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Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains β-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors. [ABSTRACT FROM AUTHOR]

Whether individuals with insulin resistance (IR) but without criteria for diabetes exhibit reduced mitochondrial oxidative capacity is unclear; addressing this question could guide research for new therapeutics. We investigated 248 participants without diabetes from the Baltimore Longitudinal Study of Aging (BLSA) to determine whether impaired mitochondrial capacity is associated with prediabetes, IR, and duration and severity of hyperglycemia exposure. Mitochondrial capacity was assessed as the postexercise phosphocreatine recovery time constant (τPCr) by 31P-magnetic resonance spectroscopy, with higher τPCr values reflecting reduced capacity. Prediabetes was defined using the American Diabetes Association criteria from fasting and 2-h glucose measurements. IR and sensitivity were calculated using HOMA-IR and Matsuda indices. The duration and severity of hyperglycemia exposure were estimated as the number of years from prediabetes onset and the average oral glucose tolerance test (OGTT) 2-h glucose measurement over previous BLSA visits. Covariates included age, sex, body composition, physical activity, and other confounders. Higher likelihood of prediabetes, higher HOMA-IR, and lower Matsuda index were associated with longer τPCr. Among 205 participants with previous OGTT data, greater severity and longer duration of hyperglycemia were independently associated with longer τPC In conclusion, in individuals without diabetes a more impaired mitochondrial capacity is associated with greater IR and a higher likelihood of prediabetes. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Optimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin-secreting β-cells in the islets of Langerhans. Insulin itself positively regulates β-cell proliferation in an autocrine manner through the insulin receptor (IR) signaling pathway. It is now coming to light that cannabinoid 1 receptor (CBIR) agonism/antagonism influences insulin action in insulin-sensitive tissues. However, the cells on which the CBIRs are expressed and their function in islets have not been firmly established. We undertook the current study to investigate if intraislet endogenous cannabinoids (ECs) regulate β-cell proliferation and if they influence insulin action. RESEARCH DESIGN AND METHODS--We measured EC production in isolated human and mouse islets and β-cell line in response to glucose and KC1. We evaluated human and mouse islets, several β-cell lines, and CB1R-null (CB1R-/-) mice for the presence of a fully functioning EC system. We investigated if ECs influence β-cell physiology through regulating insulin action and demonstrated the therapeutic potential of manipulation of the EC system in diabetic (db/db) mice. RESULTS--ECs are generated within β-cells, which also express CBIRs that are fully functioning when activated by ligands. Genetic and pharmacologic blockade of CBIR results in enhanced IR signaling through the insulin receptor substrate 2-AKT pathway in β-cells and leads to increased β-cell proliferation and mass. CBIR antagonism in db/db mice results in reduced blood glucose and increased β-cell proliferation and mass, coupled with enhanced IR signaling in β-cells. Furthermore, CBIR activation impedes insulin-stimulated IR autophosphorylation on β-cells in a Gαi-dependent manner. CONCLUSIONS--These findings provide direct evidence for a functional interaction between CB1R and IR signaling involved in the regulation of β-cell proliferation and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and proliferation in diabetes. [ABSTRACT FROM AUTHOR]

Objective: Glucose-dependent insulinotropic polypeptide (GIP), unlike glucagon-like peptide (GLP)-1, lacks glucose-lowering properties in patients with type 2 diabetes. We designed this study to elucidate the underlying pathophysiology.Research Design and Methods: Twenty-two insulin-naïve subjects with type 2 diabetes were given either synthetic human GIP (20 ng x kg(-1) x min(-1)) or placebo (normal saline) over 180 min, starting with the first bite of a mixed meal (plus 1 g of acetaminophen) on two separate occasions. Frequent blood samples were obtained over 6 h to determine plasma GIP, GLP-1, glucose, insulin, glucagon, resistin, and acetaminophen levels.Results: Compared with placebo, GIP induced an early postprandial increase in insulin levels. Intriguingly, GIP also induced an early postprandial augmentation in glucagon, a significant elevation in late postprandial glucose, and a decrease in late postprandial GLP-1 levels. Resistin and acetaminophen levels were comparable in both interventions. By immunocytochemistry, GIP receptors were present on human and mouse alpha-cells. In alphaTC1 cell line, GIP induced an increase in intracellular cAMP and glucagon secretion. CONCLUSIONS; GIP, given to achieve supraphysiological plasma levels, still had an early, short-lived insulinotropic effect in type 2 diabetes. However, with a concomitant increase in glucagon, the glucose-lowering effect was lost. GIP infusion further worsened hyperglycemia postprandially, most likely through its suppressive effect on GLP-1. These findings make it unlikely that GIP or GIP receptor agonists will be useful in treating the hyperglycemia of patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Glucose-dependent insulinotropic polypeptide (GIP), unlike glucagon-like peptide (GLP)-1, lacks glucose-lowering properties in patients with type 2 diabetes. We designed this study to elucidate the underlying pathophysiology. RESEARCH DESIGN AND METHODS--Twenty-two insulin-naïve subjects with type 2 diabetes were given either synthetic human GIP (20 ng ⋅ kg-1 ⋅ min-1) or placebo (normal saline) over 180 min, starting with the first bite of a mixed meal (plus 1 g of acetaminophen) on two separate occasions. Frequent blood samples were obtained over 6 h to determine plasma GIP, GLP-1, glucose, insulin, glucagon, resistin, and acetaminophen levels. RESULTS--Compared with placebo, GIP induced an early postprandial increase in insulin levels. Intriguingly, GIP also induced an early postprandial augmentation in glucagon, a significant elevation in late postprandial glucose, and a decrease in late postprandial GLP-1 levels. Resistin and acetaminophen levels were comparable in both interventions. By immunocytochemistry, GIP receptors were present on human and mouse α-cells. In αTC1 cell line, GIP induced an increase in intracellular cAMP and glucagon secretion. CONCLUSIONS--GIP, given to achieve supraphysiological plasma levels, still had an early, short-lived insulinotropic effect in type 2 diabetes. However, with a concomitant increase in glucagon, the glucose-lowering effect was lost. GIP infusion further worsened hyperglycemia postprandially, most likely through its suppressive effect on GLP-1. These findings make it unlikely that GIP or GIP receptor agonists will be useful in treating the hyperglycemia of patients with type 2 diabetes. Diabetes 58:1342-1349, 2009 [ABSTRACT FROM AUTHOR]

Diabetes resulting from heterozygosity for an inactivating mutation of the homeodomain transcription factor insulin promoter factor 1 (IPF-1) is due to a genetic defect of beta-cell function referred to as maturity-onset diabetes of the young 4. IPF-1 is required for the development of the pancreas and mediates glucose-responsive stimulation of insulin gene transcription. To quantitate islet cell responses in a family harboring a Pro63fsdelC mutation in IPF-1, we performed a five-step (1-h intervals) hyperglycemic clamp on seven heterozygous members (NM) and eight normal genotype members (NN). During the last 30 min of the fifth glucose step, glucagon-like peptide 1 (GLP-1) was also infused (1.5 pmol x kg(-1) x min(-1)). Fasting plasma glucose levels were greater in the NM group than in the NN group (9.2 vs. 5.9 mmol/l, respectively; P < 0.05). Fasting insulin levels were similar in both groups (72 vs. 105 pmol/l for NN vs. NM, respectively). First-phase insulin and C-peptide responses were absent in individuals in the NM group, who had markedly attenuated insulin responses to glucose alone compared with the NN group. At a glucose level of 16.8 mmol/l above fasting level, GLP-1 augmented insulin secretion equivalently (fold increase) in both groups, but the insulin and C-peptide responses to GLP-1 were sevenfold less in the NM subjects than in the NN subjects. In both groups, glucagon levels fell during each glycemic plateau, and a further reduction occurred during the GLP-1 infusion. Sigmoidal dose-response curves of glucose clearance versus insulin levels during the hyperglycemic clamp in the two small groups showed both a left shift and a lower maximal response in the NM group compared with the NN group, which is consistent with an increased insulin sensitivity in the NM subjects. A sharp decline occurred in the dose-response curve for suppression of nonesterified fatty acids versus insulin levels in the NM group. We conclude that the Pro63fsdelC IPF-1 mutation is associated with a severe impairment of beta-cell sensitivity to glucose and an apparent increase in peripheral tissue sensitivity to insulin and is a genetically determined cause of beta-cell dysfunction. [ABSTRACT FROM AUTHOR]

Diabetes is caused by a failure of the pancreas to produce insulin in amounts sufficient to meet the body's needs. A hallmark of diabetes is an absolute (type 1) or relative (type 2) reduction in the mass of pancreatic beta-cells that produce insulin. Mature beta-cells have a lifespan of approximately 48-56 days (rat) and are replaced by the replication of preexisting beta-cells and by the differentiation and proliferation of new beta-cells (neogenesis) derived from the pancreatic ducts. Here, we show that the insulinotropic hormone glucagon-like peptide (GLP)-1, which is produced by the intestine, enhances the pancreatic expression of the homeodomain transcription factor IDX-1 that is critical for pancreas development and the transcriptional regulation of the insulin gene. Concomitantly, GLP-1 administered to diabetic mice stimulates insulin secretion and effectively lowers their blood sugar levels. GLP-1 also enhances beta-cell neogenesis and islet size. Thus, in addition to stimulating insulin secretion, GLP-1 stimulates the expression of the transcription factor IDX-1 while stimulating beta-cell neogenesis and may thereby be an effective treatment for diabetes. [ABSTRACT FROM AUTHOR]

The choroid plexus (CP) produces cerebrospinal fluid (CSF) and forms a blood-CSF barrier. We already reported that mouse CP epithelial (CPE) cells synthesize and secrete insulin in response to serotonin but not glucose. We now report that insulin expression is present in human postmortem CP samples of humans - nondiabetic, T2DM, and T1DM - by RT-qPCR, in situ hybridization and LC-MS/MS based Selected Reaction Monitoring assay. Human CPE contains all the INS splicing machinery and the major INS splicing variants that are present in islets. Known β-cell specific transcription factors are also expressed in CPE, except for PDX1 and PAX4, which are absent. The SRM assay detected INS A-chain, B-chain, and C-peptide at similar levels in nondiabetic, T2DM, and T1DM islets and CP postmortem samples. Islet amyloid polypeptide (IAPP) expression was not detected in CPE in contrast to human islets where INS and IAPP mRNA, as expected, colocalize in β-cells. Furthermore, INS colocalizes with transthyretin (TTR) mRNA in CPE cells but in islets, TTR is expressed in α-cells and not β-cells. We found less or no expression of T1DM autoantigens in CPE compared to β-cells, with the exception of ICA1 (islet cell autoantigen1), which has similar expression in both tissues. In conclusion, human CPE expresses and synthesizes insulin, it is protected from autoimmune destruction and from IAPP amyloidogenic fibril formation. Disclosure: Q. Liu: None. M. Zhu: None. C. Mazucanti: None. S. Marenco: None. J. M. Egan: None. Funding: National Institute on Aging [ABSTRACT FROM AUTHOR]

Investigates whether glucagon-like peptide 1 (GLP-1) and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Effects of GLP-1 and exendin-4 on islet hormone expression; Change in amylase concentration upon incubation of AR42J cells with dexamethasone; Influence of GLP-1 on amylase release from dexamethasone-treated cells.

Circulating insulin levels are routinely measured using Enzyme-linked Immunosorbent Assay (ELISA). However, ELISA is not sensitive enough to measure cerebrospinal fluid (CSF) insulin levels which are 10-100 times lower than those of plasma: yet ELISA results are often reported for insulin analysis in CSF where it serves as a surrogate for brain interstitial fluid insulin levels. We developed a novel LC-MS/MS based selected reaction monitoring (SRM) proteomics assay for quantitative analyses targeting predetermined peptides derived from trypsin digests of insulin; each peptide depends on specific SRM transitions. We determined insulin levels using our novel insulin SRM proteomics method in the matching plasma and CSF samples that were from individuals fasting ≥12 hours and individuals after 2 hours of steady state hyperglycemia by clamp technology (2 hr SS HG). All samples were obtained in individuals with normal fasting glucose and HbA1C levels. Statistically significant differences were observed in both plasma and CSF between fasting and 2 hr SS HG by two tailed and unpaired student t test and 2-way matched ANOVA as shown in Table below. Our results confirmed that peripheral endogenous insulin appears in CSF at least 2 hours after steady state hyperglycemia in subjects with normal glucose tolerance. Our SRM insulin tryptic peptide panel reliably quantified insulin peptide levels in human CSF samples. Disclosure: Q. Liu: None. M. Zhu: None. C.W. Chia: None. J.F. O'Connell: None. P. Zhang: None. J.M. Egan: None. Funding: National Institute on Aging [ABSTRACT FROM AUTHOR]

Approximately 1.25 million Americans have type 1 diabetes mellitus (T1DM), resulting from autoimmune β-cell destruction: there is no known way to prevent such destruction of β-cells. We have already reported that activation of cannabinoid 1 receptor (CB1R) contributes to the pathogenesis of pancreatic β-cell dysfunction during high fat feeding. To uncover if CB1R signaling might influence toxin-induced β-cell injury, we induced β-cell destruction in wild type mice (WT: n=8) and in mice in which CB1R was genetically nullified in β-cells only (β-CB1R-/-: n=8) in adulthood by five daily injections of streptozotocin (STZ; 50 mg/kg intraperitoneally [IP]), a toxin selective for β-cells. We found that β-CB1R-/- mice had random blood glucose (BG) levels in the range of 137-220 mg/dL throughout the study period (28 days following STZ) as well as preserved insulin secretion with superior glucose tolerance after a glucose challenge (2g/kg) at day 8, 15 or 27, relative to their WT littermates (random BG 500-600 mg/dL). CB1R-deficient β-cells exhibited significant increases in autophagy flux following STZ. We determined that mice or β-cells lacking CB1R had reduced mTOR activation and increased nuclear translocation of TFEB, a key prerequisite for lysosome biogenesis and autophagy. Importantly, we found that CB1R deletion protected against oxidative stress, endoplasmic reticulum stress, and prevented the induction of apoptosis by STZ. These anti-stress functions were accompanied by increased phosphorylation of AKT (ser) 473 and ERK1/2 in β-cells, which reveals a previously unrecognized route for mTOR/AKT axis to regulate autophagy that coordinately is permissive to β-cell survival. We conclude that CB1R nullification confers resistance to pancreatic β-cell dysfunction by upregulating autophagy and subsequently antagonizing the decline in lysosomal activity. Altogether, we propose that CB1R is a potential therapeutic target during the honeymoon phase of T1DM. Disclosure: K. Aseer: None. J.F. O'Connell: None. J.M. Egan: None. [ABSTRACT FROM AUTHOR]

Approximately 1.25 million Americans have type 1 diabetes mellitus (T1DM), resulting from β-cell destruction. There is no known way to prevent it or to stop destruction of any remaining β-cells. Although the cannabinoid system, specifically cannabinoid 1 receptor (CB1R), is implicated in obesity and T2DM, its role, if any, in T1DM has not been studied. To address this, we induced β-cell destruction in wild type mice (WT: n=8) and mice in which CB1R was genetically nullified in β-cells only (β-CB1R-/-: n=8) in adulthood by five injections of streptozotocin (50 mg/kg intraperitoneally [IP]), a toxin selective for β-cells. We found that β-CB1R-/- mice had random blood glucose (BG) levels in the range of 137-289 mg/dL throughout the study period (28 days) as well as preserved insulin secretion with superior glucose tolerance after IP glucose (2g/kg) at day 8, 15 or 27, relative to their (random BG 500-600 mg/dL) WT littermates. CB1R knockout drove significant increases in pancreatic islet autophagic activity based on increased accumulation of autophagosome hallmarks (LC3B, Beclin-1, ATG5 and ATG7) and reduced p62 expression. Insulin and LC3B were colocalized in CB1R-deficient β-cells. Remarkably, the islets did not show the upregulation of ER unfolded protein response, which was present in β-cells of WT mice, while exerting the induction of protective chaperones. These anti-stress functions were accompanied by increased phosphorylation of AKT (ser) 473 and ERK in β-cells, which reveals a previously unrecognized route for PI3K-AKT pathway to activate the anti-apoptotic NFkB/p65 signaling that coordinately is permissive to β-cell survival. We conclude that CB1R nullification restores autophagy machinery as a novel signaling pathway to counteract ER stress-mediated cell death and confers resistance to β-cell destruction. We propose that CB1R is a potential therapeutic target during the honeymoon phase of T1DM. Disclosure: K. Aseer: None. J.M. Egan: None. [ABSTRACT FROM AUTHOR]

Cannabinoid 1 receptors (CB1Rs) are G protein-coupled receptors that are present in peripheral organs such as liver, pancreas, adipose tissue and skeletal muscle where they are involved in fine-tuning many metabolic functions. It was previously reported that liver-specific genetic deletion of CB1R (hCNR1-/-) mice fed high fat diets (HFD) had similar body weight to HFD-fed hCNR1+/+ mice, but they retained insulin sensitivity comparable to normal chow-fed (NCD) hCNR1+/+ mice. Therefore, this study was undertaken to uncover how hCB1Rs impact the insulin signaling pathway. Male hCNR1-/- and hCNR1+/+ mice were fed with a high fat high sugar diet (HFSD: N = 8-12) for 15 weeks. In contrast with the previous study of HFD only, HFSD-fed hCNR1-/- mice had less body weight gain than HFSD-fed hCNR1+/+ mice (25.1±2.7 vs. 19.8±1.7g), less fat accumulation in the liver, 1.3-fold increase in glucose disposal and 1.7-fold increase in insulin sensitivity compared to HFSD-fed hCNR1+/+ mice. Further study demonstrated that the lack of hepatic CB1R resulted in upregulated phosphorylation in liver of protein kinase B (AKT), causing activation of downstream target molecules, such as proline-rich AKT substrate 40 (PRAS40) in the mammalian target of rapamycin complex 1 (mTORC1). Similarly, primary hepatocytes isolated from hCNR1-/- mice had increased amounts of phosphorylated AKT and PRAS40 in comparison to hepatocytes from hCNR1+/+ mice. The co-immunoprecipitation studies clearly revealed hepatic CB1R modulates insulin signaling by the association or dissociation of the components of mTORC1. In support of these data, the deficiency of hepatic CB1R leads to upregulation of phosphoinositide 3-kinase (PI3K) class IB, p110γ, resulting in increased association between p110γ and G-protein beta. These findings indicate that genetic deletion of hepatic CB1R contributes to improved glucose homeostasis. Therefore, modulation of CB1R activity in liver may be a useful therapeutic in obese and diabetic individuals. Disclosure: Y. Kim: None. J.M. Egan: None. [ABSTRACT FROM AUTHOR]

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin secreted from the enteroendocrine K-cells in response to food. Unlike the other incretin, glucagon-like peptide-1 (GLP-1), GIP is reported to have lost its insulinotropic effect and not have glucose-lowering properties in patients with type 2 diabetes mellitus (T2DM). We attempted to gain insight into the pathophysiology underlying the seeming lack of effects of GIP on glucose homeostasis in T2DM. We therefore administered synthetic human GIP (with a meal) to subjects who had T2DM, and then, for 6hr, took frequent blood samples to measure various factors known to be involved in glucose homeostasis. 22 insulin-naive subjects with T2DM (ages 52.6±9.3years; BMI 36.7±7.3kg/m²; HbA[sub 1c] 7.4±1.5%; fasting plasma glucose 155±46mg/dl; diabetes duration 4.3±4.2years; f/m ratio 13/9) that had been treated with diet alone, metformin and/or sulfonylurea were recruited. They stopped oral hypoglycemic medications for 5 days prior to testing and fasted for 8hr prior to 2 study visits, each visit separated by not less than 6 weeks. After 3 baseline blood draws, the subjects received continuous intravenous administration of either placebo (0.9%NaCl) or GIP (20ng/kg/min) for 180min starting with the first bite of a 550calorie mixed-meal, and 1g of acetaminophen to study gastric emptying. Frequent blood sampling took place over the subsequent 6hr (every 5min for the first 75min then every 15min until 180min and every 20min until 360min). Compared to placebo, GIP induced an increase in plasma insulin from 5-30min but a transient decrease in plasma glucose at the 10min time-point only (p<0.05). Intriguingly, and hitherto undescribed, GIP induced a later augmentation in glucagon secretion with a peak level at approximately 30min (p<0.05), a significant elevation in plasma glucose from 120-220min (p<0.05) and a decrease in GLP-I secretion at 135min that lasted for the remainder of the 6hr study period (p<0.05). Based on plasma acetaminophen levels, GIP did not affect gastric emptying and NEFAs were similar during both studies. In T2DM therefore, GIP, given at a pharmacological dose with a meal, still has an early, short-lived insulinotropic effect, resulting in a transient, clinically irrelevant plasma glucose drop. However, this was subsequently followed by increased plasma glucagon levels and worsening hyperglycemia. These findings make it unlikely that GIP or GIP receptor agonists will ever be useful as glucose-lowering agents. [ABSTRACT FROM AUTHOR]