Hyperinsulinemic-euglycemic Clamp Experiments Research Articles

Evaluation of Clinical Metrics for Identifying Defective Physiologic Responses to Hypoglycemia in Long-Standing Type 1 Diabetes.

Repeated hypoglycemia exposure leads to impaired awareness of hypoglycemia (IAH) and the development of defective counterregulatory responses. To date, only pancreas or islet transplantation has demonstrated normalization of hypoglycemia awareness and the endogenous glucose production (EGP) response to defend against insulin-induced hypoglycemia in long-standing type 1 diabetes (T1D). This study aims to validate clinical metrics of IAH (Clarke score), hypoglycemia severity (HYPO score), glycemic lability (lability index), and continuous glucose monitoring (CGM) as predictors of absent autonomic symptom (AS) recognition and defective glucose counterregulation during insulin-induced hypoglycemia, thus enabling early identification of individuals with compromised physiologic defense against clinically significant hypoglycemia. Forty-three subjects with mean ± standard deviation age 43 ± 13 years and T1D duration 28 ± 13 years, including 32 with IAH and 11 with hypoglycemia awareness (Aware), and 12 nondiabetic control subjects, underwent single-blinded randomized-paired hyperinsulinemic-euglycemic and hypoglycemic clamp experiments. Receiver operating characteristic (ROC) curves and sensitivity analyses were performed to assess metric prediction of absent AS recognition and defective EGP responses to hypoglycemia. Clarke score and CGM measures of hypoglycemia exposure demonstrated good ability to predict absent AS recognition (area under the curve ≥0.80). A composite threshold of IAH-Clarke ≥4 with ROC curve-derived thresholds for CGM measures of hypoglycemia exposure showed high specificity and predictive value in identifying an absent AS response during the hypoglycemic clamp. Metrics demonstrated poor ability to predict defective glucose counterregulation by the EGP response, which was impaired even in the Aware group. Screening for IAH alongside assessment of CGM data can increase the specificity for identifying individuals with absent hypoglycemia symptom recognition who may benefit from further intervention.

Obesity-Linked PPARγ S273 Phosphorylation Promotes Insulin Resistance through Growth Differentiation Factor 3

The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn't been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling invitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3.

ApolipoproteinM overexpression through adeno-associated virus gene transfer improves insulin secretion and insulin sensitivity in Goto-Kakizaki rats.

Aims/ObjectiveThe development of type 2 diabetes is a result of insulin resistance in various tissues, including skeletal muscle and liver. Apolipoprotein M (ApoM) plays an important role in the function of high‐density lipoprotein, and also affects hepatic lipid and glucose metabolism. In this study, we aimed to investigate whether ApoM overexpression modulates glucose metabolism and improves insulin sensitivity.Materials and MethodsThe Goto‐Kakizaki (GK) rats were transfected with adeno‐associated virus (AAV) encoding rat ApoM gene or control blank. The oral glucose tolerance test (OGTT) and hyperinsulinemic‐euglycemic clamp (HEC) experiment were used to assess the insulin sensitivity of GK rats.ResultsThe results show that ApoM messenger ribonucleic acid and protein were significantly overexpressed in the pancreatic tissues. Overexpression of ApoM decreased fasting blood glucose and random blood glucose, improved glucose tolerance, and increased bodyweight and insulin levels in GK rats. The glucose infusion rate of rats in the AAV encoding rat ApoM gene group during HEC test was 1.04‐, 1.23‐ and 1.95‐fold higher than that in the AAV control blank group at 1–3 weeks after injection of AAV, respectively. A Wes‐ProteinSimple assay and quantification was carried out to assess phosphorylated protein kinase B/protein kinase B protein levels in the muscle tissues of ApoM‐overexpressing GK rats, and they were found to be higher than those of the control group at the seventh week after AAV injection.ConclusionsApoM overexpression through adeno‐associated virus gene transfer might improve insulin secretion and insulin sensitivity in GK rats.

Normalization of metabolic flux data during clamp studies in humans

BackgroundThere is no consensus in the field regarding the optimal method for the expression of metabolic flux data, such as glucose disposal rates during hyperinsulinemic-euglycemic clamp experiments. Several normalization methods are in use today, but their impact on study outcomes is rarely discussed. MethodsWe illustrate this issue using clamp data from 92 lean and 66 obese subjects. Glucose kinetics and insulin sensitivity were determined during hyperinsulinemic-euglycemic clamp studies using [6,6-2H2]glucose. From this single dataset, we calculated 21 expression methods for the glucose disposal rate during hyperinsulinemic conditions. Results and discussionWith most normalization methods, the obese subjects demonstrated reduced insulin-stimulated glucose disposal as compared to the lean subjects. However, depending on the normalization method, glucose disposal rates in obese subjects ranged from 26 ± 1% to 207 ± 10% of glucose disposal rates in lean subjects. We conclude that data normalization methods greatly impacted metabolic flux outcomes in our dataset of lean and obese subjects. There is no compelling evidence to select one method over the other, but we encourage authors in the metabolic arena to think about, and provide a rationale for, the best normalization method for their specific research questions.

Samidorphan mitigates olanzapine-induced weight gain and metabolic dysfunction in rats and non-human primates.

Background:Olanzapine, regarded as one of the most efficacious antipsychotic medications for the treatment of schizophrenia, is associated with a high risk of weight gain and metabolic dysfunction. ALKS 3831, a clinical candidate for treatment of schizophrenia, is a combination of olanzapine and samidorphan, an opioid receptor antagonist. The addition of samidorphan is intended to mitigate weight gain and the metabolic dysregulation associated with the use of olanzapine.Methods:Non-clinical studies were conducted to assess the metabolic effects of olanzapine and samidorphan alone and in combination at clinically relevant exposure levels.Results:Chronic olanzapine administration in male and female rats shifted body composition by increasing adipose mass, which was accompanied by an increase in the rate of weight gain in female rats. Co-administration of samidorphan normalized body composition in both sexes and attenuated weight gain in female rats. In hyperinsulinemic euglycemic clamp experiments conducted prior to measurable changes in weight and/or body composition, olanzapine decreased hepatic insulin sensitivity and glucose uptake in muscle while increasing uptake in adipose tissue. Samidorphan appeared to normalize glucose utilization in both tissues, but did not restore hepatic insulin sensitivity. In subsequent studies, samidorphan normalized olanzapine-induced decreases in whole-body glucose clearance following bolus insulin administration. Results from experiments in female monkeys paralleled the effects in rats.Conclusions:Olanzapine administration increased weight gain and adiposity, both of which were attenuated by samidorphan. Furthermore, the combination of olanzapine and samidorphan prevented olanzapine-induced insulin insensitivity. Collectively, these data indicate that samidorphan mitigates several metabolic abnormalities associated with olanzapine in both the presence and the absence of weight gain.

116-OR: Hypoglycemia Leads to a Delayed Increase in Platelet and Coagulation Markers in Subjects with Type 2 Diabetes Treated with Metformin Only—Results from an Experimental Stepwise Hypoglycemic Clamp Study

Objective: Although hypoglycemia is associated with increased risk for subsequent cardiovascular events, experimental data investigating the effect of hypoglycemia on coagulation and platelet activation in type 2 diabetes are limited. Research Design and Methods: This monocentric, open, single arm, trial included 14 subjects with type 2 diabetes (10 male/4 female, age 55±7 years, HbA1c 51±7 mmol/mol, diabetes duration 5±4 years) and metformin treatment only. A stepwise hyperinsulinemic hypoglycemic clamp was performed, aiming to investigate parameters of platelet function during predefined plateaus of hypoglycemia (at 63 and 45 mg/dl, for 30 minutes respectively), 24 hours and 7 days later. Additionally, all subjects underwent a hyperinsulinemic euglycemic clamp experiment. Results: While platelet activation assessed by light transmittance aggregometry did not significantly increase following a hypoglycemic clamp, flow cytometry based platelet activation assessment demonstrated a significant increase 24 hours (PAC1CD62PCD63pos p<0.01 compared to baseline, and p<0.001 for PAC1CD63Ppos, respectively) and 7 days after the hypoglycemic clamp (p<0.001 for PAC1CD62Ppos, and PAC1CD63Ppos, and p<0.01 for PAC1CD62PCD63pos in comparison to baseline). In addition, coagulation markers like fibrinogen, PAI-1 and factor VIII also increased, with highest levels 24 hours after the hypoglycemic clamp (p<0.001 for fibrinogen and factor VIII and <0.01 for PAI-1). No platelet or coagulation activation was observed during the hyperinsulinemic, euglycemic clamp. Conclusions: One single event of insulin induced hypoglycemia led to an increase in markers of platelet activation and coagulation, however, the activation occurred with a delay and was evident 24 hours and 7 days after the actual hypoglycemia. Disclosure F. Aberer: None. P.N. Pferschy: None. N.J. Tripolt: None. C. Sourij: None. H. Kojzar: None. B. Prietl: None. S. Kofler: None. M. Brunner: None. R. Riedl: None. E. Novak: None. A.M. Obermayer: None. P. Reitbauer: None. T. Stojakovic: None. F. Aziz: None. A. Oulhaj: None. F. Pruller: None. H. Scharnagl: Research Support; Self; Abbott, Amgen Inc. Speaker's Bureau; Self; Sanofi-Aventis Deutschland GmbH. H. Sourij: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Novo Nordisk A/S, Novo Nordisk A/S, Sanofi-Aventis. Speaker's Bureau; Self; Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis.

InVivo Decoding Mechanisms of the Temporal Patterns of Blood Insulin by the Insulin-AKT Pathway in the Liver.

Cells respond to various extracellular stimuli through a limited number of signaling pathways. One strategy to process such stimuli is to code the information into the temporal patterns of molecules. Although we showed that insulin selectively regulated molecules depending on its temporal patterns using Fao cells, the invivo mechanism remains unknown. Here, we show how the insulin-AKT pathway processes the information encoded into the temporal patterns of blood insulin. We performed hyperinsulinemic-euglycemic clamp experiments and found that, in the liver, all temporal patterns of insulin are encoded into the insulin receptor, and downstream molecules selectively decode them through AKT. S6K selectively decodes the additional secretion information. G6Pase interprets the basal secretion information through FoxO1, while GSK3β decodes all secretion pattern information. Mathematical modeling revealed the mechanism via differences in network structures and from sensitivity and time constants. Given that almost all hormones exhibit distinct temporal patterns, temporal coding may be a general principle of system homeostasis by hormones.

Dietary fat drives whole-body insulin resistance and promotes intestinal inflammation independent of body weight gain

BackgroundThe obesogenic potential of high-fat diets (HFD) in rodents is attenuated when the protein:carbohydrate ratio is increased. However, it is not known if intake of an HFD irrespective of the protein:carbohydrate ratio and in the absence of weight gain, affects glucose homeostasis and the gut microbiota. MethodsWe fed C57BL6/J mice 3 different HFDs with decreasing protein:carbohydrate ratios for 8weeks and compared the results to a LFD reference group. We analyzed the gut microbiota composition by 16S rDNA amplicon sequencing and the intestinal gene expression by real-time PCR. Whole body glucose homeostasis was evaluated by insulin and glucose tolerance tests as well as by a hyperinsulinemic euglycemic clamp experiment. ResultsCompared with LFD-fed reference mice, HFD-fed mice, irrespective of protein:carbohydrate ratio, exhibited impaired glucose tolerance, whereas no differences were observed during insulin tolerance tests. The hyperinsulinemic euglycemic clamp revealed tissue-specific effects on glucose homeostasis in all HFD-fed groups. HFD-fed mice exhibited decreased insulin-stimulated glucose uptake in white but not in brown adipose tissue, and sustained endogenous glucose production under insulin-stimulated conditions. We observed no impairment of insulin-stimulated glucose uptake in skeletal muscles of different fiber type composition. HFD-feeding altered the gut microbiota composition paralleled by increased expression of pro-inflammatory cytokines and genes involved in gluconeogenesis in intestinal epithelial cells of the jejunum. ConclusionsIntake of a HFD profoundly affected glucose homeostasis, gut inflammatory responses, and gut microbiota composition in the absence of fat mass accretion.

Oral treatment with Eubacterium hallii improves insulin sensitivity in db/db mice.

An altered intestinal microbiota composition is associated with insulin resistance and type 2 diabetes mellitus. We previously identified increased intestinal levels of Eubacterium hallii, an anaerobic bacterium belonging to the butyrate-producing Lachnospiraceae family, in metabolic syndrome subjects who received a faecal transplant from a lean donor. To further assess the effects of E. hallii on insulin sensitivity, we orally treated obese and diabetic db/db mice with alive E. hallii and glycerol or heat-inactive E. hallii as control. Insulin tolerance tests and hyperinsulinemic-euglycemic clamp experiments revealed that alive E. hallii treatment improved insulin sensitivity compared control treatment. In addition, E. hallii treatment increased energy expenditure in db/db mice. Active E. hallii treatment was found to increase faecal butyrate concentrations and to modify bile acid metabolism compared with heat-inactivated controls. Our data suggest that E. hallii administration potentially alters the function of the intestinal microbiome and that microbial metabolites may contribute to the improved metabolic phenotype.

A potent and selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, SKI2852, ameliorates metabolic syndrome in diabetic mice models

11β-Hydroxysteroid dehydrogenase type 1 (11βHSD1) has been targeted for new drugs to treat type 2 diabetes and metabolic syndrome. In this study, we determined whether the inhibition of 11βHSD1 with a new selective inhibitor, SKI2852, could improve lipid profiles, glucose levels, and insulin sensitivity in type 2 diabetic and obese conditions. SKI2852 showed a potent inhibition of cortisone to cortisol conversion for over 80% in both liver and adipose tissue ex vivo from orally administered C57BL/6 mice, and in vivo analysis results were consistent with this. Repeated oral administrations of SKI2852 in diet-induced obesity (DIO) and ob/ob mice revealed a partially beneficial effect of SKI2852 in improving levels of cholesterols, triglycerides, free fatty acids, postprandial glucose, and/or blood hemoglobinA1c. SKI2852 significantly reduced body weight increase in ob/ob mice, and efficiently suppressed hepatic mRNA levels of gluconeogenic enzymes in DIO mice. Moreover, SKI2852 enhanced hepatic and whole body insulin sensitivities in hyperinsulinemic-euglycemic clamp experiment in DIO mice. In conclusion, these results indicate that selective and potent inhibition of 11βHSD1 by SKI2852, thus blockade of active glucocorticoid conversion, may improve many aspects of metabolic parameters in type 2 diabetes and metabolic diseases, mainly by inhibitions of hepatic gluconeogenesis and partial improvements of lipid profiles. Our study strongly support that SKI2852 may have a great potential as a novel candidate drug for the treatment of diabetes and metabolic diseases.

SIRT3 Is Crucial for Maintaining Skeletal Muscle Insulin Action and Protects Against Severe Insulin Resistance in High-Fat-Fed Mice.

Protein hyperacetylation is associated with glucose intolerance and insulin resistance, suggesting that the enzymes regulating the acetylome play a role in this pathological process. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has been linked to energy homeostasis. Thus, it is hypothesized that the dysregulation of the mitochondrial acetylation state, via genetic deletion of SIRT3, will amplify the deleterious effects of a high-fat diet (HFD). Hyperinsulinemic-euglycemic clamp experiments show, for the first time, that mice lacking SIRT3 exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake. Permeabilized muscle fibers from HFD-fed SIRT3 knockout (KO) mice showed that tricarboxylic acid cycle substrate–based respiration is decreased while fatty acid–based respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function.

Transient receptor potential vanilloid type-1 channel regulates diet-induced obesity, insulin resistance, and leptin resistance.

Insulin resistance is a major characteristic of obesity and type 2 diabetes, but the underlying mechanism is unclear. Recent studies have shown a metabolic role of capsaicin that may be mediated via the transient receptor potential vanilloid type-1 (TRPV1) channel. In this study, TRPV1 knockout (KO) and wild-type (WT) mice (as controls) were fed a high-fat diet (HFD), and metabolic studies were performed to measure insulin and leptin action. The TRPV1 KO mice became more obese than the WT mice after HFD, partly attributed to altered energy balance and leptin resistance in the KO mice. The hyperinsulinemic-euglycemic clamp experiment showed that the TRPV1 KO mice were more insulin resistant after HFD because of the ∼40% reduction in glucose metabolism in the white and brown adipose tissue, compared with that in the WT mice. Leptin treatment failed to suppress food intake, and leptin-mediated hypothalamic signal transducer and activator of transcription (STAT)-3 activity was blunted in the TRPV1 KO mice. We also found that the TRPV1 KO mice were more obese and insulin resistant than the WT mice at 9 mo of age. Taken together, these results indicate that lacking TRPV1 exacerbates the obesity and insulin resistance associated with an HFD and aging, and our findings further suggest that TRPV1 has a major role in regulating glucose metabolism and hypothalamic leptin's effects in obesity.

Transcription factor TIP27 regulates glucose homeostasis and insulin sensitivity in a PI3-kinase/Akt-dependent manner in mice.

Juxtaposed with another zinc-finger gene 1 (TIP27 or JAZF1) is a 27-kDa transcription factor, and genome-wide association studies have recently revealed TIP27 to be associated with type 2 diabetes. However, little is known about its role in the regulation of metabolism. In this study, we investigated the effects of TIP27 overexpression on glucose homeostasis and insulin signaling in high-fat diet (HFD)-fed TIP27 transgenic (TIP27-Tg) mice and db/db mice. We assessed the effects of TIP27 overexpression in both TIP27-Tg mice and db/db mice on glucose metabolism and changes in insulin sensitivity during glucose (GTT) and insulin (ITT) tolerance tests. A hyperinsulinemic-euglycemic clamp was performed on TIP27-Tg mice. Real-time quantitative PCR and western blotting were used to assess mRNA and protein expressions. TIP27 overexpression in TIP27-Tg mice and in db/db mice led to reduced total cholesterol and fasting plasma insulin levels, and enhanced glucose tolerance and insulin sensitivity during GTT and ITT. Hyperinsulinemic-euglycemic clamp experiments demonstrated that HFD-fed TIP27-Tg mice had lower hepatic glucose production and higher insulin sensitivity compared with nontransgenic littermates. In addition, the hepatic expressions of phosphoenolpyruate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) mRNAs and proteins were significantly decreased, whereas the phosphorylation of insulin receptor, insulin receptor substrate-1, adenosine monophosphate-activated protein kinase and Akt kinase (Akt) in the liver was significantly increased in HFD-fed TIP27-Tg mice compared with nontransgenic littermates. Adenovirus-mediated TIP27 overexpression in db/db mice also decreased the expression of gluconeogenic genes and increased the phosphorylation of insulin signaling molecules in the liver compared with controls. Finally, LY294002, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, abolished the suppressive effect of TIP27 overexpression on PEPCK and G6Pase expression. TIP27 has an important role in glucose homeostasis through the regulation of hepatic glucose metabolism and insulin sensitivity. Furthermore, this regulation requires activation of PI3-kinase.

Tranilast Alleviates Endothelial Dysfunctions and Insulin Resistance via Preserving Glutathione Peroxidase 1 in Rats Fed a High-Fat Emulsion

We investigated the effects of treatment with tranilast on vascular and metabolic dysfunction induced by a high-fat emulsion intragastric administration. Wistar rats were randomized to receive water or high-fat emulsion with or without tranilast treatment (400 mg/kg per day) for 4 weeks. Insulin sensitivity was determined with a hyperinsulinemic-euglycemic clamp experiment and short insulin tolerance test. Vascular reactivity was evaluated using aortic rings in organ chambers. Glutathione peroxidase 1 (GPX1) expressions, eNOS phosphorylation and activity, MCP-1, H2O2 formation, and NO production were determined in vascular or soleus tissues. Tranilast treatment was found to prevent alterations in vascular reactivity and insulin sensitivity and to prevent increases in plasma glucose and insulin noted in the high-fat emulsion-treated rats. These were associated with increased antioxidant enzyme GPX1 expression, eNOS phosphorylation and activity, and NO production, but reductions in H2O2 accumulation. Moreover, tranilast preserved GPX1 expression in palmitic acid (PA)-treated endothelial cells with a consequent decreased ROS formation and increased eNOS phosphorylation and NO production. Therefore, oxidative stress induced by a relatively short-term high-fat diet could cause the early development of vascular and metabolic abnormalities in rats, and tranilast has a beneficial effect in vascular dysfunctions and insulin resistance via preserving GPX1 and alleviating oxidative stress.

The Full Capacity of AICAR to Reduce Obesity-Induced Inflammation and Insulin Resistance Requires Myeloid SIRT1

Chronic Inflammation is a key link between obesity and insulin resistance. We previously showed that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. AMPK is also a molecular target of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which has been shown to reduce insulin resistance in various animal models. Here we aim to determine whether the therapeutic effects of AICAR against insulin resistance involve its anti-inflammatory function, which requires macrophage SIRT1. Long-term administration of low-dose AICAR significantly suppressed adipose inflammation in established diet-induced obese mice. This was associated with improved glucose homeostasis and insulin sensitivity without changes of body weight. In contrast, SIRT1 deletion in myeloid SIRT1 knockout (MSKO) mice increased infiltration of classically activated M1 macrophages and decreased alternatively activated M2 macrophages in adipose tissue. As a result, MSKO mice on high fat (HF) diets exhibited impaired insulin signaling in skeletal muscle, fat, and liver, and developed systemic insulin resistance in glucose tolerance tests, insulin tolerance tests, and hyperinsulinemic-euglycemic clamp experiments. Interestingly, the beneficial effects of AICAR on adipose inflammation and insulin sensitivity were absent in MSKO mice fed HF diets, suggesting that the full capacity of AICAR to antagonize obesity-induced inflammation and insulin resistance requires myeloid SIRT1. In summary, AICAR negatively regulates HF diet-induced inflammation, which requires myeloid SIRT1, thereby contributing to the protection against insulin resistance. Myeloid SIRT1 is a therapeutic target of the anti-inflammatory and insulin-sensitizing effects of AICAR.

Circulating insulin stimulates fatty acid retention in white adipose tissue via KATP channel activation in the central nervous system only in insulin-sensitive mice

Insulin signaling in the central nervous system (CNS) is required for the inhibitory effect of insulin on glucose production. Our aim was to determine whether the CNS is also involved in the stimulatory effect of circulating insulin on the tissue-specific retention of fatty acid (FA) from plasma. In wild-type mice, hyperinsulinemic-euglycemic clamp conditions stimulated the retention of both plasma triglyceride-derived FA and plasma albumin-bound FA in the various white adipose tissues (WAT) but not in other tissues, including brown adipose tissue (BAT). Intracerebroventricular (ICV) administration of insulin induced a similar pattern of tissue-specific FA partitioning. This effect of ICV insulin administration was not associated with activation of the insulin signaling pathway in adipose tissue. ICV administration of tolbutamide, a K(ATP) channel blocker, considerably reduced (during hyperinsulinemic-euglycemic clamp conditions) and even completely blocked (during ICV administration of insulin) WAT-specific retention of FA from plasma. This central effect of insulin was absent in CD36-deficient mice, indicating that CD36 is the predominant FA transporter in insulin-stimulated FA retention by WAT. In diet-induced insulin-resistant mice, these stimulating effects of insulin (circulating or ICV administered) on FA retention in WAT were lost. In conclusion, in insulin-sensitive mice, circulating insulin stimulates tissue-specific partitioning of plasma-derived FA in WAT in part through activation of K(ATP) channels in the CNS. Apparently, circulating insulin stimulates fatty acid uptake in WAT but not in BAT, directly and indirectly through the CNS.

Regulation of Adipose Tissue Metabolism in Humans: Analysis of Responses to the Hyperinsulinemic-Euglycemic Clamp Experiment

The suppression of lipolysis is one of the key metabolic responses of the adipose tissue during hyperinsulinemia. The failure to respond and resulting increase in plasma fatty acids could contribute to the development of insulin resistance and perturbations in the fuel homeostasis in the whole body. In this study, a mechanistic, computational model of adipose tissue metabolism in vivo has been enhanced to simulate the physiological responses during hyperinsulinemic-euglycemic clamp experiment in humans. The model incorporates metabolic intermediates and pathways that are important in the fed state. In addition, it takes into account the heterogeneity of triose phosphate pools (glycolytic vs. glyceroneogenic), within the adipose tissue. The model can simulate not only steady-state responses at different insulin levels, but also concentration dynamics of major metabolites in the adipose tissue venous blood in accord with the in vivo data. Simulations indicate that (1) regulation of lipoprotein lipase (LPL) reaction is important when the intracellular lipolysis is suppressed by insulin; (2) intracellular diglyceride levels can affect the regulatory mechanisms; and (3) glyceroneogenesis is the dominant pathway for glycerol-3-phosphate synthesis even in the presence of increased glucose uptake by the adipose tissue. Reduced redox and increased phosphorylation states provide a favorable milieu for glyceroneogenesis in response to insulin. A parameter sensitivity analysis predicts that insulin-stimulated glucose uptake would be more severely affected by impairment of GLUT4 translocation and glycolysis than by impairment of glycogen synthesis and pyruvate oxidation. Finally, simulations predict metabolic responses to altered expression of phosphoenolpyruvate carboxykinase (PEP-CK). Specifically, the increase in the rate of re-esterification of fatty acids observed experimentally with the overexpression of PEPCK in the adipose tissue would be accompanied by the up-regulation of acyl Co-A synthase.

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.

Increased β-Cell Mass by Islet Transplantation and PLAG1 Overexpression Causes Hyperinsulinemic Normoglycemia and Hepatic Insulin Resistance in Mice

OBJECTIVEIt is believed that an organism remains normoglycemic despite an increase in the β-cell mass because of decreased insulin production by β-cells on a per-cell basis. However, some transgenic mouse models with β-cell hyperplasia suggest that insulin production remains excessive and that normoglycemia is maintained by insulin resistance.METHODSHere, we investigated the effect of an increased β-cell mass on glycemia and insulin resistance by grafting excess normal islets in normoglycemic mice, as well as using targeted PLAG1 expression in β-cells, which leads to β-cell expansion.RESULTSIn both models, fasting plasma insulin levels were increased, even though animals were normoglycemic. After an intraperitoneal glucose tolerance test, plasma insulin levels increased, which was associated with improved glucose clearing. Under these conditions, normoglycemia is maintained by hepatic insulin resistance as demonstrated by hyperinsulinemic euglycemic clamp experiments.CONCLUSIONSIn conclusion, we demonstrate that when excess β-cells are grafted, insulin production on a per β-cell basis is not sufficiently decreased, leading to hyperinsulinemia and hepatic insulin resistance. This observation might be important for the design of stem cell-based islet replacement therapies.

A Central Role of RLIP76 in Regulation of Glycemic Control

OBJECTIVEPathology associated with oxidative stress frequently results in insulin resistance. Glutathione (GSH) and GSH-linked metabolism is a primary defense against oxidative stress. Electrophilic lipid alkenals, such as 4-hydroxy-t-2-nonenal (4HNE), generated during oxidative stress are metabolized primarily to glutathione electrophile (GS-E) conjugates. Recent studies show that RLIP76 is the primary GS-E conjugate transporter in cells, and a regulator of oxidative-stress response. Because RLIP76−/− mice are hypoglycemic, we studied the role of RLIP76 in insulin resistance.RESEARCH DESIGN AND METHODSBlood glucose, insulin, lipid measurements, and hyperinsulinemic-euglycemic and hyperglycemic clamp experiments were performed in RLIP76+/+ and RLIP76−/− C57B mice, using Institutional Animal Care and Use Committee–approved protocols. Time-resolved three-dimensional confocal fluorescence microscopy was used to study insulin endocytosis.RESULTSThe plasma insulin/glucose ratio was ordered RLIP76−/− < RLIP76+/− < RLIP76+/+; administration of purified RLIP76 in proteoliposomes to RLIP76+/+ animals further increased this ratio. RLIP76 was induced by oxidative or hyperglycemic stress; the concomitant increase in insulin endocytosis was completely abrogated by inhibiting the transport activity of RLIP76. Hydrocortisone could transiently correct hypoglycemia in RLIP76−/− animals, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fructose 1,6-bisphosphatase, in RLIP76−/−.CONCLUSIONSThe GS-E conjugate transport activity of RLIP76 mediates insulin resistance by enhancing the rate of clathrin-dependent endocytosis of insulin. Because RLIP76 is induced by oxidative stress, it could play a role in insulin resistance seen in pathological conditions characterized by increased oxidative stress.