Pancreatic α cells have been proposed as an intra-islet source of intact GLP-1, thereby constituting a gut-independent paracrine incretin system. However, it is debated whether human α cells contain intact GLP-1 and if this relates to the presence of diabetes. The aim of this study was to determine the presence of proglucagon-derived peptides, including GLP-1 isoforms, in pancreas biopsies obtained during partial pancreatectomy from metabolically profiled human donors, stratified according to preoperative glucose tolerance. We enrolled 61 individuals with no known history of type 2 diabetes (31F/30M, age 64.6±10.6 yrs., BMI 24.2±3.68 kg/m2) scheduled for partial pancreatectomy for periampullary neoplasm. Preoperative differences in glucose tolerance and insulin secretion/sensitivity were assessed using preoperative 2h OGTT and a 4h-Mixed Meal Test; subjects were subsequently classified according to normal glucose tolerance (NGT, n=19), impaired glucose tolerance (IGT, n=20) or newly diagnosed diabetes (DM) (n=22). We measured total GLP-1, intact GLP-1, glucagon, insulin, and C-peptide in pancreas biopsies and plasma from these individuals and correlated the results to patients’ secretory and metabolic parameters. For each test we derived functional parameters describing the main features of insulin secretion rate (ISR): glucose sensitivity, glucose-standardized at 5.5 mmol ISR (5.5@ISR). Extractable levels of total GLP-1 were 23.9±2.66 pmol/g, while intact GLP-1 were 1.15±0.18 pmol/g. When subjects were classified according to glucose tolerance and proglucagon-derived peptide levels (adjusted for glucagon levels), we observed similar levels of total GLP-1, while intact GLP-1 was significantly increased in IGT (p=0.15) and DM groups (p=0.01) and inversely associated to beta-cell glucose sensitivity (r=-0.31, p=0.02) and 5.5@IRS in vivo (r=-0.38, p<0.01). Our data show that development of glucose intolerance and β-cell dysfunction are significantly associated with increased levels of intra-islet intact GLP-1, providing evidence for a paracrine role of this hormone in the pathophysiology of type 2 diabetes.