Intermittent Hypoxia Exacerbates Pancreatic β-Cell Dysfunction in A Mouse Model of Diabetes...

SLEEP, Vol. 36, No. 12, 2013 1849 Intermittent Hypoxia and β-cell Dysfunction—Sherwani et al

INTRODUCTIONObstructive sleep apnea (OSA) has been associated with

metabolic abnormalities including altered glucose metabo-lism that is independent of obesity.1-4 While it remains unclear whether OSA is a causal factor in the development of type 2 diabetes mellitus (T2DM), the two conditions often coexist in clinic patients.5 OSA is characterized by repetitive upper airway obstruction resulting in cyclic intermittent hypoxia (IH) during sleep in affected individuals. In healthy human volunteers, acute IH exposure impairs insulin sensitivity, glucose effectiveness, and insulin secretion, suggesting that IH affects the pancreatic function.6 Two previous studies conducted in lean wild type mice have revealed that four days of IH exposure causes pancreatic β-cell proliferation and cell death,7 and a combination of hyperglycemia and IH appear to have synergistic effects on pancreatic β-cell apoptosis.8 However, the effects of IH on pancreatic function in diabetic animals are unclear.

Most common forms of T2DM in humans follow poly-genic inheritance9 with the monogenic forms accounting for

INTERMITTENT HYPOXIA EXACERBATES PANCREATIC β-CELL DYSFUNCTIONhttp://dx.doi.org/10.5665/sleep.3214

Intermittent Hypoxia Exacerbates Pancreatic β-Cell Dysfunction in A Mouse Model of Diabetes MellitusShariq I. Sherwani, MS, MBA1,2; Carolyn Aldana, BS1,2; Saif Usmani, BS1,2; Christopher Adin, DVM3; Sainath Kotha, MD1,2; Mahmood Khan, PhD2,4; Timothy Eubank, PhD1,2; Philipp E. Scherer, PhD5; Narasimham Parinandi, PhD1,2; Ulysses J. Magalang, MD1,2

1Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Wexner Medical Center, The Ohio State University, Columbus, OH; 2Dorothy M. Davis Heart and Lung Research Institute, Wexner Medical Center, The Ohio State University, Columbus, OH; 3Department of Veterinary Clinical Sciences,College of Veterinary Medicine, The Ohio State University, Columbus, OH; 4Department of Emergency Medicine, Wexner Medical Center, The Ohio State University, Columbus, OH; 5Touchstone Diabetes Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX

Submitted for publication December, 2012Submitted in final revised form July, 2013Accepted for publication July, 2013Address correspondence to: Ulysses J. Magalang, MD, Division of Pul-monary, Allergy, Critical Care, and Sleep Medicine, 201 Davis Heart and Lung Research Institute, The Ohio State University, 473 West 12th Av-enue, Columbus, OH 43210; Tel: (614) 247-7701; Fax: (614) 293-4799; E-mail: [email protected]

Study Objectives: The effects of intermittent hypoxia (IH) on pancreatic function in the presence of diabetes and the underlying mechanisms are unclear. We hypothesized that IH would exacerbate pancreatic β-cell dysfunction and alter the fatty acids in the male Tallyho/JngJ (TH) mouse, a rodent model of type 2 diabetes.Design: TH mice were exposed for 14 d to either 8 h of IH or intermittent air (IA), followed by an intraperitoneal glucose tolerance test (IPGTT) and tissue harvest. The effect of IH on insulin release was determined by using a β3-adrenergic receptor (AR) agonist.Measurements and Results: During IH, pancreatic tissue pO2 decreased from 20.4 ± 0.9 to 5.7 ± 2.6 mm Hg, as determined by electron paramagnetic resonance oximetry. TH mice exposed to IH exhibited higher plasma glucose levels during the IPGTT (P < 0.001) while the insulin levels tended to be lower (P = 0.06). Pancreatic islets of the IH group showed an enhancement of the caspase-3 staining (P = 0.002). IH impaired the β-AR agonist-mediated insulin release (P < 0.001). IH increased the levels of the total free fatty acids and saturated fatty acids (palmitic and stearic acids), and decreased levels of the monounsaturated fatty acids in the pancreas and plasma. Ex vivo exposure of pancreatic islets to palmitic acid suppressed insulin secretion and decreased islet cell viability.Conclusions: Intermittent hypoxia increases pancreatic apoptosis and exacerbates dysfunction in a polygenic rodent model of diabetes. An increase in free fatty acids and a shift in composition towards long chain saturated fatty acid species appear to mediate these effects.Keywords: Intermittent hypoxia, diabetes mellitus, β-cell function, fatty acidsCitation: Sherwani SI; Aldana C; Usmani S; Adin C; Kotha S; Khan M; Eubank T; Scherer PE; Parinandi N; Magalang UJ. Intermittent hypoxia exacerbates pancreatic β-cell dysfunction in a mouse model of diabetes mellitus. SLEEP 2013;36(12):1849-1858.

only 1% to 2% of all cases of T2DM.9 Although increasing body weight is a known risk factor for T2DM, most patients with diabetes are not morbidly obese.10,11 Unlike previously described monogenic animal models of diabesity, such as the ob/ob and db/db mice, the Tallyho/JngJ (TH) is a newly established spontaneous polygenic mouse model of T2DM that is characterized by moderate obesity (body weight less than twice the weight of wild type mice of similar age).12-14 Overt hyperglycemia in the TH mouse is male-limited and develops at 16 weeks of age.13

IH has been reported to alter both the levels of circulating free fatty acids (also known as non-esterified fatty acids, NEFAs)15 and the molecular species of fatty acids.16 The latter study has reported a relative increase in circulating monounsaturated fatty acids in lean mice but not in the obese leptin-deficient mice exposed to IH.16 Hypoxia has also been reported to increase the tissue levels of long-chain saturated fatty acids.17-19 Long-chain saturated fatty acids (palmitic and stearic) can cause loss of secretory function of pancreatic β-cells by a process called “lipotoxicity.”20,21 Saturated fatty acids have been shown to induce pancreatic β-cells apoptosis21 and increase oxidative stress,22 and these effects are magni-fied under hyperglycemic conditions.23 However, the effects of IH on the composition of fatty acids in the presence of T2DM are unclear.

Therefore, the aim of this study was to examine the effects of IH on glucose tolerance in a polygenic animal model of T2DM with moderate obesity, the male TH mice. We hypoth-esized that IH would worsen glucose tolerance, exacerbate pancreatic β-cell dysfunction, and alter the composition of the


circulating and pancreatic tissue levels of fatty acids in the male TH mouse.

MATERIALS AND METHODS

AnimalsTH mice (Jackson Labs, Bar Harbor ME) at 16 weeks of

age were used in all experiments. They were acclimated to a custom-made Plexiglas chamber of dimensions 31×18.5×17 cm for 48 h prior to the start of IH or IA exposure. A 12 h light/dark cycle was regulated throughout the course of the experiment, and animals had free access to fresh chow and water ad libitum, except when fasting was needed for the glucose and insulin tests. All protocols were approved by the Ohio State University Institutional Animal Care and Use Committee (IACUC).

IH ExposureMice were placed in custom made-sealed chambers and

exposed to either IH or IA. A gas control switching system was used to expose the animals to IH at a rate of 15 cycles/h for 14 d. To simulate the cyclic hypoxemia in OSA, IH exposure consisted of 80 s of progressive hypoxia, followed by a return to IA over a 160-s period, 8 h/d starting at 08:00. Decreases in FiO2 levels were achieved by delivering 100% nitrogen at an appropriate flow rate to lower the oxygen content in each cage to 5%-6% for 6-8 s. This was immediately followed by a switch to room air oxygen levels prior to the next hypoxia exposure. The use of multiple inputs into the cage was used to produce a uniform nadir of FiO2 level throughout the cage. Six mice were housed continuously in each custom-made Plexiglas chamber, and cages were cleaned daily after the IH or IA exposure. The same timed solenoid and flow regulator set-up was used for IA control, except the gas source was always compressed air. The O2 and CO2 concentrations inside the chamber were continu-ously monitored using gas analyzers (CWE, Inc., Ardmore, PA) and recorded in a computer using a data acquisition system (Biopac Systems, Inc., Goleta, CA) during the 8-h exposure.

Pancreatic Tissue pO2 Measurement Using Electron Paramagnetic Resonance (EPR) Oximetry

EPR oximetry has been utilized to directly measure absolute values of pO2 in intact biological tissue.24 EPR oximetry has high sensitivity and specificity to pO2, and the probes are non-toxic allowing for repeated measurements in vivo.25 In a sepa-rate group of TH mice, we leveraged these advantages of EPR oximetry to directly measure pancreatic tissue pO2 in one cycle of the same IH stimulus that we used in the 14 d exposure as described. In 3 male TH animals, a midline abdominal incision was made under ketamine (100 mg/kg) / xylazine (10 mg/kg) anesthesia to expose the pancreas; 15 µL of sonicated oxygen-sensing microcrystals of LiNc-BuO in saline (100 µg/mL) was then injected into the pancreatic tissue (Figure S1C) using a 28.5-gauge needle. The sensitivity of the EPR linewidth of the LiNc-BuO probe to oxygen was calibrated as described previously.26 The animal was then placed in the supine posi-tion in the L-band (~1.2 GHz) EPR spectrometer (Magnettech, Germany) (Figure S1A) with the pancreas placed adjacent to the loop of the surface coil resonator. The head of the animal was positioned such that the nose and mouth were inside a

small plexiglas cylinder (customized to fit in the EPR spectrom-eter) that allowed changes in FiO2 level using the same timed solenoid and flow regulator set-up as above (Figure S1B). The peak-to-peak linewidth was used to calculate the pO2 using the standard calibration curve as previously described.27 pO2 values were collected every 30 s during one cycle of IH (240 s).

Intraperitoneal Glucose Tolerance Test (IPGTT)On the 14th day of IH or IA exposure, a 1g/kg glucose load was

given to each mouse by intraperitoneal (IP) injection after a 4-h fast. At time points of 0 (prior to the glucose injection), 5, 15, 30, 60, and 120 min, blood samples (50 μL/time point) were collected without anesthesia for determination of plasma glucose and insulin levels. Glucose levels were determined using a glucose oxidase assay (Sigma-Aldrich, St. Louis, MO) and insulin levels were determined using an ELISA assay (Millipore, Inc., Billerica, MA). The areas under the curve (AUC) of the glucose and insulin levels were calculated.28 In initial experiments, the IPGTT after IH or IA exposures was performed in a separate group of TH mice that were given IP injections of midazolam (0.4 mg/mouse) and fentanyl (0.02 mg/mouse) to obtain retro-orbital blood samples before and after the glucose load injection.

Pancreatic β-Cell ApoptosisAfter the IPGTT, the animals were sacrificed and the

pancreas harvested. Immunohistochemistry (IHC) for active caspase 3 (aCasp-3) was done on 4-µm sections of the pancre-atic tissues to quantitate the degree of apoptosis. Digital images of ≥ 4 pancreatic islets per animal were captured, and staining for aCasp-3 was quantified using histogram analysis in Adobe Photoshop CS5 software for the appropriate pixels according to the previously published method.29 The histogram analysis was made by an investigator blinded to experimental design (TE).

β3-Adrenergic Receptor (AR) Agonist Sensitivity TestIn a separate group of TH mice exposed to 14 d of IH or IA,

we performed β3-AR agonist sensitivity test to assess for impair-ments in β3-AR agonist-mediated pancreatic β-cell insulin release between the 2 groups.30,31 Tail vein plasma samples for insulin and NEFAs were obtained from non-fasted TH mice before and 5, 15, and 60 min after an IP injection of 1 mg/kg CL 316,243 (Sigma- Aldrich, St Louis, MO), a β3 AR-specific agonist and a potent stimulus for insulin secretion.

Lipid Extraction and Fatty Acid AnalysisTotal NEFA levels were determined in the plasma and

pancreatic tissues by commercially available assays using spec-trophotometry (Biovision, Inc., Milpitas, CA). To determine the fatty species, total lipids of plasma and pancreatic tissue were extracted by Folch extraction method according to Parinandi et al.32,33 Extracted lipids were taken to dryness under a stream of nitrogen, redissolved in a known volume of chloroform/meth-anol (2/1, vol/vol) and used for further analysis. Non-esterified free fatty acids (NEFAs) and esterified fatty acids in the lipid extracts were analyzed by gas chromatography-mass spectrom-etry (GC-MS, Shimadzu Scientific Instruments, Columbia, MD) equipped with Restek column according to Lepage and Roy,34 Kodavanti et al.,35 and Hinzey et al.36 NEFAs were derivatized into their methyl esters by a direct methylation procedure with


heptadecanoic acid (C17:0) as the internal standard in 5.0 mL of methanol-acetyl chloride 50:1 (v/v) according to Lepage and Roy.34 For the analysis of esterified fatty acids, lipids were subjected to alkaline methanolysis and the resulting fatty acid methyl esters were analyzed by GC-MS with the C17:0 as an internal standard according to Kodavanti et al.35 and Hinzey et al.36 Fatty acid species levels were normalized to mg of total protein of plasma or tissue.

We restricted the analysis of the different species of fatty acids to the saturated palmitic (C16:0) and stearic (C18:0) acids, and the unsaturated palmitoleic (C16:1), oleic (C18:1), and linoleic (C18:2) acids because these species have been reported to contribute differentially to the decline in pancreatic β-cell function, with the saturated fatty acids contributing to β-cell dysfunction while the unsaturated fatty acids appearing to have protective effects.20

Ex vivo Fatty Acid Exposure of TH Pancreatic IsletsA separate group of TH mice that were not previously

exposed to either IH or IA stimulus were anesthetized using isoflurane and euthanized by cervical dislocation. Pancreatic digestion was performed by injection of collagenase P (Sigma-Aldrich, St. Louis, MO) into the common bile duct and islets were isolated with a Ficoll density gradient separation tech-nique as previously described.37,38 Islets were plated at a density of 100 islets in 1 mL of standard RPMI 1640 media (InVitrogen, Carlsbad, CA) with 10% fetal bovine serum and 1% Pen-Strep before incubation at 37°C and 5% CO2. After 24 h, media was replaced and supplemented with either 100 μL vehicle (BSA), 0.3 mM, or 0.4 mM palmitic acid that were all freshly prepared. These concentrations were based on prior studies that show that even these relatively low concentrations of palmitic acid result in pancreatic β-cell apoptosis.23 Incubation was repeated for 24 h. At the termination of the experiment, islets were concen-trated by gravity sedimentation and re-plated to allow assess-ment of islet viability. Conditioned islet media was frozen at -80°C for determination of insulin concentrations. Islet cell viability was determined based on propidium iodide (PI) exclu-sion. Islets were examined using epifluorescent photomicros-copy after incubating with Hoechst 33258 stain and PI. Images were analyzed using NIH Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997-2011), and the percentage of PI positive cells present in each islet was calculated using a custom islet macro as previously described.38

Statistical AnalysisDifferences in glucose and insulin levels during the IPGTT

were evaluated using repeated measures ANOVA as well as comparisons of the corresponding AUCs using t-test. The insulin and NEFA levels during the β3-AR agonist sensitivity testing were also analyzed using repeated measures ANOVA. The Holm-Sidak method was used for the multiple comparison procedure. All other comparisons between groups were made using either t-test, ANOVA, or the corresponding nonpara-metric test. In the analysis of fatty acid species, the P value was adjusted using the Holm’s procedure for multiple compari-sons. The P values < 0.05 were considered significant. Data are presented as means ± SEM.

RESULTS

EPR OximetryFigure 1 shows the changes in FiO2 (Panel A) during a cycle

of the IH stimulus and the corresponding pancreatic tissue pO2 (Panel B). Consistent with previously published values,39,40 at baseline the pancreatic tissue pO2 was 20.4 ± 0.9 mm Hg. During the IH cycle, pancreatic tissue pO2 nadir was 5.7 ± 2.6 mm Hg.

Glucose Tolerance and Pancreatic β Cell ApoptosisTH male mice exposed to IH decreased their body weight

(baseline: 39.7 ± 0.7 vs. final weight: 34.4 ± 0.9 gm; P < 0.001), while there was no change in mice exposed to IA (base-line: 40.6 ± 0.4 vs. final weight: 40.1 ± 0.7gm; P = 0.263). Figure 2 shows the IPGTT results. TH mice exposed to IH had significantly higher plasma glucose values than TH mice exposed to IA (P < 0.001) and the values were different at all time periods. Despite the marked hyperglycemia, the IH exposed mice had lower insulin levels but this did not achieve statistical significance when all time points were included (P = 0.06). The AUC of the glucose levels was significantly higher in the IH exposed mice. Despite the higher glucose values, the AUC of the insulin levels were lower in the TH mice exposed to IH, although this did not reach statistical significance (P = 0.056). The results were similar in the initial experiments wherein the IPGTT was performed under anesthesia with blood samples

Figure 1—Changes in the FiO2 (A) during a cycle of the IH stimulus used in our experiments and the corresponding pancreatic tissue pO2 (B) measured by electron paramagnetic resonance (EPR) oximetry (n = 3). At baseline the pancreatic tissue pO2 was 20.4 ± 0.9 mm Hg. During the IH cycle pancreatic tissue pO2 nadir was 5.7 ± 2.6 mm Hg.


obtained via retro-orbital bleed (see Figure S2). In these experi-ments, the glucose values were significantly higher in TH mice exposed to IH (P < 0.001), but the insulin values were lower (P < 0.001). Figure 3 shows that the pancreatic islets of the TH mice exposed to IH had increased aCasp-3 staining compared to the IA exposed mice.

β3-AR Agonist Sensitivity TestAs shown in Figure 4A, TH mice exposed to IH showed an

impaired β-AR agonist-mediated insulin release (P < 0.001). As expected, CL 316,243 increased insulin levels that were main-tained even after 60 min in both groups. The plasma insulin levels of the IH group were lower compared to the IA group at all time points after the IP injection of CL 316,243, a selec-tive β3-AR agonist. Overall, the IH group also exhibited an impaired β3-AR agonist-induced release of NEFAs (Figure 4B). A multiple comparison procedure showed that the NEFA levels

were significantly different between the 2 groups only at 60 min after the IP injection of CL 316,243.

Fatty Acid Analysis

Pancreatic Tissue Fatty AcidsIH exposure (Figure 5A) caused a significant increase

in the total NEFA levels in the pancreas (IA: 2.50 ± 0.28 vs IH: 4.04 ± 0.29 mmol/gm protein, P = 0.002). Analysis of the fatty acid species of the total (esterified and non-esterified) fatty acids (Figure 5B) showed that the long-chain saturated fatty acid, palmitic (C16:0) and stearic (C18:0) acids were significantly increased in IH exposed mice after adjustments for multiple comparisons (P < 0.001 and 0.009, respectively). Similar results were obtained in the analysis of the fatty acid species of the NEFAs in the pancreas (Figure 5C). IH caused a significant increase in palmitic (C16:0) and stearic (C18:0)

Figure 2—IPGTT in TH mice. TH mice exposed to IH (n = 6) had significantly higher plasma glucose values compared to the TH mice exposed to IA (n = 6) and the multiple comparison procedure showed that the values were different at all time periods. The IH exposed mice had lower insulin levels compared to the IA exposed mice but the analysis did not achieve statistical significance when all time points were included (P = 0.06). The AUC analysis is shown in the right panel. The AUC of the glucose levels was significantly higher in the IH exposed mice (P < 0.001). Despite the higher glucose values, the AUC of the insulin levels was lower in the TH mice exposed to IH, although this did not reach statistical significance (P = 0.056). The results obtained in the experiments (n = 12) wherein the IPGTT was performed under anesthesia were similar except that the insulin values were significantly lower in the TH mice exposed to IH (see supplemental material). *P < 0.05


acids after adjustments for multiple comparisons (P = 0.007 and 0.009, respectively). Additionally, in the NEFA fraction, IH also caused a significant decrease in the monounsaturated fatty acid, palmitoleic (C16:1; P = 0.009).

Plasma Fatty AcidsIH exposure (Figure 5D) caused a significant increase in

the total NEFA levels in the plasma (IA: 1.02 ± 0.05 vs IH: 1.18 ± 0.05 mM, P = 0.04). Analysis of the fatty acid species of the total (esterified and non-esterified) fatty acids (Figure 5E) showed that the long-chain saturated fatty acids, palmitic (C16:0) and stearic (C18:0) acids, were significantly increased in IH exposed mice after adjustments for multiple comparisons (P = 0.011 and 0.005, respectively). Analysis of the fatty acid species of the NEFAs in the plasma (Figure 5F) showed that IH caused a significant increase only in stearic (C18:0) acid (P = 0.004), while the tendency for an increased circulating palmitic (C16:0) acid was not statistically significant. Addition-ally, in the NEFA fraction, IH also caused a significant decrease in the monounsaturated fatty acids, palmitoleic (C16:1) and oleic (C18:1) (P < 0.001 and 0.007, respectively).

Ex Vivo StudiesThese studies were performed in view of the results above to

show that exogenously treated palmitic acid (a long-chain satu-rated fatty acid) would cause dysfunction in the pancreatic islet

through lipotoxicity. After 24 h of incubation with palmitic acid (C16:0), there was a dose-dependent decrease in the amount of secreted insulin by the isolated pancreatic islets (Figure 6A). Pancreatic islets isolated from TH mice and exposed to different concentrations of the long-chain saturated fatty acid, palmitic acid (C16:0), showed a significant increase in PI-positive cells (Figure 6B) indicating reduced islet cell viability (necrosis/apoptosis). A representative example showing dual staining of islets using PI (pink) and Hoeschst (blue) stains is shown in Figure 6C, with the islet cells exposed to palmitic acid exhib-iting more PI staining.

DISCUSSIONThe main findings of this study were that: (i) IH expo-

sure worsened glucose tolerance in the male TH mouse, a polygenic mouse model of T2DM, (ii) the worsened glucose tolerance in IH exposed mice was associated with impaired pancreatic β-cell function as shown by the reduction in insulin

Figure 3—Pancreatic islet active caspase 3 (aCasp-3) IHC. After the IPGTT, TH mice were sacrificed and their pancreas harvested. TH mice exposed to IH (n = 6) had increased aCasp-3 brown staining (arrow) compared to IA exposed mice (n = 6) indicating increased apoptosis in pancreatic islet cells *P = 0.002. Figure 4—β3-AR agonist sensitivity test. In the non-fasted state, TH mice

exposed to IH (n = 6) showed an impaired β-AR agonist-mediated insulin release compared to the TH mice exposed to IA (n = 6). The plasma insulin levels of the IH group were lower compared to the IA group at all time points after the IP injection of 1 mg/kg CL 316,243, a selective β3-AR agonist. Overall, TH mice exposed to IH also exhibited an impaired β3-AR agonist-induced release of NEFAs. A multiple comparison procedure showed that the NEFA levels were significantly different between groups only at 60 min after the IP injection of CL 316,243. *P < 0.05


response to glucose loading, impairment in β3-AR-mediated insulin secretion, and increased apoptosis in pancreatic islet cells, (iii) IH increased the levels of NEFA in the circulation as well as pancreatic tissue, (iv) IH altered the circulating and pancreatic tissue fatty acid composition causing increased levels of long-chain saturated fatty acids and a corresponding decrease in the long-chain monounsaturated fatty acids in both the esterified and non-esterified fractions of fatty acids, and (v) ex vivo exposure of TH pancreatic islet cells to the long-chain saturated fatty acid, palmitic acid, caused a decrease in insulin secretion and decreased islet cell viability. Taken together, our results suggest, but do not definitively establish that both an increase in the levels of NEFA and a shift in composition towards saturated fatty acids, in part, mediate the effects of IH on pancreatic β-cell function under conditions of T2DM.

Two prior studies have shown that even a relatively short exposure to IH (4 d) in lean mice (FVB and C57BL/J strains) increases pancreatic β-cell apoptosis.7,8 In addition, it has been

reported that the combination of IH and hyperglycemia produces the highest rate of β-cell apoptosis in non-diabetic mice.8 In the leptin-deficient (C57BL/6J-Lepob) obese mice, IH exposure also has been shown to worsen glucose tolerance but increased fasting insulin levels, suggesting that IH accelerates the progression of insulin resistance with a compensatory increase in pancreatic insulin secretion.41 Non-diabetic rats exposed to 35 days of IH had a significantly and progressively reduced insulin response to a glucose load.42 Our study extended these findings to a polygenic mouse model of T2DM with only moderate obesity. The male TH mouse is a recently identified mouse model that has been originally derived by selective inbreeding of Theiller Original mice, based on the hyperglycemic phenotype of the original deviant stock having both polyuria and glucosuria.12 Hyperglycemia in the TH mouse is associated with a reces-sive non-insulin-dependent diabetes mellitus 1 (Tanidd1) locus on Chromosome 19. Tanidd1 interacts with other gene loci, including Tanidd2 on Chromosome 13, Tanidd3 on Chromo-some 15, TH-associated body weight (Tabw1) on Chromosome

Figure 5—Effects of IH on fatty acids. (A) IH exposure caused a significant increase in the total NEFA levels in the pancreas (P = 0.002). (B) Analysis of the fatty acid species of the total (esterified and non-esterified) fatty acids showed that the long chain saturated fatty acids, palmitic (C16:0) and stearic (C18:0), were significantly increased in IH exposed mice (P < 0.001 and 0.009, respectively). (C) Similar results were obtained in the analysis of the fatty acid species of the NEFAs in the pancreas (P = 0.007 and 0.009, respectively). Additionally, in the NEFA fraction, IH also caused a significant decrease in the monounsaturated fatty acid, palmitoleic (C16:1) (P = 0.009). (D) IH exposure caused a significant increase in the total NEFA levels in the plasma (P = 0.04). (E) Analysis of the fatty acid species of the total (esterified and non-esterified) fatty acids showed that the long chain saturated fatty acids, palmitic (C16:0) and stearic (C18:0), were significantly increased in IH exposed mice (P = 0.011 and 0.005, respectively). (F) Analysis of the fatty acid species of the NEFAs in the plasma showed that IH caused a significant increase only in stearic (C18:0) acid (P = 0.004), while the tendency for an increased circulating palmitic (C16:0) acid did not achieve statistical significance. Additionally, in the NEFA fraction, IH also caused a significant decrease in the monounsaturated fatty acids, palmitoleic (C16:1) and oleic (C18:1) (P < 0.001 and 0.007, respectively).


7, and TH-associated fat pad (Tafat) on Chromo-some 6.43

Our current study revealed three lines of evidence showing that IH exacerbated pancreatic dysfunction in the male TH mice. Firstly, there was an abnormal response to glucose loading as shown by an impaired insulin response (Figure 2 and Figure S2) to the marked hyperglycemia. In our experiments, TH mice were fasted for 4 h prior to the IPGTT protocol44 as overnight fasting results in a significantly suppressed basal plasma glucose and insulin levels, indi-cating that such prolonged fasting in the mice is a considerably long period of time before an IPGTT.45 Secondly, there was evidence of increased apoptosis (Figure 3) in the pancreas. Thirdly, there was a lower β3-AR-mediated insulin secretion in mice exposed to IH. Previous studies have demonstrated that acute treatment of rodents with β3-AR-selective agonist reliably increases serum insulin levels by 10-100-fold, an effect directly mediated by the actions of the agonist in the adipose tissue.30,31 Although the exact nature of the signal that is transmitted from the adipocytes by β3-AR-selective stimulation is currently unknown, we observed a reduced NEFA level response in TH mice exposed to IH, which was only significant at 60 min after CL 316,243 treatment. This observation suggested that the reduced insulin response to β3-AR stimulation was due to the pancreatic dysfunction, although there might also be a concom-itant adipose tissue dysfunction because of the lower NEFA response at 60 min post-injection of CL 316,243. The effects of IH on adipose tissue in the TH mice need to be studied further.

In view of the previous report showing increased oxidative stress in pancreatic islet cells of lean mice,7 it could be surmised that IH by itself had direct effects on pancreatic β-cells leading to increased apoptosis as observed in the current study. However, the results of our current study suggested another mechanism involving the elevation of NEFAs as well as a shift in their composition towards saturated fatty acids, which could also contribute to the pancreatic dysfunction induced by IH. In contrast to acute exposure of the pancreatic β-cells to NEFA which results in an increase of insulin release, chronic expo-sure results in desensitization and suppression of insulin secre-tion.46 In our study, we demonstrated that IH elevated NEFAs both in the circulation as well as in pancreatic tissue. Elevation

of circulating NEFA by IH has been shown in other rodent strains,15 but not uniformly, as other studies show no effect, particularly in lean mice.16 Our report is the first showing the elevated levels of NEFAs in pancreatic tissue of the hypergly-cemic mice exposed to IH. Accumulation of NEFAs has been implicated in pancreatic dysfunction.20 In the current study, we observed that exposure of isolated TH pancreatic islets to the long chain saturated fatty acid, palmitic acid, caused a decrease in insulin secretion and an increase in islet cell necrosis/apop-tosis. Both palmitic acid (C16:0) and stearic acid (C18:0) have been shown to induce apoptosis in rodents, with the mono-unsaturated fatty acids, palmitoleic acid (C16:1) and oleic acid (C18:1), having protective effects.20,21,23 Our results also suggested that a decrease in fatty acid desaturation might be another pathway in which IH could have increased apoptosis in the pancreas, since an increase in fatty acid desaturation has been shown to have protective effects on pancreatic β-cells from lipoapoptosis.47

Although an increase in the levels of long-chain saturated fatty acids induced by IH in pancreatic tissue as observed in the current study is consistent with the earlier reported studies, which showed that exposure to hypoxia has similar effects on fatty acid composition in the brain and the liver,17-19 the mecha-nism by which IH induces this is unclear. The existing condition of hyperglycemia presents a condition of excess acetyl-CoA, which may explain the elevated palmitic acid as this is the first fatty acid synthesized from acetyl-CoA. Palmitic acid can then

Figure 6—Ex vivo studies. Pancreatic islets were isolated from TH mice and the isolated islets were exposed to different concentrations of the long chain unsaturated FA, palmitic acid (C16:0). (A) After 24 h of incubation with C16:0, secreted insulin level was significantly reduced (P < 0.001). (B) Exposure to C16:0 caused a significant increase in islet cell death (P = 0.002). (C) Representative images are shown that illustrate the increased cell death seen in islets incubated with C16:0 compared to control. *Significantly different from control, P < 0.05; #significantly different from 0.3 mM condition, P < 0.05.


be modified to give rise to other fatty acids. Nevertheless, a prior study has reported that IH does not increase de novo fatty acid biosynthesis.16 However, that study has been conducted in lean mice without T2DM, in which IH exposure does not cause marked hyperglycemia, as opposed to our current observations in the TH mice.

Our current study had certain limitations. The IH exposure of the TH mice was relatively short at 14 d and adaptive mecha-nisms may play a role with a longer exposure. However, two prior studies have shown that even shorter exposure of IH in lean mice (5 d) is sufficient to cause apoptosis in pancreatic islets.7,8 Although we have shown that an increased in NEFA levels as well as a change in saturated fatty acid composi-tion both in the circulation and pancreatic tissue were associ-ated with the development of impaired pancreatic function, further studies would be needed to determine if lowering of NEFA levels and changing the composition of fatty acids (for example, towards the mono-unsaturated species) would protect from the development of pancreatic dysfunction induced by IH in TH mice. Nonetheless, our ex vivo results offered convincing evidence in favor of a direct link between NEFA and its compo-sition in the development of IH-induced pancreatic dysfunc-tion in TH mice. It should also be emphasized that the exact mechanism(s) of elevation of NEFA and long-chain fatty acid levels in pancreas induced by IH under hyperglycemic condi-tions in TH mice warrant thorough investigation. IH may also increase catecholamine and corticosterone levels and future studies are needed to determine the role of these hormones in mediating the effects of IH on pancreatic dysfunction. Another potential confounder with the NEFA measures is that the IH exposed mice lost weight during the14 day exposure. Poten-tial mechanisms for the weight reduction that was observed in the IH exposed mice include a reduction in food intake as well as an increase in lipolytic effect on adipose tissue through increased sympathetic nervous system activity.48 We did not measure food intake during our experiments and a reduced food intake caused by IH could certainly confound the NEFA measurements, as “starvation” is known to increase NEFA levels. However, a recent study showed that NEFA levels return to baseline after 6 days of starvation.49 Furthermore, six days of starvation was reported to have opposite effects to our find-ings in the molecular species of NEFA in the blood; starvation decreased the levels of C18:0 and increased C18:1.49 Therefore, it is unlikely that reduced food intake could explain our find-ings. IH exposure reduces the amount of slow wave and rapid eye movement (REM) sleep and increases wake time during the major sleep period in mice.50 We did not obtain electroencepha-lographic data during the IH exposure. However, we exposed the TH mice during their usual sleep period during the daytime, a protocol that has been successfully used by others in many studies to simulate the hypoxic stress of OSA.7,8,15,16 We also cannot exclude the possibility that a concomitant increase in insulin resistance induced by IH, as has been shown in previous studies,51 contributed to the marked hyperglycemia observed in the TH mice exposed to IH.

The IH stimulus used in our experiments represents a moderate to severe form of hypoxic stimulus. A previous study by Reinke and colleagues52 using a more frequent IH stimulus (60 cycles/h) but similar FiO2 nadir of 5%, showed a

comparable degree of tissue pO2 reduction in muscle tissue in lean mice (37.7 ± 7.3 to 11.2 ± 2.1 mm Hg) with what we have observed in pancreatic tissue (20.4 ± 0.9 to 5.7 ± 2.6 mm Hg) using a less frequent IH stimulus of 15 cycles/hour. However, as in our experiments, measurement of tissue oxygen levels in the study by Reinke et al. were performed in the anesthetized animal, and tissue pO2 levels would be expected to be higher in the awake state during the 14-d exposure because ventilation increases massively during IH exposure. Indeed, an IH regimen of 60 cycles/h with the same FiO2 nadir of 5% to 6% (similar nadir to our IH stimulus) was found to decrease arterial pO2 to 47 ± 2 mm Hg and oxyhemoglobin saturation to 85% ± 2% in conscious unrestrained mice, a degree of hypoxic stress that occurs in moderate to severe clinical OSA.53

The effects of OSA treatment with continuous positive airway pressure (CPAP) on glycemic control in patients with T2DM are conflicting,54-56 with a recent study suggesting that there may be a benefit.57 However, the effects of OSA treat-ment on pancreatic function and β-cell mass have not been systematically studied in human subjects. The results of the current study suggested further research in patients with OSA and T2DM is warranted.

In summary, our current study revealed that IH exacer-bated the pancreatic dysfunction in a polygenic rodent model of T2DM, the TH mice. Although other mechanisms could be likely involved, our results suggested that an increase in NEFA and a shift in composition of fatty acids towards long-chain saturated fatty acid species in the pancreatic tissue, partly medi-ated the effects of IH in causing the pancreatic β-cell dysfunc-tion. Our current study also suggested that the pancreatic β-cell dysfunction was mostly likely to occur under the conditions of uncontrolled hyperglycemia.

ACKNOWLEDGMENTThe authors thank Dr. P. Kuppusamy for generously

providing the LiNc-BuO EPR probe.

DISCLOSURE STATEMENTThis was not an industry supported study. This work was funded

by NIH grant HL093463 (to Drs. Parinandi and Magalang), the Tzagournis Medical Research Endowment Funds of The Ohio State University (to Dr. Magalang), and the Juvenile Diabetes Association JDRF 17-2012-366 (to Dr. Scherer). The authors have indicated no financial conflicts of interest.

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SLEEP, Vol. 36, No. 12, 2013 1858A Intermittent Hypoxia and β-cell Dysfunction—Sherwani et al

SUPPLEMENTAL MATERIAL

Figure S1—Electron paramagnetic resonance (EPR) oximetry set-up. EPR oximetry was used to directly measure adipose tissue pO2 in one cycle of the same IH stimulus that we used in the 14 d exposure. (A) The animal was placed in the supine position in the L-band (~1.2 GHz) EPR spectrometer after a midline abdominal incision was made under anesthesia to expose the pancreas. 15 µL of sonicated oxygen-sensing microcrystals of LiNc-BuO was injected into the pancreatic tissue using a small needle as shown by the black arrow in (C). (B) A Plexiglas cylinder (white arrows) was customized to fit in the EPR spectrometer and allowed changes in FiO2 level using the same timed solenoid and flow regulator set-up as described. The head of the animal (C57BL/6 mouse is shown as an example) was positioned such that the nose and mouth were inside the small cylinder. pO2 values were collected every 30 s during one cycle of IH.

SLEEP, Vol. 36, No. 12, 2013 1858B Intermittent Hypoxia and β-cell Dysfunction—Sherwani et al

Figure S2—IPGTT in TH mice. In these initial experiments, the IPGTT was performed under anesthesia with blood samples obtained via retro-orbital bleed. TH mice exposed to IH (n = 6) had significantly higher plasma glucose values compared to the TH mice exposed to IA (n = 6) and the multiple comparison procedure showed that the values were different at all time periods. The IH exposed mice had lower insulin levels compared to the IA exposed mice and the multiple comparison procedure showed that the values were different at all time periods. The AUC analysis is shown in the right panel.

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