Hyperglycemic clamp

Male Sprague-Dawley rats (3 mo old, n=10, 5/group) were subjected to 2 h of moderate hyperglycemia (11 mM). Briefly, 25% glucose was infused intravenously to raise the plasma glucose concentration acutely to 11 mM. The glucose infusion rate (GIR) was then varied to maintain the plasma glucose concentration at this level for 2 h. A total dose of 60 μg of HNGF6A (sequence: MAPRGASCLLLLTGEIDLPVKRRA; Genemed Custom Peptide Synthesis, San Antonio, TX, USA) was administered per animal; one-third was given as a bolus, and the remainder was administered as a continuous infusion at the rate of the 0.07 mg/kg/h over 2 h. Plasma samples for insulin were obtained at 2-min intervals for the first 10 min and at 10 min intervals throughout the study. Samples for C peptide were collected every 30 min. The area under the curve (AUC) of the first-phase insulin response was calculated by the trapezoid rule, using 0-, 2-, 4-, 6-, 8-, and 10-min samples with the formula (i₀ + i₂)/2 × 0.5 + (i₂ + i₄)/2 × 0.5 + (i₄ + i₆)/2 × 0.5 + (i₆ + i₈)/2 × 0.5 + (i₈ + i₁₀)/2 × 0.5. Insulin levels and GIRs were averaged over the last 1 h of the clamp.

Intravenous glucose tolerance test (IVGTT) to assess first-phase insulin secretion

Indwelling catheters were placed in C57BL6/N mice (n=6/group) in the jugular vein for collection of blood samples without stress during the IVGTT study. After recovery, mice were subjected to i.v. injection of vehicle or HNGF6A (2 mg/kg body weight) 10 min before the injection of 2 g/kg glucose load, and blood samples were collected from mice at 0, 2, 5, 10, 20, and 30 min for estimation of blood glucose and insulin.

Analytical procedures for in vivo studies

Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II; Beckman Instruments, Inc., Palo Alto, CA, USA), and plasma insulin was measured by ELISA using rat or mouse insulin standards. Plasma C-peptide levels were assayed using ELISA kit from Millipore (Billerica, MA, USA).

Islet isolation

Islets from 25- to 30-g male C57BL6/J wild-type (WT) and diabetic db/db mice were isolated using standard collagenase digestion, as described previously (25), and were cultured overnight in RPMI medium supplemented with 10% FBS plus antibiotics. Prior to experiments, islets were transferred to MilliCell-PCF culture plate filter inserts (Millipore) at a density of ~10–20 islets/insert. The inserts were placed within individual wells of a 24-well cell culture plate, and each well was filled with 1 ml of DME and 5 mM glucose. After a 6 h preincubation at 5 mM glucose, the inserts were transferred to new wells containing 0.5 ml volume of medium, and islets were challenged with glucose (5 or 16 mM) and HNGF6A (50 ng/ml) as indicated. Medium was collected from beneath inserts; islets were floated by a rapid application of 0.5 ml of PBS added to the inserts. Islets were pelleted and then lysed by sonication as described previously (26). An antiprotease cocktail containing aprotinin (1 mU/ml), leupeptin (0.1 mM), pepstatin (10 μM), EDTA (5 mM), and diisopropylfluorophospbate (1 mM) was added to the collected medium and cell lysates. Insulin levels in the medium were measured using the Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc., Downers Grove, IL, USA). The repeatability of all findings was confirmed by performing each experiment ≥3 times.

Cell culture

Mouse pancreatic β cells (βTC3 line) were maintained in DMEM containing 10% heat-inactivated FBS, d-glucose (4500 μg/ml), and glutamine (4 mM) at 37°C in a humidified environment with 5% CO₂. The initial studies probing the effects of HNGF6A were done at glucose concentrations of 0, 5 and 16 mM glucose levels. Subsequent studies were all done at 16 mM glucose levels. For the dose-response study, βTC3 cells were treated with either 24-aa scrambled peptide (SP; sequence: FRGGETRARAMPLIDLSPLCLLKV) or varying doses of HNGF6A between 5 and 500 ng/ml, and insulin in the medium was assessed at 2 h. For the time-course study, βTC3 cells were treated for the indicated times with 50 ng/ml of either SP or HNGF6A. Insulin levels were assayed in the media using the Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc., Downers Grove, IL, USA).

Western blot

βTC3 cells and mouse pancreas were homogenized in 1× RIPA buffer (50 mM Tris, pH7.4; 150 mM NaCl; 1% Triton X-100; and 0.1% SDS) containing 1× proteinase inhibitors (Roche, Indianapolis, IN, USA). Proteins (20 μg) were resolved on precast gradient SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBS-T) for 1 h at room temperature. Primary antibodies against CNTFRα, GP130, WSX-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Rattin (GeneTex, Irvine, CA, USA) and β-actin (Sigma, St. Louis, MO, USA) diluted in TBS-T containing 5% of BSA were added to the membranes and incubated overnight at 4°C. The membranes were subsequently washed with TBS-T 3 times for 10 min and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The signals were detected by the SuperSignal West Dura extended duration substrate (Thermo Scientific, Rockford, IL, USA).

Glucose transporter 2 (GLUT2) translocation

βTC3 cells were seeded on coverslips in a 6-well plate 1 d before transfection. Cells transfected with GLUT2-GFP construct (kindly provided by Dr. Jeffery Pessin, Albert Einstein College of Medicine) were incubated in medium, without glucose, overnight, and then treated with 50 ng/ml HNGF6A or SP in 16 mM glucose for 15 min. After 2 washes with cold PBS, cells were fixed using 4% formaldehyde for 20 min. Images were taken using Leica SP2 confocal microscopy (Leica Microsystems, Buffalo Grove, IL, USA).

Glucokinase (GCK) activity assay

Glucose phosphorylation rate was measured by a well-established fluorimetric assay, as described previously (27). In brief, SP- or HNGF6A (50 ng/ml)-treated βTC3 cells were homogenized in 500 μl of cold lysis buffer (10 mM HEPES, pH 7.4; 250 mM sucrose; 2 mM EDTA; 1 mM DTT; 1.5 mM MgCl₂; and 10 mMKCl) by 10 passages through a 22-gauge needle. The soluble cytosolic fractions collected from centrifugation at 10⁵ g and 250 μg of this soluble cytosolic protein were added to 100 μl assay buffer (50 mM HEPES, pH 7.7; 100 mM KCl; 10 mM MgCl₂; 15 mM β-mercaptoethanol; 0.5 mM NAD⁺; 5 mM ATP; 10 μg/ml G6PDH; and 0.05% BSA) containing 0.5 or 50 mM glucose and incubated at 37°C for 1 h. A reaction mix with cytosol protein, glucose, and assay buffer without ATP was also created for background subtraction. Relative GCK activity was calculated by subtracting values of hexokinase (collected from 0.5 mM glucose reactions) from values of total kinases (collected from 50 mM glucose reactions).

Glucose oxidation assay

βTC3 cells were grown in 6-well plates for 24 h. Cells were rinsed with warm PBS twice after 60 min of treatment with SP or HNGF6A and then incubated with 1 ml of incubation buffer (M199 medium without phenol red, complemented with 5 mM glucose and 2 mM pyruvate) containing 1 uCi/ml of ¹⁴C-U-glucose for 1 h. After incubation, 900 μl of medium was collected, and CO₂ in the medium was trapped using 200 μl of 3 N NaOH for 3 h. The ¹⁴CO₂ was measured by scintillation counting. Oxidation of glucose is expressed in nanomoles of oxidized glucose per incubation time per milligram of protein.

Measurement of mitochondrial transmembrane potential (ΔΨ_m)

Mitochondrial transmembrane potential was investigated using the fluorochrome dye JC-10 (Enzo Life Sciences, Ann Arbor, MI, USA). βTC3 cells were seeded in 96-well plates and grown to ~70% confluence. The cells were treated for 30 min at 37°C, 10% CO₂ with growth medium with or without HNGF6A (50 ng/ml) and H₂O₂ (40 μM) to challenge the β cell. This medium was aspirated and replaced with 100 μl/well of 1× Hanks buffered salt solution containing 20 mM HEPES and 20 μM JC-10, and the plate was incubated for an additional 30 min at 37°C. The wells were rinsed 3× with a wash buffer (1× HBSS, 10 mM HEPES, and 0.02 mg/100 ml d-glucose). The final aliquot of wash buffer was left in the wells, and the fluorescent signal was read on a Molecular Devices SpectraMax M5e scanning spectrophotometer, using 490 and 525 nm as green excitation and emission wavelengths, respectively (cutoff 515 nm), and 490 and 590 nm as red excitation and emission wavelengths, respectively (cutoff 570 nm). The ratio of the reading at 590 nm to that at 525 nm (590:525 ratio) was considered as the relative ΔΨ_m value.

Bioluminescent assay for cellular ATP

βTC3 cells were treated for the indicated time with or without HNGF6A (50 ng/ml), and ATP levels were measured with a bioluminescent assay kit (FL-AA; Sigma). Briefly, cells were grown in 12-well plates, washed with PBS, and dispersed in 85 mM sodium citrate. An aliquot of each sample was removed for cell counting for normalization, and the rest was brought to 2.3% with TCA to arrest ATP metabolism and for storage of samples at 4°C. Just prior to the assay, each sample was neutralized with Tris acetate EDTA buffer (0.1 M Tris, adjusted to pH 7.75 with acetic acid, and 2 mM EDTA), boiled for 3 min, and placed on ice. Samples were placed in black, clear-bottomed 96-well plates in duplicate, and the diluted luciferase reagent was injected as luminescence readings were taken by a FluoStar Optima multimode microplate reader (BMG Labtech, Ortenberg, Germany). Cells were also studied in the presence of aminooxyacetate (AOA), a malate-aspartate shuttle inhibitor. Insulin levels were assessed at 1 h in the presence or absence of HNGF6A after preincubation for 2 h with AOA.

HNGF6A augments insulin secretion in isolated islets of WT and db/db mice

Impaired GSIS is observed in islets obtained from diabetic mice and humans (29, 30). To confirm the outcomes of the hyperglycemic clamp studies and to extend these observations to an experimental model of diabetes, for the next experiments we isolated islets from 3-mo-old WT and db/db mice. Direct addition of HNGF6A to islets was chosen to avoid any confounding variables introduced by whole-animal studies. The amount of insulin released from db/db islets was lower relative to WT controls under glucose-stimulated (16 mM) conditions (Fig. 2A). As shown, the exposure of islets to 16 mM glucose plus HNGF6A increased insulin release by 3-fold in islets from WT and 2.5-fold in islets from diabetic mice (Fig. 2A).

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Figure 2.

Effects of HNGF6A on insulin secretion in islets isolated from WT and db/db diabetic mice and βTC3 cells. A) Insulin levels in the islets isolated from 10- to 12-wk-old C57BLK6/J mice and db/db mice. B) Expression of Rattin (homologue of HN in rodents) and receptors, including GP130, CNTFRa, and WSX-1 in βTC3 cells and mouse pancreas. C) Insulin levels in βTC3 cells treated with or without HNGF6A in the presence or absence of 5 and 16 mM glucose. D) Dose-dependent effects of HNGF6A on insulin secretion. Experiments were repeated ≥3 times; data are presented as means ± sem. *P < 0.05, **P < 0.01 (n=12).

HNGF6A increases GSIS in βTC3 cells

Whole-mouse pancreas and mouse βTC3 cells express endogenous rattin (homologue of HN) in addition to the various receptor components (Fig. 2B). To test whether exogenously added HNGF6A directly affects insulin secretion from the β cells, we characterized basal- and glucose-induced insulin release in the glucose-sensitive βTC3 cells (31, 32). βTC3 cells were incubated in the presence of various concentrations of glucose, and insulin released into medium during a 2-h challenge with or without HNGF6A was measured. An HNGF6A-dependent increase in insulin secretion was seen only in the presence of 16 mM glucose and not in the absence of glucose or at normal glucose levels of 5 mM (Fig. 2C). To identify the optimal dose of HNGF6A, we tested various concentrations of HNGF6A at 16 mM glucose (Fig. 2D). We observed maximal effects at a HNGF6A concentration of 50 ng/ml (Fig. 2D). This HNGF6A concentration (50 ng/ml) was used in all subsequent in vitro experiments unless noted otherwise.

HNGF6A enhances glucose sensing in βTC3 cells

GLUTs and GCK comprise the glucose sensing system, allowing β cells must sense a change in extracellular glucose and initiate GSIS. GLUT2 is the major isoform responsible for glucose transport in β cells, and glucose phosphorylation is the rate-limiting step. To test whether HNGF6A affects GLUT2 translocation, we performed GLUT2 translocation assays in cultured βTC3 cells. Within 15 min, HNGF6A markedly increased membrane-localized GLUT2 (Fig. 3A). This effect was dependent on the presence of glucose (Fig. 3A). GCK activity in HNGF6A-treated β cells was 1.5-fold higher within 15 min of treatment compared to the control β cells (Fig. 3B).

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Figure 3.

Effects of HNGF6A on glucose metabolism. A) GLUT2 translocation in βTC3 cells treated with or without HNGF6A in the presence of no glucose (0 mM) or high (16 mM) concentrations of glucose. B) Effects of HNGF6A on glucose phosphorylation rate (GCK activity) in βTC3 cells. C) Effects of HNGF6A on glucose oxidation in βTC3 cells. D) Effects of HNGF6A on intracellular ATP levels in βTC3 cells. E) Time-course effects of HNGF6A on insulin secretion in βTC3 cells. F, G) effects of HNGF6A on intracellular ATP levels (F) and insulin secretion (G) with the presence of AOA an inhibitor of TCA cycle. H) Effects of HNGF6A on mitochondrial membrane potential in βTC3 cells. Experiments were repeated ≥3 times (n=12); data are presented as means ± sem. *P < 0.05.

HNGF6A-induced GSIS is linked to an increase in cellular ATP production

Increased plasma glucose levels initiate increased glucose uptake, glycolysis, and mitochondrial metabolism by the β cell. Cytosolic and mitochondrial reactions generate ATP, and each of these discrete sources of ATP plays an important role in regulating insulin exocytosis (33). Mitochondrial ATP generation, in particular, plays a key role in coupling glucose metabolism with insulin secretion (34). To further explore the potential mechanisms by which HNGF6A may regulate insulin secretion, we analyzed glucose oxidation and ATP production in βTC3 cells. As shown in Fig. 3C, overall glucose oxidation rate, as estimated using ¹⁴C-U-glucose, was enhanced by 60% (P<0.05) with HNGF6A treatment in BTC3 cells compared to controls, and a significant increase in ATP production (Fig. 3D) was noted at 60 min incubation (Fig. 3D). Consistent with the insulin secretion data, no significant increase was found in ATP levels when β cells were incubated in 5 mM glucose (data not shown).

To determine the relationship between ATP production and insulin secretion, we studied the time-dependent effects of HNGF6A on insulin exocytosis. Glucose and HNGF6A concentrations were adjusted to 16 mM and 50 mg/ml, respectively, and the insulin released during time periods ranging from 15 to 60 min was measured (Fig. 3E). Although a trend toward HNGF6A-augmented insulin secretion was seen at 15 and 30 min, significant increase was observed at the 60-min time point (Fig. 3E), coinciding with the time point when marked increases in ATP levels were noted (Fig. 3D). Mitochondrial metabolism, as evidenced by increased ATP, is associated with production of reactive oxygen species that could affect mitochondrial integrity. We determined mitochondrial membrane potential (MMP) as a measure of mitochondrial membrane integrity and demonstrate that HNGF6A preserved MMP in the β cells (Fig. 3F).

To investigate the role of mitochondria in HNGF6A-mediated effects, βTC3 cells were pretreated with AOA, an inhibitor of aspartate aminotransferases of the malate-aspartate NADH shuttle (35). A 2-h pretreatment of βTC3 cells with AOA attenuated ATP production in both control and HNGF6A treated cells (Fig. 3G). Along with a reduction in ATP production, AOA also blocked HNGF6A-dependent effects on insulin secretion (Fig. 3H).

The authors thank Dr. Natalia V. Cheshenko (Department of Pediatrics, Albert Einstein College of Medicine) for assistance with determination of intracellular calcium concentrations.

This work was supported by grants from the U.S. National Institutes of Health, K08-AG-027462;, K08-AG-027462-03S1;, and R01-AG-035114; to R.H.M.; P30-AG-038072; to R.K.; R01-AG-618381; and P30-AG-038072; to N.B.; K99/R00; award AG-037574 to D.M.H.; and by the Core laboratories of the Albert Einstein Diabetes Research and Training Center (DK 20541) and Einstein Nathan Shock Center, Cellular and Tissue Aging Core P30-AG-038072.

Disclosure: N.B. is a consultant and a shareholder of CoHBar.