Evaluation of the specific effects of intranasal glucagon on glucose production and lipid concentration in healthy men during a pancreatic clamp.

AIM: To investigate the specific effects of intranasal glucagon (ING) on plasma glucose, endogenous glucose production (EGP) and lipid concentration. METHODS: We conducted a single-blind, randomized, crossover study at our academic investigation unit. Under pancreatic clamp conditions with tracer infusion, 1 mg ING or intranasal placebo (INP) was administered to 10 healthy men. As pilot studies showed that ING transiently increased plasma glucagon, we infused intravenous glucagon for 30 minutes along with INP to ensure similar plasma glucagon concentrations between interventions. The main outcome measures were plasma glucose, EGP, free fatty acid (FFA) and triglyceride (TG) concentrations. RESULTS: In the presence of similar plasma glucagon concentrations, the increase in plasma glucose under these experimental conditions was attenuated with ING (mean plasma glucose analysis of variance P < .001) with reduction in EGP (P = .027). No significant differences were seen in plasma FFA and TG concentrations. CONCLUSION: ING raises plasma glucose but this route of administration attenuates the gluco-stimulatory effect of glucagon by reducing EGP. This observation invites speculation about a potential central nervous system effect of glucagon, which requires further investigation. If ING is developed as a treatment for hypoglycaemia, this attenuated effect on plasma glucose should be taken into account.

IKKß inhibition prevents fat-induced beta cell dysfunction in vitro and in vivo in rodents.

AIMS/HYPOTHESIS: We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase ß (IKKß), which is activated by oxidative stress, is also implicated. METHODS: Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKß inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKß) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice. RESULTS: 48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet-1 h-1) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet-1 h-1] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKß also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet-1 h-1] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate). CONCLUSIONS/INTERPRETATION: Our results demonstrate a causal role for IKKß in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.

A low-protein diet combined with low-dose endotoxin leads to changes in glucose homeostasis in weanling rats.

Severe malnutrition is a leading cause of global childhood mortality, and infection and hypoglycemia or hyperglycemia are commonly present. The etiology behind the changes in glucose homeostasis is poorly understood. Here, we generated an animal model of severe malnutrition with and without low-grade inflammation to investigate the effects on glucose homeostasis. Immediately after weaning, rats were fed diets containing 5 [low-protein diet (LP)] or 20% protein [control diet (CTRL)], with or without repeated low-dose intraperitoneal lipopolysaccharide (LPS; 2 mg/kg), to mimic inflammation resulting from infections. After 4 wk on the diets, hyperglycemic clamps or euglycemic hyperinsulinemic clamps were performed with infusion of [U-(13)C6]glucose and [2-(13)C]glycerol to assess insulin secretion, action, and hepatic glucose metabolism. In separate studies, pancreatic islets were isolated for further analyses of insulin secretion and islet morphometry. Glucose clearance was reduced significantly by LP feeding alone (16%) and by LP feeding with LPS administration (43.8%) compared with control during the hyperglycemic clamps. This was associated with a strongly reduced insulin secretion in LP-fed rats in vivo as well as ex vivo in islets but signficantly enhanced whole body insulin sensitivity. Gluconeogenesis rates were unaffected by LP feeding, but glycogenolysis was higher after LP feeding. A protein-deficient diet in young rats leads to a susceptibility to low-dose endotoxin-induced impairment in glucose clearance with a decrease in the islet insulin secretory pathway. A protein-deficient diet is associated with enhanced peripheral insulin sensitivity but impaired insulin-mediated suppression of hepatic glycogenolysis.

FFA-induced hepatic insulin resistance in vivo is mediated by PKCδ, NADPH oxidase, and oxidative stress.

Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKCδ and oxidative stress play causal roles in this process and whether this occurs via IKKß- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IκBα content, increased JNK phosphorylation (markers of IKKß and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKCδ activation. Furthermore, an antisense oligonucleotide against PKCδ prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKCδ, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA → PKCδ → NADPH oxidase and oxidative stress → IKKß/JNK → impaired hepatic insulin signaling.

Overexpression of glutathione peroxidase 4 prevents ß-cell dysfunction induced by prolonged elevation of lipids in vivo.

We have shown that oxidative stress is a mechanism of free fatty acid (FFA)-induced ß-cell dysfunction. Unsaturated fatty acids in membranes, including plasma and mitochondrial membranes, are substrates for lipid peroxidation, and lipid peroxidation products are known to cause impaired insulin secretion. Therefore, we hypothesized that mice overexpressing glutathione peroxidase-4 (GPx4), an enzyme that specifically reduces lipid peroxides, are protected from fat-induced ß-cell dysfunction. GPx4-overexpressing mice and their wild-type littermate controls were infused intravenously with saline or oleate for 48 h, after which reactive oxygen species (ROS) were imaged, using dihydrodichlorofluorescein diacetate in isolated islets, and ß-cell function was assessed ex vivo in isolated islets and in vivo during hyperglycemic clamps. Forty-eight-hour FFA elevation in wild-type mice increased ROS and the lipid peroxidation product malondialdehyde and impaired ß-cell function ex vivo in isolated islets and in vivo, as assessed by decreased disposition index. Also, islets of wild-type mice exposed to oleate for 48 h had increased ROS and lipid peroxides and decreased ß-cell function. In contrast, GPx4-overexpressing mice showed no FFA-induced increase in ROS and lipid peroxidation and were protected from the FFA-induced impairment of ß-cell function assessed in vitro, ex vivo and in vivo. These results implicate lipid peroxidation in FFA-induced ß-cell dysfunction.

A monolithic polymeric microdevice for pH-responsive drug delivery.

A drug-delivery microdevice integrating pH-responsive nano-hydrogel particles functioning as intelligent nano valves is described. The polymeric microdevices are monolithic without requiring peripheral control hardware or additional components for controlling drug-release rates. pH-responsive nanoparticles were synthesized and embedded into a composite membrane. The resulting pH-responsive composite membranes were integrated with PDMS micro reservoirs via a room-temperature transfer bonding technique to form the proof-of-concept microdevices. In vitro release characterization of the microdevices was conducted in which the release rate of Vitamin B(12) (VB(12)) as a model drug increased dramatically when the local pH value was decreased from 7.4 to 4. This device concept can serve as a platform technology for intelligent drug delivery in response to various in vivo environmental signals.

Pharmacologic or genetic activation of SIRT1 attenuates the fat-induced decrease in beta-cell function in vivo.

BACKGROUND: There is evidence that sirtuin 1 (SIRT1), a key regulator of nutrient metabolism, increases ß-cell secretory function. Excess circulating fat, as seen in obesity, has been shown to decrease ß-cell function, an effect that may involve decreased SIRT1 activity. Consequently, SIRT1 activation may increase ß-cell function in conditions of elevated plasma-free fatty acid levels. Here we attempted to attenuate the lipid-induced decrease in ß-cell function in vivo using pharmacological and genetic models of SIRT1 activation. METHODS: Our pharmacologic model involved 48 h intravenous infusion of Wistar rats with either saline or oleate with or without the SIRT1 activator resveratrol. Additionally, we used ß-cell-specific SIRT1 overexpressing (BESTO) mice and wild-type littermates infused for 48 h intravenously with either saline or oleate. In both models, the infusion period was followed by assessment of ß-cell function using the hyperglycemic clamp method. RESULTS: Lipid infusion resulted in a significant decrease in ß-cell function as expected in both rats (p < 0.05) and mice (p < 0.001). Both models of SIRT1 activation, which did not alter ß-cell function in the absence of fat, resulted in partial protection from the fat-induced decrease in ß-cell function (NS vs. control). CONCLUSION: These results suggest that SIRT1 is a therapeutic target in decreased ß-cell function specifically induced by fat.

Glucose-Induced ß-Cell Dysfunction In Vivo: Evidence for a Causal Role of C-jun N-terminal Kinase Pathway.

Prolonged elevation of glucose can adversely affect ß-cell function. Oxidative stress, which has been implicated in glucose-induced ß-cell dysfunction, can activate c-jun N-terminal kinase (JNK). However, whether JNK is causal in glucose-induced ß-cell dysfunction in vivo is unclear. Therefore, we aimed at investigating the causal role of JNK activation in in vivo models of glucose-induced ß-cell dysfunction. Glucose-induced ß-cell dysfunction was investigated in the presence or absence of JNK inhibition. JNK inhibition was achieved using either (i) the JNK-specific inhibitor SP600125 or (ii) JNK-1-null mice. (i) Rats or mice were infused intravenously with saline or glucose with or without SP600125. (ii) JNK-1 null mice and their littermate wild-type controls were infused intravenously with saline or glucose. Following the glucose infusion periods in rats and mice, ß-cell function was assessed in isolated islets or in vivo using hyperglycemic clamps. Forty-eight-hour hyperglycemia at ~20 mM in rats or 96-hour hyperglycemia at ~13 mM in mice impaired ß-cell function in isolated islets and in vivo. Inhibition of JNK using either SP600125 or JNK-1-null mice prevented glucose-induced ß-cell dysfunction in isolated islets and in vivo. Islets of JNK-1-null mice exposed to hyperglycemia in vivo showed an increase in Pdx-1 and insulin 2 mRNA, whereas islets of wild-type mice did not. Together, these data show that JNK pathway is involved in glucose-induced ß-cell dysfunction in vivo and is thus a potential therapeutic target for type 2 diabetes.

Effect of hydroxycut intake on fasted and postprandial lipemia in rats.

Hydroxycut, an herbal supplement not currently defined as a drug, is frequently sold over the counter to increase exercise performance, build muscles, and burn fat. The effects of 8 wk of hydroxycut-induced changes on blood lipid profile in rats fed with either regular or high-fat diet were evaluated. Regardless of fat content in the diet, the doses of hydroxycut used significantly decreased fasting serum concentrations of cholesterol, triacylglycerol (TAG), low-density lipoprotein (LDL) cholesterol, total apolipoprotein B (apo B), and LDL/high-density lipoprotein (HDL) cholesterol ratio. A significant increase in serum blood glucose level was observed with hydroxycut intake in the presence of a high-fat diet. No hydroxycut-related changes in serum activities of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate dehydrogenase (SGPT), lactate dehydrogenase (LDH), and creatinine phosphokinase (CPK) enzymes were noted, indicating no liver damage occurred. A decrease in liver fat content was observed with hydroxycut intake. The drug did not affect the number and composition of secreted very-low-density lipoprotein (VLDL) particles except for a decrease in VLDL TAG when the fat content in the diet was high. Hydroxycut reduced significantly LDL apo B and LDL TAG and cholesterol concentrations. Hydroxycut increased TAG and cholesterol excretion in feces. A single intragastric food load containing hydroxycut reduced significantly postprandial plasma TAG concentration in a dose-dependent manner. In conclusion, hydroxycut intake in recommended doses exerts a beneficial impact on atherosclerosis, an effect attributed to improved clearance and metabolism of lipoprotein particles, and to a lesser extent to an increased excretion of TAG and cholesterol in the feces. More studies are needed to ensure the safety of long-term use of hydroxycut.

Intranasal insulin suppresses endogenous glucose production in humans compared with placebo in the presence of similar venous insulin concentrations.

Intranasal insulin (INI) has been shown to modulate food intake and food-related activity in the central nervous system in humans. Because INI increases insulin concentration in the cerebrospinal fluid, these effects have been postulated to be mediated via insulin action in the brain, although peripheral effects of insulin cannot be excluded. INI has been shown to lower plasma glucose in some studies, but whether it regulates endogenous glucose production (EGP) is not known. To assess the role of INI in the regulation of EGP, eight healthy men were studied in a single-blind, crossover study with two randomized visits (one with 40 IU INI and the other with intranasal placebo [INP] administration) 4 weeks apart. EGP was assessed under conditions of an arterial pancreatic clamp, with a primed, constant infusion of deuterated glucose and infusion of 20% dextrose as required to maintain euglycemia. Between 180 and 360 min after administration, INI significantly suppressed EGP by 35.6% compared with INP, despite similar venous insulin concentrations. In conclusion, INI lowers EGP in humans compared with INP, despite similar venous insulin concentrations. INI may therefore be of value in treating excess liver glucose production in diabetes.

Is Insulin Action in the Brain Relevant in Regulating Blood Glucose in Humans?

PURPOSE: In addition to its direct action on the liver to lower hepatic glucose production, insulin action in the central nervous system (CNS) also lowers hepatic glucose production in rodents after 4 hours. Although CNS insulin action (CNSIA) modulates hepatic glycogen synthesis in dogs, it has no net effect on hepatic glucose output over a 4-hour period. The role of CNSIA in regulating plasma glucose has recently been examined in humans and is the focus of this review. METHODS AND RESULTS: Intransal insulin (INI) administration increases CNS insulin concentration. Hence, INI can address whether CNSIA regulates plasma glucose concentration in humans. We and three other groups have sought to answer this question, with differing conclusions. Here we will review the critical aspects of each study, including its design, which may explain these discordant conclusions. CONCLUSIONS: The early glucose-lowering effect of INI is likely due to spillover of insulin into the systemic circulation. In the presence of simultaneous portal and CNS hyperinsulinemia, portal insulin action is dominant. INI administration does lower plasma glucose independent of peripheral insulin concentration (between ∼3 and 6 h after administration), suggesting that CNSIA may play a role in glucose homeostasis in the late postprandial period when its action is likely greatest and portal insulin concentration is at baseline. The potential physiological role and purpose of this pathway are discussed in this review. Because the effects of INI are attenuated in patients with type 2 diabetes and obesity, this is unlikely to be of therapeutic utility.

Evaluation of the Effect of Enteral Lipid Sensing on Endogenous Glucose Production in Humans.

Administration of lipids into the upper intestine of rats has been shown to acutely decrease endogenous glucose production (EGP) in the preabsorptive state, postulated to act through a gut-brain-liver axis involving accumulation of long-chain fatty acyl-CoA, release of cholecystokinin, and subsequent neuronal signaling. It remains unknown, however, whether a similar gut-brain-liver axis is operative in humans. Here, we infused 20% Intralipid (a synthetic lipid emulsion) or saline intraduodenally for 90 min at 30 mL/h, 4 to 6 weeks apart, in random order, in nine healthy men. EGP was assessed under pancreatic clamp conditions with stable isotope enrichment techniques. Under these experimental conditions, intraduodenal infusion of Intralipid, compared with saline, did not affect plasma glucose concentration or EGP throughout the study period. We conclude that Intralipid infusion into the duodenum at this rate does not elicit detectable effects on glucose homeostasis or EGP in healthy men, which may reflect important interspecies differences between rodents and humans with respect to the putative gut-brain-liver axis.

Susceptibility to fatty acid-induced ß-cell dysfunction is enhanced in prediabetic diabetes-prone biobreeding rats: a potential link between ß-cell lipotoxicity and islet inflammation.

ß-Cell lipotoxicity is thought to play an important role in the development of type 2 diabetes. However, no study has examined its role in type 1 diabetes, which could be clinically relevant for slow-onset type 1 diabetes. Reports of enhanced cytokine toxicity in fat-laden islets are consistent with the hypothesis that lipid and cytokine toxicity may be synergistic. Thus, ß-cell lipotoxicity could be enhanced in models of autoimmune diabetes. To determine this, we examined the effects of prolonged free fatty acids elevation on ß-cell secretory function in the prediabetic diabetes-prone BioBreeding (dp-BB) rat, its diabetes-resistant BioBreeding (dr-BB) control, and normal Wistar-Furth (WF) rats. Rats received a 48-h iv infusion of saline or Intralipid plus heparin (IH) (to elevate free fatty acid levels ~2-fold) followed by hyperglycemic clamp or islet secretion studies ex vivo. IH significantly decreased ß-cell function, assessed both by the disposition index (insulin secretion corrected for IH-induced insulin resistance) and in isolated islets, in dp-BB, but not in dr-BB or WF, rats, and the effect of IH was inhibited by the antioxidant N-acetylcysteine. Furthermore, IH significantly increased islet cytokine mRNA and plasma cytokine levels (monocyte chemoattractant protein-1 and IL-10) in dp-BB, but not in dr-BB or WF, rats. All dp-BB rats had mononuclear infiltration of islets, which was absent in dr-BB and WF rats. In conclusion, the presence of insulitis was permissive for IH-induced ß-cell dysfunction in the BB rat, which suggests a link between ß-cell lipotoxicity and islet inflammation.

In vitro and in vivo testing of glucose-responsive insulin-delivery microdevices in diabetic rats.

We have developed glucose-responsive implantable microdevices for closed-loop delivery of insulin and conducted in vivo testing of these devices in diabetic rats. The microdevices consist of an albumin-based bioinorganic membrane that utilizes glucose oxidase (GOx), catalase (CAT) and manganese dioxide (MnO(2)) nanoparticles to convert a change in the environmental glucose level to a pH stimulus, which regulates the volume of pH-sensitive hydrogel nanoparticles and thereby the permeability of the membrane. The membrane is integrated with microfabricated PDMS (polydimethylsiloxane) structures to form compact, stand-alone microdevices, which do not require tethering wires or tubes. During in vitro testing, the microdevices showed glucose-responsive insulin release over multiple cycles at clinically relevant glucose concentrations. In vivo, the microdevices were able to counter hyperglycemia in diabetic rats over a one-week period. The in vitro and in vivo testing results demonstrated the efficacy of closed-loop biosensing and rapid response of the 'smart' insulin delivery devices.