Time-dependent changes in glucose and insulin regulation during intermittent hypoxia and continuous hypoxia.

Hypoxia manifests in many forms including the short repetitive intermittent hypoxia (IH) of sleep apnoea and the continuous hypoxia (CH) of altitude, both of which may impact metabolic function. Based on our own previous studies and the available literature, we hypothesized that whereas acute exposure to IH and CH would lead to comparable metabolic dysfunction, with longer-term exposure, metabolism would normalize to a greater extent with CH than IH. Studies were conducted in lean C57BL/6J mice exposed to either IH or CH for 1 day or 4 weeks and compared to either intermittent air (IA) or unhandled (UN) controls, respectively. We utilized the frequently sampled intravenous glucose tolerance test and minimal model analyses to determine insulin-dependent (insulin sensitivity; S (I)) and insulin-independent (glucose effectiveness; S (g)) glucose disposal, as well as the insulin response to glucose (acute insulin response to glucose; AIR(g)). Our data show that 1-day exposure impaired the glucose tolerance and caused reductions in S (g) and AIR(g) in both the IH and CH groups, but only IH caused a significant decrease in S (I) (7.5 ± 2.7 vs. 17.0 ± 5.3 µU ml(-1) min(-1); p < 0.05). After 4-week exposure, there was evidence of metabolic adaptation in both hypoxic groups, however, in the CH group, there was a supranormal increase in S (I) relative to both UN and IH groups. We conclude that in lean mice, the marked metabolic dysfunction that occurs with acute exposure to hypoxia is reversed to a greater extent with chronic CH exposure than chronic IH exposure.

Chronic intermittent hypoxia exposure improves left ventricular contractility in transgenic mice with heart failure.

We previously reported the unexpected finding that 4 wk of exposure to intermittent hypoxia (IH), which simulates the hypoxic stress of obstructive sleep apnea, improved LV cardiac function in healthy, lean C57BL/6J mice. The purpose of the present study was to assess the impact of 4 wk of IH on cardiac function in a transgenic murine model that exhibits a natural history of heart failure. We hypothesized that IH exposure would exacerbate cardiac decompensation in heart failure. Adult male FVB (wild type) and transgenic mice with cardiac overexpression of tumor necrosis factor α (TNF-αTG) at 10-12 wk of age were exposed to 4 wk of IH (nadir inspired oxygen 5-6% at 60 cycles/h for 12 h during light period) or intermittent air (IA) as control. Cardiac function was assessed by echocardiography and pressure-volume loop analyses, and mRNA and protein expression were performed on ventricular homogenates. TNF-αTG mice exposed to IA exhibited impaired LV contractility and increased LV dilation associated with markedly elevated cardiac expression of atrial natriuretic peptide and brain natriuretic peptide compared with wild-type mice. When wild-type FVB mice were exposed to IH, they exhibited increases in arterial pressure and dP/dt(max), consistent with our previous report in C57BL/6J mice. Surprisingly, we found that TNF-αTG mice exposed to IH showed a reduction in end-diastolic volume (38.7 ± 3.8 to 22.2 ± 2.1 ul; P < 0.01) and an increase in ejection fraction (29.4 ± 2.5 to 41.9 ± 3.1%; P < 0.05). In contrast to our previous study in C56Bl/6J mice, neither FVB nor TNF-αTG mice exhibited an upregulation in ß-adrenergic expression or cAMP in response to IH exposure. We conclude that 4 wk of exposure to IH in mice induces adaptive responses that improve cardiac function in not only healthy animals but also in animals with underlying heart failure.

Free fatty acids block glucose-induced ß-cell proliferation in mice by inducing cell cycle inhibitors p16 and p18.

Pancreatic ß-cell proliferation is infrequent in adult humans and is not increased in type 2 diabetes despite obesity and insulin resistance, suggesting the existence of inhibitory factors. Free fatty acids (FFAs) may influence proliferation. In order to test whether FFAs restrict ß-cell proliferation in vivo, mice were intravenously infused with saline, Liposyn II, glucose, or both, continuously for 4 days. Lipid infusion did not alter basal ß-cell proliferation, but blocked glucose-stimulated proliferation, without inducing excess ß-cell death. In vitro exposure to FFAs inhibited proliferation in both primary mouse ß-cells and in rat insulinoma (INS-1) cells, indicating a direct effect on ß-cells. Two of the fatty acids present in Liposyn II, linoleic acid and palmitic acid, both reduced proliferation. FFAs did not interfere with cyclin D2 induction or nuclear localization by glucose, but increased expression of inhibitor of cyclin dependent kinase 4 (INK4) family cell cycle inhibitors p16 and p18. Knockdown of either p16 or p18 rescued the antiproliferative effect of FFAs. These data provide evidence for a novel antiproliferative form of ß-cell glucolipotoxicity: FFAs restrain glucose-stimulated ß-cell proliferation in vivo and in vitro through cell cycle inhibitors p16 and p18. If FFAs reduce proliferation induced by obesity and insulin resistance, targeting this pathway may lead to new treatment approaches to prevent diabetes.

Simultaneous measurement of insulin sensitivity, insulin secretion, and the disposition index in conscious unhandled mice.

Of the parameters that determine glucose disposal and progression to diabetes in humans: first-phase insulin secretion, glucose effectiveness (Sg), insulin sensitivity (Si), and the disposition index (DI), only Si can be reliably measured in conscious mice. To determine the importance of the other parameters in murine glucose homeostasis in lean and obese states, we developed the frequently sampled intravenous glucose tolerance test (FSIVGTT) for use in unhandled mice. We validated the conscious FSIVGTT against the euglycemic clamp for measuring Si in lean and obese mice. Insulin-resistant mice had increased first-phase insulin secretion, decreased Sg, and a reduced DI, qualitatively similar to humans. Intriguingly, although insulin secretion explained most of the variation in glucose disposal in lean mice, Sg and the DI more strongly predicted glucose disposal in obese mice. DI curves identified individual diet-induced obese (DIO) mice as having compensated or decompensated insulin secretion. Conscious FSIVGTT opens the door to apply mouse genetics to the determinants of in vivo insulin secretion, Sg, and DI, and further validates the mouse as a model of metabolic disease.

Cardiac-specific leptin receptor deletion exacerbates ischaemic heart failure in mice.

AIMS: the obesity-related adipokine, leptin, has multiple actions on peripheral organs, including the mitigation of adverse cardiovascular outcomes after myocardial infarction (MI). Although we recently demonstrated that leptin, its receptor, and downstream signalling are up-regulated in the heart after MI, the significance of intact cardiomyoctye leptin signalling is unknown. Therefore, our objective was to generate a cardiomyocyte-specific leptin receptor knock-out (ObRKO) mouse to determine whether worse cardiac outcomes after MI result from impaired leptin signalling in cardiomyocytes. METHODS AND RESULTS: tamoxifen-inducible ObRKO mice were subjected to experimental MI or sham surgeries and studied after 1 month. After MI, ObRKO mice displayed a loss of cardiac signal transducer and activator of transcription (STAT) 3 and adenosine monophosphate-activated protein kinase (AMPK) signalling. Worse survival and cardiac morbidity were also seen in the ObRKO mouse post-MI, including decreased contractile function and glycolytic metabolism, and increased left ventricular dilation, hypertrophy, collagen deposition, matrix metalloproteinase activity, apoptosis, and inflammation. Treatment of ObRKO mice post-MI with an ObR-independent AMPK activator improved cardiac function and restored many of these maladaptive processes to wild-type levels. CONCLUSION: these data indicate that leptin signalling mitigates cardiac injury in the post-MI failing heart by acting directly on cardiomyocytes to increase STAT3 and AMPK activation, to decrease cardiac hypertrophy, apoptosis, and inflammation, and to limit deleterious changes in cardiac structure, function, and glycolytic metabolism.

Hyperinsulinemia predicts survival in a hyperglycemic mouse model of critical illness.

OBJECTIVES: : The mechanisms by which correcting hyperglycemia with exogenous insulin improves mortality and morbidity in critically ill patients remain unclear. We designed this study to test the hypothesis that relative endogenous insulin deficiency is associated with adverse outcomes in critical illness related to hyperglycemia. DESIGN: : Prospective controlled animal study. SETTING: : University research laboratory. SUBJECTS: : Male C57BL/6J mice, 8-12 wks old. INTERVENTIONS: : Spontaneously breathing mice were instrumented with chronic indwelling arterial and venous catheters. After a postoperative recovery period, endotoxemia was initiated with intra-arterial lipopolysaccharide (1 mg/kg) in the presence of dextrose infusion (100 microL/hr). Insulin secretion was blocked with diazoxide (2.5-30 mg/kg/day). Mice were monitored continuously for 48 hrs with blood sampled serially for blood glucose and plasma insulin determinations. MEASUREMENTS AND MAIN RESULTS: : In both saline- and glucose-infused mice, lipopolysaccharide administration induced transient hemodynamic instability without significant impact on mortality. In the saline-infused group, lipopolysaccharide administration caused a transient reduction in blood glucose and in circulating insulin. However, in glucose-infused mice, lipopolysaccharide induced a large and unexpected increase in circulating insulin without significant alteration in blood glucose. Blockade of insulin secretion in response to lipopolysaccharide in the presence of exogenous glucose precipitated marked hyperglycemia and resulted in >90% mortality. In a subanalysis of animals matched for the degree of hyperglycemia, nonsurvivors had markedly lower insulin levels compared with survivors (3.5 +/- 0.8 ng/dL vs. 9.3 +/- 1.4 ng/dL; p < .004). CONCLUSIONS: : Endogenous insulin deficiency in the face of hyperglycemia is associated with mortality in a mouse model of lipopolysaccharide-induced critical illness.

Chronic intermittent hypoxia increases left ventricular contractility in C57BL/6J mice.

Intermittent hypoxia (IH) commonly occurs in patients with obstructive sleep apnea and can cause a wide range of pathology, including reduced left ventricular (LV) ejection fraction in rats as determined by echocardiography, in rodent models. We utilized echocardiography and pressure-volume (PV) loop analyses to determine whether LV contractility was decreased in inbred C57BL/6J mice exposed to IH and whether blockade of beta-adrenergic receptors modified the response to hypoxia. Adult male 9- to 10-wk-old mice were exposed to 4 wk of IH (nadir inspired O(2) 5-6% at 60 cycles/h for 12 h during the light period) or intermittent air (IA) as control. A second group of animals were exposed to the same regimen of IH or IA, but in the presence of nonspecific beta-blockade with propranolol. Cardiac function was assessed by echocardiography and PV loop analyses, and mRNA and protein expression in ventricular homogenates was determined. Contrary to our expectations, we found with PV loop analyses that LV ejection fraction (63.4 +/- 3.5 vs. 50.5 +/- 2.6%, P = 0.015) and other measures of LV contractility were increased in IH-exposed animals compared with IA controls. There were no changes in contractile proteins, atrial natriuretic peptide levels, LV posterior wall thickness, or heart weight with IH exposure. However, cAMP levels were elevated after IH, and propranolol administration attenuated the increase in LV contractility induced by IH exposure. We conclude that, contrary to our hypothesis, 4 wk of IH exposure in C57BL/6J mice causes an increase in LV contractility that occurs independent of ventricular hypertrophy and is, in part, mediated by activation of cardiac beta-adrenergic pathways.

Leptin signalling reduces the severity of cardiac dysfunction and remodelling after chronic ischaemic injury.

AIMS: Leptin is elevated under conditions of both obesity and heart failure (HF), and activation of leptin receptor (ObR) signalling is known to increase in vivo cardiac contractility and to have anti-hypertrophic effects on the left ventricle (LV). However, it is unknown whether ObR signalling is altered in cardiomyocytes after myocardial infarction (MI) leading to HF, or if a deficiency in ObR signalling leads to worse HF. METHODS AND RESULTS: In separate experimental protocols, C57BL/6J and leptin-deficient (ob/ob) mice underwent open-chest surgery to induce permanent left coronary artery ligation (CAL) or had a sham operation. Subgroups of ob/ob mice examined were lean (food-restricted), obese (food-ad libitum), and leptin repleted. Four weeks post-surgery, cardiac structure and function was examined by echocardiography, and the activation of cardiac leptin signalling was characterized through quantitative PCR, western blotting, and DNA-binding activities. CAL produced echocardiographic evidence of HF in C57BL/6J mice, elevated circulating leptin, increased cardiomyocyte leptin and ObR expression, and activated myocardial signal transducer and activator of transcription-3 (STAT3). In leptin-deficient ob/ob mice, whether lean or obese, CAL caused increased hypertrophy and dilation, decreased contractility of the LV, and worsened survival relative to wildtype or leptin-repleted mice after CAL. In ob/ob mice, activation of cardiac STAT3 signalling after CAL is enhanced in the presence of leptin and parallels the induction of the STAT3-responsive genes, tissue-inhibitor of metalloproteinase-1 and heat shock protein-70. CONCLUSION: These data demonstrate that HF increases ObR signalling in cardiomyocytes and that activation of ObR signalling improves functional outcomes in chronic ischaemic injury leading to HF.

Intermittent hypoxia reverses the diurnal glucose rhythm and causes pancreatic beta-cell replication in mice.

Obstructive sleep apnoea (OSA) and type 2 diabetes frequently co-exist and potentially interact haemodynamically and metabolically. However, the confounding effects of obesity have obscured the examination of any independent or interactive effects of the hypoxic stress of OSA and the hyperglycaemia of type 2 diabetes on haemodynamic and metabolic outcomes. We have developed a chronically catheterized, unhandled, lean murine model to examine the effects of intermittent hypoxic (IH) exposure and exogenous glucose infusion on the diurnal pattern of arterial blood pressure and blood glucose, as well as pancreatic beta-cell growth and function. Four experimental groups of adult male C57BL/J mice were exposed to 80 h of (1) either IH (nadir of inspired oxygen 5-6% at 60 cycles h(-1) for 12 h during light period) or intermittent air (IA; control) and (2) continuous infusion of either 50% dextrose or saline (control). IH exposure during saline infusion caused a sustained increase in arterial blood pressure of 10 mmHg (P < 0.0001), reversed the normal diurnal rhythm of blood glucose (P < 0.03), doubled corticosterone levels (P < 0.0001), and increased replication of pancreatic beta-cells from 1.5 +/- 0.3 to 4.0 +/- 0.8% bromodeoxyuridine (BrdU)-positive) beta-cells. The combined stimulus of IH exposure and glucose infusion attenuated the hypertension, exacerbated the reversed diurnal glucose rhythm, and produced the highest rates of apoptosis in beta-cells, without any additive effects on beta-cell replication. We conclude that, in contrast to the development of sustained hypertension, IH impaired glucose homeostasis only during periods of hypoxic exposure. IH acted as a stimulus to pancreatic beta-cell replication, but the presence of hyperglycaemia may increase the hypoxic susceptibility of beta-cells. This model will provide a basis for future mechanistic studies as well as assessing the metabolic impact of common comorbities in OSA, including obesity, insulin resistance and type 2 diabetes.