cured by limited surgery. (18) F DOPA-PET/CT is an established non-invasive approach to differentiate focal from nonfocal CHI. OBJECTIVE: The purpose of this study was to identify possible limitations of (18) F DOPA-PET/CT scan in patients with focal forms nonfocal CHI. DESIGN: A retrospective chart review of 32 patients (from 2008 through 2013) who underwent (18) F DOPA-PET/CT and partial pancreatectomy for focal CHI at the reference centres in Berlin, Germany and London, UK. RESULTS: In most cases (n = 29, 90·7%), (18) F DOPA-PET/CT was sufficient to localize the complete focal lesion. However, in some patients (n = 3, 9·3%), (18) F DOPA-PET/CT wrongly visualized only a small portion of the focal lesion. In this group of patients, a so-called 'giant focus' was detected in histopathological analysis during the surgery. CONCLUSIONS: Our data show that in most patients with focal CHI (18) F DOPA-PET/CT correctly predicts the size and anatomical localisation of the lesion. However, in those patients with a 'giant focal' lesion (18) F DOPA-PET/CT is unreliable for correct identification of 'giant focus' cases.

Novel biological action of the dipeptidylpeptidase-IV inhibitor, sitagliptin, as a glucagon-like peptide-1 secretagogue.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted into the circulation by the intestinal L cell. The dipeptidylpeptidase-IV (DPP-IV) inhibitor, sitagliptin, prevents GLP-1 degradation and is used in the clinic to treat patients with type 2 diabetes mellitus, leading to improved glycated hemoglobin levels. When the effect of sitagliptin on GLP-1 levels was examined in neonatal streptozotocin rats, a model of type 2 diabetes mellitus, a 4.9 ± 0.9-fold increase in basal and 3.6 ± 0.4-fold increase in oral glucose-stimulated plasma levels of active GLP-1 was observed (P < 0.001), in association with a 1.5 ± 0.1-fold increase in the total number of intestinal L cells (P < 0.01). The direct effects of sitagliptin on GLP-1 secretion and L cell signaling were therefore examined in murine GLUTag (mGLUTag) and human hNCI-H716 intestinal L cells in vitro. Sitagliptin (0.1-2 µM) increased total GLP-1 secretion by mGLUTag and hNCI-H716 cells (P < 0.01-0.001). However, MK0626 (1-50 µM), a structurally unrelated inhibitor of DPP-IV, did not affect GLP-1 secretion in either model. Treatment of mGLUTag cells with the GLP-1 receptor agonist, exendin-4, did not modulate GLP-1 release, indicating the absence of feedback effects of GLP-1 on the L cell. Sitagliptin increased cAMP levels (P < 0.01) and ERK1/2 phosphorylation (P < 0.05) in both mGLUTag and hNCI-H716 cells but did not alter either intracellular calcium or phospho-Akt levels. Pretreatment of mGLUTag cells with protein kinase A (H89 and protein kinase inhibitor) or MAPK kinase-ERK1/2 (PD98059 and U0126) inhibitors prevented sitagliptin-induced GLP-1 secretion (P < 0.05-0.01). These studies demonstrate, for the first time, that sitagliptin exerts direct, DPP-IV-independent effects on intestinal L cells, activating cAMP and ERK1/2 signaling and stimulating total GLP-1 secretion.

Mechanisms underlying metformin-induced secretion of glucagon-like peptide-1 from the intestinal L cell.

Glucagon-like peptide-1(7-36NH2) (GLP-1) is secreted by the intestinal L cell in response to both nutrient and neural stimulation, resulting in enhanced glucose-dependent insulin secretion. GLP-1 is therefore an attractive therapeutic for the treatment of type 2 diabetes. The antidiabetic drug, metformin, is known to increase circulating GLP-1 levels, although its mechanism of action is unknown. Direct effects of metformin (5-2000 µm) or another AMP kinase activator, aminoimidazole carboxamide ribonucleotide (100-1000 µm) on GLP-1 secretion were assessed in murine human NCI-H716, and rat FRIC L cells. Neither agent stimulated GLP-1 secretion in any model, despite increasing AMP kinase phosphorylation (P < 0.05-0.01). Treatment of rats with metformin (300 mg/kg, per os) or aminoimidazole carboxamide ribonucleotide (250 mg/kg, sc) increased plasma total GLP-1 over 2 h, reaching 37 ± 9 and 29 ± 9 pg/ml (P < 0.001), respectively, compared with basal (7 ± 1 pg/ml). Plasma activity of the GLP-1-degrading enzyme, dipeptidylpeptidase-IV, was not affected by metformin treatment. Pretreatment with the nonspecific muscarinic antagonist, atropine (1 mg/kg, iv), decreased metformin-induced GLP-1 secretion by 55 ± 11% (P < 0.05). Pretreatment with the muscarinic (M) 3 receptor antagonist, 1-1-dimethyl-4-diphenylacetoxypiperidinium iodide (500 µg/kg, iv), also decreased the GLP-1 area under curve, by 48 ± 8% (P < 0.05), whereas the antagonists pirenzepine (M1) and gallamine (M2) had no effect. Furthermore, chronic bilateral subdiaphragmatic vagotomy decreased basal secretion compared with sham-operated animals (7 ± 1 vs. 13 ± 1 pg/ml, P < 0.001) but did not alter the GLP-1 response to metformin. In contrast, pretreatment with the gastrin-releasing peptide antagonist, RC-3095 (100 µg/kg, sc), reduced the GLP-1 response to metformin, by 55 ± 6% (P < 0.01) at 30 min. These studies elucidate the mechanism underlying metformin-induced GLP-1 secretion and highlight the benefits of using metformin with dipeptidylpeptidase-IV inhibitors in patients with type 2 diabetes.

Essential role for protein kinase Cζ in oleic acid-induced glucagon-like peptide-1 secretion in vivo in the rat.

Luminal monounsaturated long-chain fatty acids [e.g. oleic acid (OA)] increase secretion of the incretin, glucagon-like peptide-1 (GLP-1) from the ileocolonic L cell. However, it is not known whether OA ingestion causes a sufficient increase in distal luminal concentrations to directly enhance GLP-1 secretion. Furthermore, we have demonstrated that protein kinase Cζ (PKCζ) is required for OA-induced GLP-1 secretion in vitro; however, the physiological relevance of this finding remains unknown. Therefore, we have determined luminal OA concentrations in OA-fed rats and examined the effects of direct OA stimulation on GLP-1 secretion using a novel model of intestinal-specific PKCζ knockdown. Murine GLUTag L cells express numerous fatty acid transport proteins and take up OA in a saturable manner. Oral administration of OA increased the ileal chyme content of OA by 140-fold over 60-120 min (P < 0.05-0.01), peaking at 105 ± 50 µmol/g. To evaluate the direct effects of OA on GLP-1 secretion, 125 mm OA was rectally infused into the colon and terminal ileum of rats. Plasma bioactive GLP-1 increased from 20 ± 6 to 102 ± 21 pg/ml at 60 min (P < 0.01). However, pretreatment with ileocolonic adenoviral PKCζ small interfering RNA resulted in a 68 ± 8% reduction in the GLP-1 response to rectal OA (P < 0.001). The results of these studies indicate that OA levels in the rat terminal gut after oral ingestion are sufficient to induce GLP-1 secretion and that PKCζ is necessary for the effects of OA on GLP-1 secretion in vivo. PKCζ may therefore serve as a novel therapeutic target to enhance GLP-1 levels in patients with type 2 diabetes.

Carcinogenic effects of exogenous and endogenous glucagon-like peptide-2 in azoxymethane-treated mice.

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent intestinotropic hormone that promotes intestinal growth, via increased intestinal proliferation and decreased apoptosis, as well as increases in nutrient absorption and barrier function. The long-acting analog h(Gly(2))GLP-2[1-33] is currently being tested for treatment of short bowel syndrome and Crohn's disease. However, the role of GLP-2 in colon carcinogenesis is controversial. To assess the intestinotropic effects of exogenous and endogenous GLP-2, C57BL6/J mice were injected with 1 microg h(Gly(2))GLP-2[1-33]; 30 or 60 ng hGLP-2[3-33], a GLP-2 receptor antagonist; or PBS (4 wk, twice a day, sc). Chronic h(Gly(2))GLP-2[1-33] increased small intestinal weight/body weight (P < 0.001), villus height (P < 0.001), crypt depth (P < 0.001), and crypt cell proliferation, as measured by expression of the proliferative marker Ki67 (P < 0.05-0.01). In contrast, chronic hGLP-2[3-33] decreased small intestinal weight/body weight (P < 0.05) and colon weight/body weight (P < 0.05). To assess the carcinogenic effects of endogenous and exogenous GLP-2, separate mice were injected with azoxymethane (10 mg/kg, 4 wk, every 7 d, ip), followed by 1.5 microg h(Gly(2))GLP-2[1-33], 30 ng hGLP-2[3-33], or PBS (4 wk, twice a day, sc) 2 or 12 wk thereafter. At 10 or 46 wk after azoxymethane treatment, the numbers of aberrant crypt foci increased with h(Gly(2))GLP-2[1-33] (P < 0.001) and decreased with hGLP-2[3-33] (P < 0.01-0.05) treatment. Furthermore, mucin-depleted aberrant foci, consistent with progressive dysplasia, were almost exclusively present in h(Gly(2))GLP-2[1-33]-treated mice (P < 0.01-0.001). Additionally, adenocarcinomas developed in h(Gly(2))GLP-2[1-33]-treated mice but not in those receiving hGLP-2[3-33] or PBS. Taken together, these studies indicate that chronic treatment with GLP-2 enhances colon carcinogenesis, whereas antagonism of the GLP-2 receptor decreases dysplasia, with possible implications for human therapy.

GPR119 is essential for oleoylethanolamide-induced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L-cell.

OBJECTIVE: Intestinal L-cells secrete the incretin glucagon-like peptide-1 (GLP-1) in response to ingestion of nutrients, especially long-chain fatty acids. The Galphas-coupled receptor GPR119 binds the long-chain fatty acid derivate oleoylethanolamide (OEA), and GPR119 agonists enhance GLP-1 secretion. We therefore hypothesized that OEA stimulates GLP-1 release through a GPR119-dependent mechanism. RESEARCH DESIGN AND METHODS: Murine (m) GLUTag, human (h) NCI-H716, and primary fetal rat intestinal L-cell models were used for RT-PCR and for cAMP and GLP-1 radioimmunoassay. Anesthetized rats received intravenous or intraileal OEA, and plasma bioactive GLP-1, insulin, and glucose levels were determined by enzyme-linked immunosorbent assay or glucose analyzer. RESULTS: GPR119 messenger RNA was detected in all L-cell models. OEA treatment (10 micromol/l) of mGLUTag cells increased cAMP levels (P < 0.05) and GLP-1 secretion (P < 0.001) in all models, with desensitization of the secretory response at higher concentrations. GLP-1 secretion was further enhanced by prevention of OEA degradation using the fatty acid amide hydrolase inhibitor, URB597 (P < 0.05-0.001 vs. OEA alone), and was abolished by H89-induced inhibition of protein kinase A. OEA-induced cAMP levels and GLP-1 secretion were significantly reduced in mGLUTag cells transfected with GPR119-specific small interfering RNA (P < 0.05). Application of OEA (10 micromol/l) directly into the rat ileum, but not intravenously, increased plasma bioactive GLP-1 levels in euglycemic animals by 1.5-fold (P < 0.05) and insulin levels by 3.9-fold (P < 0.01) but only in the presence of hyperglycemia. CONCLUSIONS: The results of these studies demonstrate, for the first time, that OEA increases GLP-1 secretion from intestinal L-cells through activation of the novel GPR119 fatty acid derivate receptor in vitro and in vivo.

Immune restoration in head and neck cancer patients after in vivo COX-2 inhibition.

PURPOSE: To determine the immunomodulatory effects of in vivo COX-2 inhibition on leukocyte infiltration and function in patients with head and neck cancer. EXPERIMENTAL DESIGN: Patients with squamous cell carcinoma of the head and neck preoperatively received a specific COX-2 inhibitor (rofecoxib, 25 mg daily) orally for 3 weeks. Serum and tumor specimens were collected at the start of COX-2 inhibition (day 0) and again on the day of surgery (day 21). Adhesion to peripheral blood monocytes to ICAM-1 was examined. Percentages of tumor-infiltrating monocytes (CD68, CCR5) and lymphocytes (CCR5, CD4, CD8 and CD25) were determined by immunohistochemistry. RESULTS: Monocytes obtained from untreated cancer patients showed lower binding to ICAM-1 compared to monocytes of healthy donors but significantly regained adhesion affinity following incubation in sera of healthy donors. Conversely, sera of cancer patients inhibited adhesion of healthy donors' monocytes. Tumor monocyte adhesion to ICAM-1 was increased (P<0.001) after 21 days of COX-2 inhibition, and concomitant increases in tumor infiltrating monocytes (CD68+), lymphocytes (CD68- CCR5+, CD4+ and CD8+) and activated (CD25+) T cells were observed. CONCLUSIONS: Short-term administration of a COX2 inhibitor restored monocyte binding to ICAM-1 and increased infiltration into the tumor of monocytes and Th1 and CD25+ activated lymphocytes. Thus, in vivo inhibition of the COX-2 pathway may be useful in potentiating specific active immunotherapy of cancer.

Granulomatous Pneumocystis carinii pneumonia in a non-AIDS patient: an atypical presentation.

We present the computed tomographic findings of pulmonary involvement by granulomatous Pneumocystis carinii pneumonia in a 73-year-old woman recently tapered from a high-dose long-term systemic corticosteroid therapy for Factor VII deficiency.

Impaired monocyte function in cancer patients: restoration with a cyclooxygenase-2 inhibitor.

Epidemiological data and animal models have provided evidence that nonsteroidal antiinflammatory drugs (NSAIDs) have an anticancer effect. However, the molecular mechanisms underlying these antineoplastic effects are not well understood. We described previously that expression levels of the chemokine receptor, CCR5, and the beta2-integrin, Mac-1, were down-regulated on primary monocytes after incubation in supernatants from human carcinoma cell lines, and that this down-regulation resulted in impaired monocyte function with respect to migration and adhesion. We now demonstrate that these impairments are also present in vivo. Monocytes from cancer patients displayed significantly reduced CCR5 levels and migration capacities in comparison to cells from healthy donors. Because migration is necessary for the antitumor activity of monocytes/macrophages, these deficits may contribute to the suppressed immune system seen in cancer patients. In a clinical study, we analyzed the effect of a selective COX-2 inhibitor, Rofecoxib, on the migration of monocytes derived from cancer patients. The results revealed significant improvement in migration equal to those levels seen in healthy donors. We conclude that in patients with cancer, the intake of Rofecoxib for 3 wk leads to significant restoration of monocyte function. These data may, at least in part, help explain the anticancer effects of NSAIDs.