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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]

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]

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]

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]

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]

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]