Glucagon stimulates hepatic glucose production through its direct effects on the liver but may indirectly inhibit this process by acting on the brain. This was tested by delivering glucagon via the cerebral circulatory system. Central nervous system glucagon action reduced liver gluconeogenic flux, but glycogenolysis increased, resulting in no net change in hepatic glucose production. Surprisingly, brain glucagon also appeared to suppress lipolysis (plasma free fatty acid and glycerol levels were reduced).

The purpose of this study was to assess insulin-stimulated gene expression in canine skeletal muscle with a particular focus on NPPC, the gene that encodes C-type natriuretic peptide, a key hormonal regulator of cardiometabolic function. Four conscious canines underwent hyperinsulinemic, euglycemic clamp studies. Skeletal muscle biopsy and arterial plasma samples were collected under basal and insulin-stimulated conditions. Bulk RNA sequencing of muscle tissue was performed to identify differentially expressed genes between these 2 steady-state conditions. Our results showed that NPPC was the most highly expressed gene in skeletal muscle in response to insulin infusion, rising 4-fold between basal and insulin-stimulated conditions. In support of our RNA sequencing data, we found that raising the plasma insulin concentration 15-fold above basal elicited a 2-fold (P = 0.0001) increase in arterial plasma concentrations of N-terminal prohormone C-type natriuretic peptide. Our data suggest that insulin may play a role in stimulating secretion of C-type natriuretic peptide by skeletal muscle. In this context, C-type natriuretic peptide may act in a paracrine manner to facilitate muscle-vascular bed crosstalk and potentiate insulin-mediated vasodilation. This could serve to enhance insulin and glucose delivery, particularly in the postprandial absorptive state.

Glucagon’s effect on hepatic glucose production (HGP), under hyperglycemic conditions, is time dependent such that after an initial burst of HGP, it slowly wanes. It is not known whether this is also the case under hypoglycemic conditions, where an increase in HGP is essential. This question was addressed using adrenalectomized dogs to avoid the confounding effects of other counterregulatory hormones. During the study, infusions of epinephrine and cortisol were given to maintain basal levels. Somatostatin and insulin (800 µU·kg−1·min−1) were infused to induce hypoglycemia. After 30 min, glucagon was infused at a basal rate (1 ng·kg−1·min−1, baGGN group, n = 5 dogs) or a rate eightfold basal (8 ng·kg−1·min−1, hiGGN group, n = 5 dogs) for 4 h. Glucose was infused to match the arterial glucose levels between groups (≈50 mg/dL). Our data showed that glucagon has a biphasic effect on the liver despite hypoglycemia. Hyperglucagonemia stimulated a rapid, transient peak in HGP (4-fold basal production) over ~60 min, which was followed by a slow reduction in HGP to a rate 1.5-fold basal. During the last 2 h of the experiment, hiGGN stimulated glucose production at a rate fivefold greater than baGGN (2.5 vs. 0.5 mg·kg−1·min−1, respectively), indicating a sustained effect of the hormone. Of note, the hypoglycemia-induced rises in norepinephrine and glycerol were smaller in hiGGN compared with the baGGN group despite identical hypoglycemia. This finding suggests that there is reciprocity between glucagon and the sympathetic nervous system such that when glucagon is increased, the sympathetic nervous response to hypoglycemia is downregulated.

Peripheral hyperinsulinemia resulting from subcutaneous insulin injection is associated with metabolic defects which include abnormal glucose metabolism. The first aim of this study was to quantify the impairments in liver and muscle glucose metabolism that occur when insulin is delivered via a peripheral vein compared to when it is given through its endogenous secretory route (the hepatic portal vein) in overnight fasted conscious dogs. The second aim was to determine if peripheral delivery of a hepato-preferential insulin analog could restore the physiologic response to insulin that occurs under meal feeding conditions. This study is the first to show that hepatic glucose uptake correlates with insulin's direct effects on the liver under hyperinsulinemic-hyperglycemic conditions. In addition, glucose uptake was equally divided between the liver and muscle when insulin was infused into the portal vein, but when it was delivered into a peripheral vein the percentage of glucose taken up by muscle was 4-times greater than that going to the liver, with liver glucose uptake being less than half of normal. These defects could not be corrected by adjusting the dose of peripheral insulin. On the other hand, hepatic and non-hepatic glucose metabolism could be fully normalized by a hepato-preferential insulin analog.

With the recent interest in sympathetic modulation as a way to improve insulin sensitivity and insulin secretion, it is important to assess the safety of the procedure. Five dogs were fed a high fat high fructose diet for 5-week before undergoing a hyperinsulinemic hypoglycemic clamp (insulin infused IV at a rate of 1.5 mU/kg/min). Then the dogs underwent a common hepatic artery surgical denervation (CHAD n=5) and were studied 1, 2 and 3 months post-surgery with the same hypoglycemic challenge. At the end of the protocol, the norepinephrine content in the liver (7±3 ng/g) and pancreas (91±34 ng/g) were significantly lower than levels in the livers and pancreas of 4 sham denervated animals (460±100 and 590±93 ng/g respectively), indicating successful denervation. There were no significant differences in fasting insulin, glucose or glucagon concentrations over the 3 months (see Table). The insulin and glucose concentrations during the last 90 min of the hypoglycemic challenge prior to and post-CHAD were also not different. The peak in plasma glucagon and epinephrine levels occurred at 30 and 120 min respectively and were similar in magnitude pre- and post-CHAD. Four sham denervated animals had the same response to the insulin induced hypoglycemic challenge as CHAD dogs. In conclusion, the response to hypoglycemia was not affected by sympathetic denervation of the common hepatic artery. Disclosure G. Kraft: Consultant; Self; Metavention. L. Moore: None. B. Farmer: None. M.F. Scott: None. D.S. Edgerton: Speaker's Bureau; Self; Novo Nordisk A/S. P.E. Williams: None. B. Azamian: Employee; Self; Metavention. A.D. Cherrington: Board Member; Self; Biocon, Fractyl Laboratories, Inc., Metavention, Sensulin, LLC., vTv Therapeutics, Zafgen, Inc. Consultant; Self; Abvance, Boston Scientific Corporation, California Institute for Biomedical Research, Galvani Bioelectronics Limited, MedImmune, Novo Nordisk Inc., Thetis Pharmaceuticals LLC. Research Support; Self; Abvance, Diasome Pharmaceuticals, Inc., Novo Nordisk Inc. Stock/Shareholder; Self; Abvance, Biocon, Fractyl Laboratories, Inc., Metavention, Novo Nordisk Inc., Sensulin, LLC., Thetis Pharmaceuticals LLC, Zafgen, Inc. Funding Metavention, Inc.

In heathy individuals the liver is a primary site of glucose disposal, it is in a state of glucose uptake most of the day, and this process is impaired in diabetes. Insulin stimulates hepatic glucose uptake (HGU) but the importance of its indirect effects (at fat, brain and pancreatic alpha cells) in driving HGU is unclear. Thus, the aim of this study was to determine the impact of insulin’s indirect mechanisms of control of HGU. Postprandial conditions were simulated for 4-hour in conscious dogs (n=7/group) by infusing somatostatin, intraportal insulin (1.8 mU/kg/min) and glucose (4 mg/kg/min), and leg vein glucose (to clamp arterial glucose at 2-fold basal). In one group insulin’s direct and indirect effects on the liver were both present (D+I) while in the other insulin’s indirect effects were blocked (D-only). In D+I the basal intraportal glucagon infusion rate was decreased to create a progressive decline in glucagon over time, FFA levels were allowed to fall, and brain insulin action was allowed to increase. In D-only glucagon and FFA levels were maintained at basal levels by infusions of intraportal glucagon and leg vein lipid emulsion. Hypothalamic insulin action was blocked by 3rd ventricle infusion of an insulin receptor antagonist. In the D+I and D-only groups, respectively, hepatic insulin increased during the experimental period (from 20±3 to 88±8 and 21±3 to 89±8 µU/ml), hepatic glucagon fell or remained basal (39±5 to 24±3 and 40±6 to 41±2 pg/ml) and arterial FFA fell or remained basal (1069±19 to 99±23 and 1195±63 to 998±99 µmol/l). HGU and net hepatic glucose uptake (NHGU) were independently measured using tracer and cold glucose balance techniques. The AUCs for HGU and NHGU (mg/kg/240 min) in D+I were 841±143 and 792±105, respectively, and 738±130 and 698±58 in D-only (P=0.6 and 0.4 between groups, respectively). Thus, in the healthy animal, HGU responds normally to insulin even in the absence of the hormone’s indirect effects. Instead HGU is driven almost exclusively by insulin’s direct effects. Disclosure D.S. Edgerton: Speaker's Bureau; Self; Novo Nordisk A/S. G. Kraft: Consultant; Self; Metavention. B. Farmer: None. M.S. Smith: None. P.E. Williams: None. A.D. Cherrington: Board Member; Self; Biocon, Fractyl Laboratories, Inc., Metavention, Sensulin, LLC., vTv Therapeutics, Zafgen, Inc. Consultant; Self; Abvance, Boston Scientific Corporation, California Institute for Biomedical Research, Galvani Bioelectronics Limited, MedImmune, Novo Nordisk Inc., Thetis Pharmaceuticals LLC. Research Support; Self; Abvance, Diasome Pharmaceuticals, Inc., Novo Nordisk Inc. Stock/Shareholder; Self; Abvance, Biocon, Fractyl Laboratories, Inc., Metavention, Novo Nordisk Inc., Sensulin, LLC., Thetis Pharmaceuticals LLC, Zafgen, Inc. Funding National Institutes of Health

We observed that a 4-h morning (AM) duodenal infusion of glucose versus saline doubled hepatic glucose uptake (HGU) and storage during a hyperinsulinemic–hyperglycemic (HIHG) clamp that afternoon (PM). To separate the effects of AM hyperglycemia versus AM hyperinsulinemia on the PM response, we used hepatic balance and tracer ([3-3H]glucose) techniques in conscious dogs. From 0 to 240 min, dogs underwent a euinsulinemic-hyperglycemic (GLC; n = 7) or hyperinsulinemic-euglycemic (INS; n = 8) clamp. Tracer equilibration and basal sampling occurred from 240 to 360 min, followed by an HIHG clamp (360–600 min; four times basal insulin, two times basal glycemia) with portal glucose infusion (4 mg ⋅ kg−1 ⋅ min−1). In the HIHG clamp, HGU (5.8 ± 0.9 vs. 3.3 ± 0.3 mg ⋅ kg−1 ⋅ min−1) and net glycogen storage (6.0 ± 0.8 vs. 2.9 ± 0.5 mg ⋅ kg−1 ⋅ min−1) were approximately twofold greater in INS than in GLC. PM hepatic glycogen content (1.9 ± 0.2 vs. 1.3 ± 0.2 g/kg body weight) and glycogen synthase (GS) activity were also greater in INS versus GLC, whereas glycogen phosphorylase (GP) activity was reduced. Thus AM hyperinsulinemia, but not AM hyperglycemia, enhanced the HGU response to a PM HIHG clamp by augmenting GS and reducing GP activity. AM hyperinsulinemia can prime the liver to extract and store glucose more effectively during subsequent same-day meals, potentially providing a tool to improve glucose control.

Epidemiologic studies have identified gastroesophageal reflux disease (GERD) as the strongest risk factor for esophageal adenocarcinoma.1Souza R.F. Dig Dis. 2016; 34: 483-490Crossref PubMed Scopus (46) Google Scholar However, it remains not well understood how gastroesophageal reflux facilitates tumor development. In this study, we investigated, for the first time, the role of isolevuglandins (isoLGs), which are formed through free radical and enzymatic cyclooxygenation of polyunsaturated fatty acids.2Salomon R.G. et al.Antioxid Redox Signal.. 2015; 22: 1703-1718Google Scholar Structurally, isoLGs are categorized as lipid-derived γ-ketoaldehydes that are highly reactive with free amines on lysine residues forming LG-lysine lactam protein adducts, protein-protein, and protein-DNA crosslinks.2Salomon R.G. et al.Antioxid Redox Signal.. 2015; 22: 1703-1718Google Scholar, 3Davies S.S. et al.FASEB J. 2002; 16: 715-717Crossref PubMed Scopus (93) Google Scholar To investigate the reflux-induced cellular alterations, we exposed esophageal cells derived from the normal esophagus (EPC2), Barrett's esophagus (CP-A), and cancer (TE-7) to acidic growth medium (pH 4.0), supplemented with 100 μM bile salts cocktail (BA/A). The composition, total bile salts concentration and pH, were selected based on previous measurements conducted in patients with GERD.4Dvorak K. et al.Gut. 2007; 56: 763-771Crossref PubMed Scopus (200) Google Scholar IsoLGs were analyzed using D11 single chain antibody. This antibody was generated by screenings of phage-display libraries and tested to specifically recognize isoLG protein adducts independently of protein amino acid sequences.5Yan H.P. et al.Free Radic Biol Med. 2017; 106: 62-68Crossref PubMed Scopus (12) Google Scholar, 6Kirabo A. et al.J Clin Invest. 2014; 124: 4642-4656Crossref PubMed Scopus (308) Google Scholar We found that treatment of esophageal cells with acidic bile salts led to significant accumulation of isoLG protein adducts compared with untreated control (Figure 1A, Supplementary Figure 1A and B). Notably, multiple proteins were adducted after treatment with BA/A, which was indicated by a strong increase in the intensities of multiple protein bands. Induction of isoLG protein adducts was also observed using immunofluorescence with D11 scFv (Supplementary Figure 2). The formation of LG-lysine lactam adducts was further verified by liquid chromatography-electrospray-ionization-tandem mass spectrometry in TE-7 cells. Our quantification analyses were based on specific transitions from the molecular ion at m/z = 479.2 to the specific fragment at m/z = 332.1. The elution of the 2 fragment ions found in BA/A-treated samples was similar to the [13C6] lysine–lactam internal standard. As an additional control we analyzed TE-7 cells treated with BA/A in the presence of the specific isoLG scavenger 2-hydroxybenzylamine (2-HOBA; 50 μM).7Davies S.S. et al.Biochemistry. 2006; 45: 15756-15767Crossref PubMed Scopus (43) Google Scholar Mass spectrometry analyses found an upregulation of LG-lysine lactam adducts after treatment of esophageal cells with BA/A. Treatment with 2-HOBA inhibited the effect of BA/A, providing further support to our findings (Supplementary Figure 1C). To investigate the formation of isoLG protein adducts in vivo, we used a mouse model of esophageal reflux injury.8Zaika E. et al.FASEB J. 2011; 25: 4406-4414Crossref PubMed Scopus (0) Google Scholar A section of the mouse jejunum was transected and then anastomosed to the esophagus resulting in increased reflux. Using immunohistochemistry with D11 antibody, the levels of isoLG protein adducts were compared in esophageal specimens collected from animals with reflux and control animals with sham surgery (n = 12). We found significant accumulation of isoLG protein adducts in the esophagus of animals affected by reflux (Figure 1B). To further investigate isoLG adducts in vivo, we conducted a small-scale study in patients with GERD. Esophageal biopsies collected from 10 GERD patients and 9 healthy individuals were immunostained with D11 antibody and analyzed for isoLG protein adducts. We found a trend toward increasing formation of isoLG protein adducts in patients with GERD (P = .07). An increased staining for the isoLG adducts was observed in 50% (5 out of 10) of patients with GERD (Figure 1C). Normal subjects primarily showed low or undetectable levels of isoLGs. Interestingly, some patients with GERD showed a strong nuclear staining showing that adducts form on nuclear proteins (Figure 1C, inset). To investigate how induction of isoLGs by reflux affects proteins, we focused our studies on the regulation of p53 protein because it plays a key tumor suppressor role in the esophagus. Given that isoLGs have strong hydrophobic properties and may cause protein aggregation,9Bi W. et al.Chem Res Toxicol. 2016; 29: 1628-1640Crossref PubMed Scopus (12) Google Scholar we separately analyzed soluble and insoluble cellular fractions, which were prepared as described in the Supplementary Methods section. Surprisingly, we found that the solubility of the p53 protein significantly decreased after treatment with BA/A, whereas the insoluble cellular fraction was enriched with p53 protein aggregates in all tested cell lines (Figure 2A, Supplementary Figure 3A and B). p53 precipitation was prevented by 2-HOBA, suggesting that isoLGs are responsible for the precipitation of p53 protein (Figure 2A, Supplementary Figure 3A and B). To directly analyze p53 protein adducts, cellular lysates were immunoprecipitated with p53-specific antibody (DO1). The immunoprecipitated p53 protein was then analyzed for the isoLG adducts by Western blotting using D11 scFv. We found that BA/A led to the formation of p53 isoLG protein adducts. Notably, 2-HOBA inhibited the formation of adducts on p53 protein further supporting our findings (Figure 2B, Supplementary Figure 3C). Then, the effect of 2-HOBA was compared with antioxidants (tempol and N-acetylcysteine), which were shown to inhibit the production of reactive oxygen species by acidic bile salts at tested concentrations.10Bhardwaj V. et al.Carcinogenesis. 2016; 37: 1161-1169PubMed Google Scholar Although both tested antioxidants had some inhibitory effect, suppression of isoLG protein adducts was significantly stronger by 2-HOBA (Supplementary Figure 4). In summary, this study revealed, for the first time, that gastroesophageal reflux leads to the formation of isoLG and accumulation of isoLG protein adducts causing precipitation and inactivation of p53 tumor suppressor. We also found that isoLG scavenger 2-HOBA efficiently suppresses accumulation of isoLG adducts in the esophagus. Human nontumorous esophageal cell line (HET-1A), human Barrett's esophageal cell line (CP-A), and human esophageal carcinoma cell line (TE-7) were purchased from American Type Culture Collection. The morphology, karyotyping, and polymerase chain reaction–based techniques were used by American Type Culture Collection to confirm the cell line identity. Human immortalized esophageal epithelial cells, EPC-2, were kindly provided by Dr. Claudia Andl, University of Central Florida. HET-1A cells were cultured in Dulbecco's modified eagle's medium (Life Technologies, Carlsbad, CA) and TE-7 in RPMI (Life Technologies) media. Both Dulbecco's modified eagle's medium and RPMI were supplemented with 10% fetal bovine serum and 100 μg/mL penicillin/streptomycin. EPC-2 and CP-A cells were cultured in keratinocyte SFM media supplemented with 40 μg/mL bovine pituitary extract, 1.0 ng/mL epidermal growth factor (Life Technologies), 5% fetal bovine serum, and 100 μg/mL penicillin/streptomycin. All cells were cultured at 37°C in an atmosphere of 5% CO2. Cells were treated in acidic Dulbecco's modified eagle's media (pH 4.0), containing bile salts cocktail at a final concentration of 100 μM. The bile salt cocktail was prepared with a combination of glycocholic, taurocholic, glycodeoxycholic, glycochenodeoxycholic, and deoxycholic sodium salts (all reagents were from Sigma-Aldrich, St. Louis, MO) at a concentration of 20 μM each. CP-A, TE-7, and HET-1A cells were treated for 30 minutes, whereas EPC-2 cells were treated for 15 minutes and then the media was replaced. The IsoLG scavenger 2-hydroxybenzylamine (2-HOBA) has been previously characterized.1Davies S.S. Brantley E.J. Voziyan P.A. Amarnath V. Zagol-Ikapitte I. Boutaud O. Hudson B.G. Oates J.A. Roberts 2nd, L.J. Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H2O2-mediated cytotoxicity.Biochemistry. 2006; 45: 15756-15767Crossref PubMed Scopus (59) Google Scholar Final concentration of 2-HOBA was 50 μM. CP-A and EPC-2 cells were treated with the antioxidants, Tempol (4-hydroxy-2, 2, 6, 6-tetramethylpiperydine-1-oxyl; Enzo Biochem, Farmingdale, NY), and N-acetylcysteine (Sigma-Aldrich), as described previously.2Bhardwaj V. Gokulan R.C. Horvat A. Yermalitskaya L. Korolkova O. Washington K.M. El-Rifai W. Dikalov S.I. Zaika A.I. Activation of NADPH oxidases leads to DNA damage in esophageal cells.Sci Rep. 2017; 7: 9956Crossref PubMed Scopus (17) Google Scholar The following antibodies were used: β-actin (Sigma Aldrich), p53 (DO- 1; Millipore, Burlington, MA), and antimouse IgG HRP (Promega, Madison, WI). D-11, an isoLG-lysyl adducts-specific scFv antibody, has been isolated from a phage display recombinant antibody library.3Davies S.S. Talati M. Wang X. Mernaugh R.L. Amarnath V. Fessel J. Meyrick B.O. Sheller J. Roberts 2nd, L.J. Localization of isoketal adducts in vivo using a single-chain antibody.Free Radic Biol Med. 2004; 36: 1163-1174Crossref PubMed Scopus (47) Google Scholar The resulting scFv antibody displays an E-tag recognized by an anti-E-tag antibody.3Davies S.S. Talati M. Wang X. Mernaugh R.L. Amarnath V. Fessel J. Meyrick B.O. Sheller J. Roberts 2nd, L.J. Localization of isoketal adducts in vivo using a single-chain antibody.Free Radic Biol Med. 2004; 36: 1163-1174Crossref PubMed Scopus (47) Google Scholar In our study, we used anti-E tag HRP-conjugated secondary antibody from Abcam (Abcam, Cambridge, UK). The binding specificity of D11 scFv was previously characterized.3Davies S.S. Talati M. Wang X. Mernaugh R.L. Amarnath V. Fessel J. Meyrick B.O. Sheller J. Roberts 2nd, L.J. Localization of isoketal adducts in vivo using a single-chain antibody.Free Radic Biol Med. 2004; 36: 1163-1174Crossref PubMed Scopus (47) Google Scholar After treatment with acidic bile salts, the cell lysates were sonicated and centrifuged at 16,000 × g at 4°C for 20 minutes. The supernatant, which contains the soluble cellular fraction, was carefully collected. The remaining cell pellet, which contains the insoluble cellular fraction, was washed twice, resuspended in phosphate-buffered saline containing protease inhibitor cocktail (Sigma Aldrich), and sonicated. Both soluble and insoluble cellular fractions were analyzed by Western blotting as described previously.4Zaika E. Wei J. Yin D. Andl C. Moll U. El-Rifai W. Zaika A.I. p73 protein regulates DNA damage repair.FASEB J. 2011; 25: 4406-4414Crossref PubMed Scopus (33) Google Scholar p53 protein was immunoprecipitated from total cell lysate using p53 (DO-1) antibody and protein G agarose (Roche, Basel, Switzerland). The immunoprecipitated samples were analyzed by Western blotting using D11 antibody. The analyzed membranes were stripped in the Restore Western Blot Stripping Buffer (ThermoFisher Scientific, Waltham, MA) at +50°C for 1 hour, washed, blocked with 5% milk, and analyzed by Western blotting with p53 (DO1) antibody. TE-7 and EPC-2 cells were grown on chamber slides, treated with BA/A, and cultured in fresh media for 18 hours. After fixation with methanol and acetone mixture at a 1:1 ratio (vol/vol) and blocking with 0.1% Tween 20, the slides were incubated with D11 antibody for 18 hours in a humidified chamber, followed by incubation with rabbit anti-E-tag and goat antirabbit AlexaFluor 594 conjugated antibody (Invitrogen, Carlsbad, CA) for 1 hour. After washing with phosphate-buffered saline, the slides were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; ThermoFisher Scientific) for 1 minute and examined under a fluorescence microscope (Olympus, Pittsburgh, PA). At least 150 cells in 3–5 randomly selected fields were analyzed. Reflux in mice was induced by esophagojejunal anastomosis, as previously described.4Zaika E. Wei J. Yin D. Andl C. Moll U. El-Rifai W. Zaika A.I. p73 protein regulates DNA damage repair.FASEB J. 2011; 25: 4406-4414Crossref PubMed Scopus (33) Google Scholar Esophagojejunostomy was performed on 14 129SV mice of 8 weeks old according to the protocol approved by the Vanderbilt University Animal Care and Use Committee. The esophageal and gastroesophageal areas from animals with esophagojejunostomy and sham surgery were harvested 3 weeks after surgery, paraffin-embedded, and analyzed by immunohistochemistry with D11 antibody. The negative control was performed by omitting the primary antibody. Indices of D11 positivity were scored and compared in test and control groups of animals. Nineteen archival esophageal biopsies collected at Vanderbilt University Medical Center from patients with GERD and healthy individuals were used for analyses. The use of all human pathology specimens for research was approved by the institutional review board. Because only deidentified tissues were included in this retrospective study, the institutional review board waived the requirements for informed consent. Immunohistochemical staining was done with D11-E tag antibody (1 μg/mL) and anti-E tag HRP-conjugated antibody. The intensity of staining was graded as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The frequency was graded according to the percentage of positive cells. Total scores were calculated by multiplying the intensity score by the percentage of positive cells. The individuals scoring the tissue were blinded. TE-7 cells were treated with acidic bile salts and collected at the indicated time points. Cells were incubated with indomethacin and pyridoxamine in phosphate-buffered saline, pH 7.4 at 4°C for 30 minutes, homogenized, and centrifugated at 18,000 × g for 10 minutes. Proteins were subjected to complete enzymatic digestion using pronase (1 mg protease per mg of protein) overnight. Samples were then heated at 98°C for 10 minutes, and after cooling, 0.3 μL aminopeptidase M (Calbiochem, EMD Chemicals, Gibbstown, NJ) was added per mg of protein. The digest was incubated at 37°C for 18 hours. The liquid chromatography/electrospray ionization/tandem mass spectrometry of LG adducts was carried out as previously described.5Zagol-Ikapitte I. Masterson T.S. Amarnath V. Montine T.J. Andreasson K.I. Boutaud O. Oates J.A. Prostaglandin H(2)-derived adducts of proteins correlate with Alzheimer's disease severity.J Neurochem. 2005; 94: 1140-1145Crossref PubMed Scopus (58) Google Scholar Statistical analysis was performed using the Student t test and Mann-Whitney test, depending on the data set. Results were shown as mean ± standard deviation and considered significant if P < .05. All experimental protocols were approved by the Institutional Animal Care and Use Committee and institutional review board of Vanderbilt University Medical Center.Supplementary Figure 2Representative images of isoLG-positive cells. Analyses were performed using immunofluorescence with D11 antibody after treatment with BA/A for 18 hours. Cell nuclei were stained with DAPI. Significant increase in isoLG positivity was found in treated EPC-2 (A) and TE-7 (B) cells. ***P < .001. At least 150 cells were assessed in each experiment. Statistical analyses were performed with Student t test. Data are represented as mean ± standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 3Acidic bile salts cause the formation of adducts on p53 protein and its precipitation. (A) p53 protein is precipitated after treatment of EPC-2 cells with BA/A. 2-HOBA prevents precipitation of p53 protein. (B) The same as A, but HET-1A cells are shown. (C) Analysis of isoLG protein adducts using D11 scFv after immunoprecipitation of p53 protein with p53-specific antibody (DO1) in EPC-2 cells. Exposure of esophageal cells to BA/A significantly increases levels of p53 protein adducts. Each analysis is representative of 3 independent experiments and values are expressed as mean ± standard deviation. D11 positivity was arbitrarily set at 1 in control samples. Statistical analyses were performed with the Student t test. *P < .05, **P < .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 4Effect of 2-HOBA, Tempol, and NAC on the formation of isoLG protein adducts. EPC-2 and CP-A cells were treated with BA/A alone or in combination with either 2-HOBA or 20 μM of Tempol or 20 μM of NAC. Treated cells were collected and analyzed for the formation of isoLG protein adducts. D11 positivity was arbitrarily set at 1 in control samples. Results from 3 independent experiments are shown. Statistical analyses were performed with the Student t test. *P < .05, **P < .01, ***P < .001. Data are represented as mean ± standard deviation. NAC, N-acetylcysteine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplemental Graphical SummaryView Large Image Figure ViewerDownload Hi-res image Download (PPT)

Oral insulin avoids the need for injections, and insulin enters via the hepatic portal vein (Po), the normal route of secretion. We examined the effect of I338, an acylated analog designed for oral use, dosed either Po to match the oral absorption profile or IV to match the steady state basal subcutaneous (SC) plasma profile, on a Po glucose challenge. To achieve steady-state concentrations in normal dogs, I338 was infused IV 45 min daily for 4 days. On day 5 a primed, continuous infusion of 3-3H glucose was given. After 90 min of equilibration and 30 min of basal sampling, a clamp was conducted (0-300 min), with somatostatin to inhibit pancreatic secretion and basal glucagon replacement. In the LOW dogs (n=5) I338 (pmol/kg/min) was infused either Po at 40 (0-45 min to mimic oral absorption) or IV at 1 (0-300 min to mimic subcutaneous [SC] delivery), approximately an equivalent daily dose (1800 vs. 1440 pmol/kg, Po vs. IV). High dose dogs (HI; n=4) received I338 at 50 (Po) or 5 (IV) pmol/kg/min. A Po glucose infusion mimicking meal absorption was given via computer algorithm from 30-252 min. All dogs were studied twice, 2 weeks apart in random order, receiving both Po and IV I338. Po vs. IV, respectively, resulted in: 1) lower glycemic levels (peak glucose LOW 6.2±0.3 and 10.3±0.6,* HI 4.5±0.3, and 7.8±0.8* mM); 2) increased net hepatic glucose uptake (AUC30-240 min Low 218.4±151.8 and -852.0±371.4,* HI 732.6±338.5 and -58.5±578.4* µmol/kg); 3) enhanced glucose clearance (AUC30-240 min LOW 481.9±29.7 and 359.4±33.7,* HI 874.3±99.8 and 495.3±25.9* mL/kg) and 4) greater peak hepatic fractional glucose extraction (LOW 0.05±0.02 and -0.01±0.01,* HI 0.10±0.and 0.04±0.02*)(*P<0.vs. corresponding Po treatment). In short, Po basal I338 delivery produced superior glucose lowering and hepatic glucose disposal during a morning “meal” compared to SC-like delivery. Thus basal oral I338 can improve the glycemic response to the first meal after dosing, potentially improving glucose disposal at subsequent meals via the 2nd meal effect. Disclosure M.C. Moore: None. E. Nishimura: Stock/Shareholder; Self; Novo Nordisk A/S. Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. C.L. Brand: Other Relationship; Self; Novo Nordisk A/S. T. Kjeldsen: Stock/Shareholder; Self; Novo Nordisk A/S. Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. P. Madsen: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. H.H. Refsgaard: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. K. Wassermann: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. S. Gram-Nielsen: Employee; Self; Novo Nordisk A/S. M.S. Smith: None. L. Moore: None. B. Farmer: None. J.R. Hastings: None. P.E. Williams: None. A.D. Cherrington: Advisory Panel; Self; Biocon. Consultant; Self; Boston Scientific Corporation. Research Support; Self; Boston Scientific Corporation. Consultant; Self; Eli Lilly and Company. Advisory Panel; Self; Fractyl Laboratories, Inc.. Stock/Shareholder; Self; Fractyl Laboratories, Inc.. Consultant; Self; Galvani Bioelectronics Limited. Research Support; Self; Galvani Bioelectronics Limited. Consultant; Self; MedImmune. Advisory Panel; Self; Metavention. Stock/Shareholder; Self; Metavention. Consultant; Self; Novo Nordisk Inc.. Research Support; Self; Novo Nordisk Inc.. Advisory Panel; Self; NuSirt Biopharma, Inc., Sensulin Labs, LLC.. Other Relationship; Self; Sensulin Labs, LLC.. Consultant; Self; Silver Lake. Research Support; Self; Silver Lake. Consultant; Self; Thermalin Diabetes, LLC., Thetis Pharmaceuticals LLC.. Stock/Shareholder; Self; Thetis Pharmaceuticals LLC.. Advisory Panel; Self; VTV Therapeutics. Consultant; Self; VTV Therapeutics. Advisory Panel; Self; Zafgen. Research Support; Self; Zafgen. Stock/Shareholder; Self; Zafgen. Consultant; Self; Abvance. Other Relationship; Self; Abvance. Consultant; Self; California Institute for Biomedical Research (Calibr). Advisory Panel; Self; These Three Medical, Inc (T3M).

AimsCurrent therapy fails to emulate rapid (first‐phase) insulin release in relation to a meal, a key defect in types 1 and 2 diabetes. We aimed to quantify the pharmacokinetic (PK) and pharmacodynamic (PD) profile of insulin tregopil, an enterically‐absorbed insulin analog that restores the normal distribution of insulin between the hepatic portal and peripheral circulations.Materials and methodsThe PK and PD profiles of insulin tregopil were studied in overnight‐fasted, catheterized, conscious canines using four approaches: (1) equimolar intraportal infusions of tregopil vs human insulin; (2) escalating doses of oral tregopil; (3) identical, consecutive enteric doses of tregopil; and (4) comparison of oral tregopil to inhaled and subcutaneous human insulin administration.ResultsEquimolar intraportal infusions of tregopil and human insulin resulted in very similar PK profiles and PD profiles were nearly identical. Enteric delivery of tregopil brought about rapid absorption with tmax = 20 minutes in most cases. Median tmax was 20 minutes for oral tregopil and inhaled insulin and 88 minutes for subcutaneous human insulin. The time required for arterial plasma insulin levels to return to baseline was approximately 90, 210 and 360 minutes for oral tregopil, inhaled insulin and subcutaneous insulin, respectively.ConclusionsEnterically delivered tregopil is rapidly absorbed and restores a portal‐to‐peripheral vascular distribution. These characteristics should improve postprandial hyperglycaemia in types 1 and 2 diabetes.

Purpose Current intra-arterial chemotherapy (IAC) drug regimens for retinoblastoma have ocular and vascular toxicities. No small-animal model of IAC exists to test drug efficacy and toxicity in vivo for IAC drug discovery. The purpose of this study was to develop a small-animal model of IAC and to analyze the ocular tissue penetration, distribution, pharmacokinetics, and treatment efficacy. Methods Following selective ophthalmic artery (OA) catheterization, melphalan (0.4 to 1.2 mg/kg) was injected. For pharmacokinetic studies, rabbits were euthanized at 0.5, 1, 2, 4, or 6 hours following intra-OA infusion. Drug levels were determined in vitreous, retina, and blood by liquid chromatography tandem mass spectrometry. To assess toxicity, angiograms, photography, fluorescein angiography, and histopathology were performed. For in situ tissue drug distribution, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was performed. The tumor model was created by combined subretinal/intravitreal injection of human WERI-Rb1 retinoblastoma cells; the tumor was treated in vivo with intra-arterial melphalan or saline; and induction of tumor death was measured by cleaved caspase-3 activity. Results OA was selectively catheterized for 79 of 79 (100%) eyes in 47 of 47 (100%) rabbits, and melphalan was delivered successfully in 31 of 31 (100%) eyes, without evidence of vascular occlusion or retinal damage. For treated eyes, maximum concentration (Cmax) in the retina was 4.95 μM and area under the curve (AUC0→∞) was 5.26 μM·h. Treated eye vitreous Cmax was 2.24 μM and AUC0→∞ was 4.19 μM·h. Vitreous Cmax for the treated eye was >100-fold higher than for the untreated eye (P = 0.01), and AUC0→∞ was ∼50-fold higher (P = 0.01). Histology-directed MALDI-IMS revealed highest drug localization within the retina. Peripheral blood Cmax was 1.04 μM and AUC0→∞ was 2.07 μM·h. Combined subretinal/intravitreal injection of human retinoblastoma cells led to intra-retinal tumors and subretinal/vitreous seeds, which could be effectively killed in vivo with intra-arterial melphalan. Conclusions This first small-animal model of IAC has excellent vitreous and retinal tissue drug penetration, achieving levels sufficient to kill human retinoblastoma cells, facilitating future IAC drug discovery.

Insulin can inhibit hepatic glucose production (HGP) by acting directly on the liver as well as indirectly through effects on adipose tissue, pancreas, and brain. While insulin's indirect effects are indisputable, their physiologic role in the suppression of HGP seen in response to increased insulin secretion is not clear. Likewise, the mechanisms by which insulin suppresses lipolysis and pancreatic α cell secretion under physiologic circumstances are also debated. In this study, insulin was infused into the hepatic portal vein to mimic increased insulin secretion, and insulin's indirect liver effects were blocked either individually or collectively. During physiologic hyperinsulinemia, plasma free fatty acid (FFA) and glucagon levels were clamped at basal values and brain insulin action was blocked, but insulin's direct effects on the liver were left intact. Insulin was equally effective at suppressing HGP when its indirect effects were absent as when they were present. In addition, the inhibition of lipolysis, as well as glucagon and insulin secretion, did not require CNS insulin action or decreased plasma FFA. This indicates that the rapid suppression of HGP is attributable to insulin's direct effect on the liver and that its indirect effects are redundant in the context of a physiologic increase in insulin secretion.

249/250 words) 24 The contribution of hormone-independent counterregulatory signals in defense of insulin25 induced hypoglycemia were determined in adrenalectomized, overnight-fasted conscious dogs 26 receiving hepatic portal vein insulin infusions at a rate 20-fold basal. Either euglycemia was 27 maintained (group 1) or hypoglycemia (≈45 mg/dL) was allowed to occur. There were 3 28 hypoglycemic groups: one in which hepatic autoregulation against hypoglycemia occurred in the 29 absence of sympathetic nervous system input (group 2), autoregulation in the presence of 30 norepinephrine (NE) signaling to fat and muscle (group 3), and autoregulation in the presence of 31 NE signaling to fat, muscle, and liver (group 4). Average net hepatic glucose balance (NHGB) 32 during the last hour for groups 1-4 was -0.7±0.1, 0.3±0.1 (p<0.01 vs. group1), 0.7±0.1 (p=0.01 33 vs. group 2), and 0.8±0.1 (p=0.7 vs group 3) mg/kg/min, respectively. Hypoglycemia per se 34 (group 2) increased NHGB by causing an inhibition of net hepatic glycogen synthesis. NE 35 signaling to fat and muscle (group 3) increased NHGB further by mobilizing gluconeogenic 36 precursors resulting in a rise in gluconeogenesis. Lowering glucose per se decreased non-hepatic 37 glucose uptake by 8.9 mg/kg/min and the addition of increased neural efferent signaling to 38 muscle and fat blocked glucose uptake further by 3.2 mg/kg/min. The addition of increased 39 neural efferent input to liver did not affect NHGB or non-hepatic glucose uptake significantly. In 40 conclusion, even in the absence of increases in counterregulatory hormones, the body can defend 41 itself against hypoglycemia using glucose autoregulation and increased neural efferent signaling 42 both of which stimulate hepatic glucose production and limit glucose utilization. 43

The contribution of hormone-independent counterregulatory signals in defense of insulin-induced hypoglycemia was determined in adrenalectomized, overnight-fasted conscious dogs receiving hepatic portal vein insulin infusions at a rate 20-fold basal. Either euglycemia was maintained ( group 1) or hypoglycemia (≈45 mg/dl) was allowed to occur. There were three hypoglycemic groups: one in which hepatic autoregulation against hypoglycemia occurred in the absence of sympathetic nervous system input ( group 2), one in which autoregulation occurred in the presence of norepinephrine (NE) signaling to fat and muscle ( group 3), and one in which autoregulation occurred in the presence of NE signaling to fat, muscle, and liver ( group 4). Average net hepatic glucose balance (NHGB) during the last hour for groups 1–4 was −0.7 ± 0.1, 0.3 ± 0.1 ( P < 0.01 vs. group 1), 0.7 ± 0.1 ( P = 0.01 vs. group 2), and 0.8 ± 0.1 ( P = 0.7 vs. group 3) mg·kg−1·min−1, respectively. Hypoglycemia per se ( group 2) increased NHGB by causing an inhibition of net hepatic glycogen synthesis. NE signaling to fat and muscle ( group 3) increased NHGB further by mobilizing gluconeogenic precursors resulting in a rise in gluconeogenesis. Lowering glucose per se decreased nonhepatic glucose uptake by 8.9 mg·kg−1·min−1, and the addition of increased neural efferent signaling to muscle and fat blocked glucose uptake further by 3.2 mg·kg−1·min−1. The addition of increased neural efferent input to liver did not affect NHGB or nonhepatic glucose uptake significantly. In conclusion, even in the absence of increases in counterregulatory hormones, the body can defend itself against hypoglycemia using glucose autoregulation and increased neural efferent signaling, both of which stimulate hepatic glucose production and limit glucose utilization.

We used hepatic balance and tracer ([3H]glucose) techniques to examine the impact of “breakfast” on hepatic glucose metabolism later in the same day. From 0–240 min, 2 groups of conscious dogs (n = 9 dogs/group) received a duodenal infusion of glucose (GLC) or saline (SAL), then were fasted from 240–360 min. Three dogs from each group were euthanized and tissue collected at 360 min. From 360–600 min, the remaining dogs underwent a hyperinsulinemic (4× basal) hyperglycemic clamp (arterial blood glucose 146 ± 2 mg/dL) with portal GLC infusion. The total GLC infusion rate was 14% greater in dogs infused with GLC than in those receiving SAL (AUC360–600min 2,979 ± 296 vs. 2,597 ± 277 mg/kg, respectively). The rates of hepatic glucose uptake (5.8 ± 0.8 vs. 3.2 ± 0.3 mg ⋅ kg−1 ⋅ min−1) and glycogen storage (4.7 ± 0.6 vs. 2.9 ± 0.3 mg ⋅ kg−1 ⋅ min−1) during the clamp were markedly greater in dogs receiving GLC compared with those receiving SAL. Hepatic glycogen content was ∼50% greater, glycogen synthase activity was ∼50% greater, glycogen phosphorylase activity was ∼50% lower, and the amount of phosphorylated glycogen synthase was 34% lower, indicating activation of the enzyme, in dogs receiving GLC compared with those receiving SAL. Thus, morning GLC primed the liver to extract and store more glucose in the presence of hyperinsulinemic hyperglycemia later in the same day, indicating that breakfast enhances the liver’s role in glucose disposal in subsequent same-day meals.

The postprandial state is characterized by a storage of nutrients in the liver, muscle, and adipose tissue for later utilization. In the case of a protein-rich meal, amino acids (AA) stimulate glucagon secretion by the α-cell. The aim of the present study was to determine the impact of the rise in glucagon on AA metabolism, particularly in the liver. We used a conscious catheterized dog model to recreate a postprandial condition using a pancreatic clamp. Portal infusions of glucose, AA, and insulin were used to achieve postprandial levels, while portal glucagon infusion was either maintained at the basal level or increased by three-fold. The high glucagon infusion reduced the increase in arterial AA concentrations compared with the basal glucagon level (-23%, P < 0.05). In the presence of high glucagon, liver AA metabolism shifted toward a more catabolic state with less protein synthesis (-36%) and increased urea production (+52%). Net hepatic glucose uptake was reduced modestly (-35%), and AA were preferentially used in gluconeogenesis, leading to lower glycogen synthesis (-54%). The phosphorylation of AMPK was increased by the high glucagon infusion (+40%), and this could be responsible for increasing the expression of genes related to pathways producing energy and lowering those involved in energy consumption. In conclusion, the rise in glucagon associated with a protein-rich meal promotes a catabolic utilization of AA in the liver, thereby, opposing the storage of AA in proteins.

Liver glycogen is important for the counterregulation of hypoglycemia and is reduced in individuals with type 1 diabetes (T1D). Here, we examined the effect of varying hepatic glycogen content on the counterregulatory response to low blood sugar in dogs. During the first 4 hours of each study, hepatic glycogen was increased by augmenting hepatic glucose uptake using hyperglycemia and a low-dose intraportal fructose infusion. After hepatic glycogen levels were increased, animals underwent a 2-hour control period with no fructose infusion followed by a 2-hour hyperinsulinemic/hypoglycemic clamp. Compared with control treatment, fructose infusion caused a large increase in liver glycogen that markedly elevated the response of epinephrine and glucagon to a given hypoglycemia and increased net hepatic glucose output (NHGO). Moreover, prior denervation of the liver abolished the improved counterregulatory responses that resulted from increased liver glycogen content. When hepatic glycogen content was lowered, glucagon and NHGO responses to insulin-induced hypoglycemia were reduced. We conclude that there is a liver-brain counterregulatory axis that is responsive to liver glycogen content. It remains to be determined whether the risk of iatrogenic hypoglycemia in T1D humans could be lessened by targeting metabolic pathway(s) associated with hepatic glycogen repletion.

This study reports the initial in vivo use of a combined radiofrequency ablation and cryo-anchoring (RFC) catheter as an alternative to surgical mitral valve (MV) leaflet resection. Radiofrequency ablation thermally shrinks enlarged collagenous tissues, providing an alternative to leaflet resection, and cryo-anchoring provides reversible attachment of a catheter to freely mobile MV leaflets. Excised porcine MVs (n = 9) were tested in a left heart flow simulator to establish treatment efficacy criteria. Resected leaflet area was quantified by tracking markers on the leaflet surface, and leaflet length reductions were directly measured on echocardiography. Leaflet area decreased by 38 ± 2.7%, and leaflet length decreased by 9.2 ± 1.8% following RFC catheter treatment. The RFC catheter was then tested acutely in healthy pigs (n = 5) under epicardial echocardiographic guidance, open-chest without cardiopulmonary bypass, using mid-ventricular free wall access. Leaflet length was quantified using echocardiography. Quantitative assessment of MV leaflet length revealed that leaflet resection was successful in 4 of 5 pigs, with a leaflet length reduction of 13.3 ± 4.6%. Histological, mechanical, and gross pathological findings also confirmed that RFC catheter treatment was efficacious. The RFC catheter significantly reduces MV leaflet size in an acute animal model, providing a possible percutaneous alternative to surgical leaflet resection.