Dapagliflozin prevents abdominal visceral and subcutaneous adipose tissue dysfunction in the insulin-resistant canine model.

OBJECTIVE: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) promote urinary glucose excretion, induce weight loss, and reduce fat accumulation. The effects of the SGLT2i dapagliflozin (DAPA) on subcutaneous (SC) and visceral (VIS) adipose tissue function remain unclear. The objective of this study is to evaluate SC and VIS adipose tissue function in an insulin-resistant canine model. METHODS: A total of 12 dogs were fed a high-fat diet (HFD) for 6 weeks and then were given a single low dose of streptozotocin (18.5 mg/kg) to induce insulin resistance. Animals were then randomized and exposed to DAPA (n = 6, 1.25 mg/kg) or placebo (n = 6) once per day for 6 weeks while remaining on the HFD. RESULTS: DAPA prevented further weight gain induced by the HFD and normalized fat mass. DAPA reduced fasting glucose and increased free fatty acids, adiponectin, and ß-hydroxybutyrate. DAPA reduced adipocyte diameter and cell distribution. Furthermore, DAPA increased genes associated with beiging, lipolysis, and adiponectin secretion and the expression of the adiponectin receptor ADR2, in SC and VIS adipose tissue. DAPA increased AMP-activated protein kinase activity and maximal mitochondrial respiratory function, especially in the SC depot. Furthermore, DAPA reduced cytokines and ceramide synthesis enzymes in SC and VIS depots. CONCLUSIONS: For the first time, to our knowledge, we identify mechanisms by which DAPA enhances adipose tissue function in regulating energy homeostasis in an insulin-resistant canine model.

Elevation of fatty acid desaturase 2 in esophageal adenocarcinoma increases polyunsaturated lipids and may exacerbate bile acid-induced DNA damage.

BACKGROUND: The risk of esophageal adenocarcinoma (EAC) is associated with gastro-esophageal reflux disease (GERD) and obesity. Lipid metabolism-targeted therapies decrease the risk of progressing from Barrett's esophagus (BE) to EAC, but the precise lipid metabolic changes and their roles in genotoxicity during EAC development are yet to be established. METHODS: Esophageal biopsies from the normal epithelium (NE), BE, and EAC, were analyzed using concurrent lipidomics and proteomics (n = 30) followed by orthogonal validation on independent samples using RNAseq transcriptomics (n = 22) and immunohistochemistry (IHC, n = 80). The EAC cell line FLO-1 was treated with FADS2 selective inhibitor SC26196, and/or bile acid cocktail, followed by immunofluorescence staining for γH2AX. RESULTS: Metabolism-focused Reactome analysis of the proteomics data revealed enrichment of fatty acid metabolism, ketone body metabolism, and biosynthesis of specialized pro-resolving mediators in EAC pathogenesis. Lipidomics revealed progressive alterations (NE-BE-EAC) in glycerophospholipid synthesis with decreasing triglycerides and increasing phosphatidylcholine and phosphatidylethanolamine, and sphingolipid synthesis with decreasing dihydroceramide and increasing ceramides. Furthermore, a progressive increase in lipids with C20 fatty acids and polyunsaturated lipids with ≥4 double bonds were also observed. Integration with transcriptome data identified candidate enzymes for IHC validation: Δ4-Desaturase, Sphingolipid 1 (DEGS1) which desaturates dihydroceramide to ceramide, and Δ5 and Δ6-Desaturases (fatty acid desaturases, FADS1 and FADS2), responsible for polyunsaturation. All three enzymes showed significant increases from BE through dysplasia to EAC, but transcript levels of DEGS1 were decreased suggesting post-translational regulation. Finally, the FADS2 selective inhibitor SC26196 significantly reduced polyunsaturated lipids with three and four double bonds and reduced bile acid-induced DNA double-strand breaks in FLO-1 cells in vitro. CONCLUSIONS: Integrated multiomics revealed sphingolipid and phospholipid metabolism rewiring during EAC development. FADS2 inhibition and reduction of the high polyunsaturated lipids effectively protected EAC cells from bile acid-induced DNA damage in vitro, potentially through reduced lipid peroxidation.

Skin Cancer-Associated S. aureus Strains Can Induce DNA Damage in Human Keratinocytes by Downregulating DNA Repair and Promoting Oxidative Stress.

Actinic keratosis (AK) is a premalignant lesion, common on severely photodamaged skin, that can progress over time to cutaneous squamous cell carcinoma (SCC). A high bacterial load of Staphylococcus aureus is associated with AK and SCC, but it is unknown whether this has a direct impact on skin cancer development. To determine whether S. aureus can have cancer-promoting effects on skin cells, we performed RNA sequencing and shotgun proteomics on primary human keratinocytes after challenge with sterile culture supernatant ('secretome') from four S. aureus clinical strains isolated from AK and SCC. Secretomes of two of the S. aureus strains induced keratinocytes to overexpress biomarkers associated with skin carcinogenesis and upregulated the expression of enzymes linked to reduced skin barrier function. Further, these strains induced oxidative stress markers and all secretomes downregulated DNA repair mechanisms. Subsequent experiments on an expanded set of lesion-associated S. aureus strains confirmed that exposure to their secretomes led to increased oxidative stress and DNA damage in primary human keratinocytes. A significant correlation between the concentration of S. aureus phenol soluble modulin toxins in secretome and the secretome-induced level of oxidative stress and genotoxicity in keratinocytes was observed. Taken together, these data demonstrate that secreted compounds from lesion-associated clinical isolates of S. aureus can have cancer-promoting effects in keratinocytes that may be relevant to skin oncogenesis.

C5b-9 Membrane Attack Complex Formation and Extracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett's Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Impact of sleep deprivation and high-fat feeding on insulin sensitivity and beta cell function in dogs.

AIMS/HYPOTHESIS: Insufficient sleep is increasingly recognised as a major risk factor for the development of obesity and diabetes, and short-term sleep loss in clinical studies leads to a reduction in insulin sensitivity. Sleep loss-induced metabolic impairments are clinically relevant, since reductions in insulin sensitivity after sleep loss are comparable to insulin sensitivity differences between healthy individuals and those with impaired glucose tolerance. However, the relative effects of sleep loss vs high-fat feeding in the same individual have not been assessed. In addition, to our knowledge no diurnal (active during the daytime) non-human mammalian model of sleep loss-induced metabolic impairment exists, which limits our ability to study links between sleep and metabolism. METHODS: This study examined the effects of one night of total sleep deprivation on insulin sensitivity and beta cell function, as assessed by an IVGTT, before and after 9 months of high-fat feeding in a canine model. RESULTS: One night of total sleep deprivation in lean dogs impaired insulin sensitivity to a similar degree as a chronic high-fat diet (HFD)(normal sleep: 4.95 ± 0.45 mU-1 l-1 min-1; sleep deprivation: 3.14 ± 0.21 mU-1 l-1 min-1; HFD: 3.74 ± 0.48 mU-1 l-1 min-1; mean ± SEM). Hyperinsulinaemic compensation was induced by the chronic HFD, suggesting adequate beta cell response to high-fat feeding. In contrast, there was no beta cell compensation after one night of sleep deprivation, suggesting that there was metabolic dysregulation with acute sleep loss that, if sustained during chronic sleep loss, could contribute to the risk of type 2 diabetes. After chronic high-fat feeding, acute total sleep deprivation did not cause further impairments in insulin sensitivity (sleep deprivation + chronic HFD: 3.28 mU-1 l-1 min-1). CONCLUSIONS/INTERPRETATION: Our findings provide further evidence that sleep is important for metabolic health and establish a diurnal animal model of metabolic disruption during insufficient sleep.

The vascular endothelium plays a role in insulin action.

The endocrine system relies on the vasculature for delivery of hormones throughout the body, and the capillary microvasculature is the site where the hormones cross from the blood into the target tissue. Once considered an inert wall, various studies have now highlighted the functions of the capillary endothelium to regulate transport and therefore affect or maintain the interstitial environment. The role of the capillary may be clear in areas where there is a continuous endothelium, yet there also appears to be a role of endothelial cells in tissues with a sinusoidal structure. Here we focused on the most common endocrine disorder, diabetes, and several of the target organs associated with the disease, including skeletal muscle, liver and pancreas. However, it is important to note that the ability of hormones to cross the endothelium to reach their target tissue is a component of all endocrine functions. It is also a consideration in organs throughout the body and may have greater impact for larger hormones with target tissues containing a continuous endothelium. We noted that the blood levels do not always equal interstitial levels, which is what the cells are exposed to, and discussed how this may change in diseases such as obesity and insulin resistance. The capillary endothelium is, therefore, an essential and understudied aspect of endocrinology and metabolism that can be altered in disease, which may be an appropriate target for treatment.

Hypothesis: Role of Reduced Hepatic Insulin Clearance in the Pathogenesis of Type 2 Diabetes.

There is wide variance among individuals in the fraction of insulin cleared by the liver (20% to 80%). Hepatic insulin clearance is 67% lower in African Americans than European Americans. Clearance is also lower in African American children 7-13 years of age. Lower hepatic insulin clearance will result in peripheral hyperinsulinemia: this exacerbates insulin resistance, which stresses the ß-cells, possibly resulting in their ultimate failure and onset of type 2 diabetes. We hypothesize that lower insulin clearance can be a primary cause of type 2 diabetes in at-risk individuals.

Activation of NPRs and UCP1-independent pathway following CB1R antagonist treatment is associated with adipose tissue beiging in fat-fed male dogs.

CB1 receptor (CB1R) antagonism improves the deleterious effects of a high-fat diet (HFD) by reducing body fat mass and adipocyte cell size. Previous studies demonstrated that the beneficial effects of the CB1R antagonist rimonabant (RIM) in white adipose tissue (WAT) are partially due to an increase of mitochondria numbers and upregulation thermogenesis markers, suggesting an induction of WAT beiging. However, the molecular mechanism by which CB1R antagonism induces weight loss and WAT beiging is unclear. In this study, we probed for genes associated with beiging and explored longitudinal molecular mechanisms by which the beiging process occurs. HFD dogs received either RIM (HFD+RIM) or placebo (PL) (HFD+PL) for 16 wk. Several genes involved in beiging were increased in HFD+RIM compared with pre-fat, HFD, and HFD+PL. We evaluated lipolysis and its regulators including natriuretic peptide (NP) and its receptors (NPRs), ß-1 and ß-3 adrenergic receptor (ß1R, ß3R) genes. These genes were increased in WAT depots, accompanied by an increase in lipolysis in HFD+RIM. In addition, RIM decreased markers of inflammation and increased adiponectin receptors in WAT. We observed a small but significant increase in UCP1; therefore, we evaluated the newly discovered UCP1-independent thermogenesis pathway. We confirmed that SERCA2b and RYR2, the two key genes involved in this pathway, were upregulated in the WAT. Our data suggest that the upregulation of NPRs, ß-1R and ß-3R, lipolysis, and SERCA2b and RYR2 may be one of the mechanisms by which RIM promotes beiging and overall the improvement of metabolic homeostasis induced by RIM.

Variability of Directly Measured First-Pass Hepatic Insulin Extraction and Its Association With Insulin Sensitivity and Plasma Insulin.

Although the ß-cells secrete insulin, the liver, with its first-pass insulin extraction (FPE), regulates the amount of insulin allowed into circulation for action on target tissues. The metabolic clearance rate of insulin, of which FPE is the dominant component, is a major determinant of insulin sensitivity (SI). We studied the intricate relationship among FPE, SI, and fasting insulin. We used a direct method of measuring FPE, the paired portal/peripheral infusion protocol, where insulin is infused stepwise through either the portal vein or a peripheral vein in healthy young dogs (n = 12). FPE is calculated as the difference in clearance rates (slope of infusion rate vs. steady insulin plot) between the paired experiments. Significant correlations were found between FPE and clamp-assessed SI (rs = 0.74), FPE and fasting insulin (rs = -0.64), and SI and fasting insulin (rs = -0.67). We also found a wide variance in FPE (22.4-77.2%; mean ± SD 50.4 ± 19.1) that is reflected in the variability of plasma insulin (48.1 ± 30.9 pmol/L) and SI (9.4 ± 5.8 × 104 dL · kg-1 · min-1 · [pmol/L]-1). FPE could be the nexus of regulation of both plasma insulin and SI.

Assessment of hepatic insulin extraction from in vivo surrogate methods of insulin clearance measurement.

Hyperinsulinemia, accompanied by reduced first-pass hepatic insulin extraction (FPE) and increased secretion, is a primary response to insulin resistance. Different in vivo methods are used to estimate the clearance of insulin, which is assumed to reflect FPE. We compared two methodologically different but commonly used indirect estimates with directly measured FPE in healthy dogs ( n = 9). The indirect methods were 1) metabolic clearance rate of insulin (MCR) during the hyperinsulinemic-euglycemic clamp (EGC), a steady-state method, and 2) fractional clearance rate of insulin (FCR) during the frequently sampled intravenous glucose tolerance test (FSIGT), a dynamic method. MCR was calculated as the ratio of insulin infusion rate to steady-state plasma insulin. FCR was calculated as the exponential decay rate constant of the injected insulin. Directly measured FPE is based on the difference in insulin measurements during intraportal vs. peripheral vein insulin infusions. We found a strong correlation between indirect FCR (min-1) and FPE (%). In contrast, we observed a poor association between MCR (ml·min-1·kg-1) and FPE (%). Our findings in canines suggest that FCR measured during FSIGT can be used to estimate FPE. However, MCR calculated during EGC appears to be a poor surrogate for FPE.

Insulin Access to Skeletal Muscle is Preserved in Obesity Induced by Polyunsaturated Diet.

OBJECTIVE: Diets high in saturated fat induce obesity and insulin resistance and impair insulin access to skeletal muscle, leading to reduced insulin levels at the muscle cell surface available to bind insulin receptors and induce glucose uptake. In contrast, diets supplemented with polyunsaturated fat improve insulin sensitivity (SI) and reduce the risk for type 2 diabetes. It was hypothesized that a diet high in polyunsaturated fat would preserve SI and insulin access to muscle, as compared with a diet high in saturated fat. METHODS: After 12 weeks of control, saturated (LARD), or polyunsaturated (salmon oil [SO]) high-fat diet feeding, muscle SI and insulin access to skeletal muscle were measured by using lymph, a surrogate of skeletal muscle interstitial fluid. RESULTS: Both high-fat diets induced similar weight gain, yet only LARD impaired SI. Hyperinsulinemia in the LARD group did not induce an increase in basal interstitial insulin, suggesting reduced insulin access to muscle after LARD, but not after SO. CONCLUSIONS: A diet high in polyunsaturated fat does not impair insulin access to muscle interstitium or induce insulin resistance as observed with a saturated fat diet, despite similar weight gain. Future studies should determine whether dietary SO supplementation improves impairments in insulin access to skeletal muscle.

Isoflurane and Sevoflurane Induce Severe Hepatic Insulin Resistance in a Canine Model.

INTRODUCTION: Anesthesia induces insulin resistance, which may contribute to elevated blood glucose and adverse post-operative outcomes in critically ill patients, and impair glycemic control in surgical patients with diabetes. However, little is known about the mechanisms by which anesthesia impairs insulin sensitivity. Here we investigate the effects of anesthesia on insulin sensitivity in metabolic tissues. METHODS: Hyperinsulinemic-euglycemic clamps were performed in 32 lean (control diet; n = 16 conscious versus n = 16 anesthetized) and 24 fat-fed (6 weeks fat-feeding; n = 16 conscious versus n = 8 anesthetized) adult male mongrel dogs in conjunction with tracer methodology to differentiate hepatic versus peripheral insulin sensitivity. Propofol was administered as an intravenous bolus (3mg/kg) to initiate anesthesia, which was then maintained with inhaled sevoflurane or isoflurane (2-3%) for the duration of the procedure. RESULTS: Anesthesia reduced peripheral insulin sensitivity by approximately 50% in both lean and fat-fed animals as compared to conscious animals, and insulin action at the liver was almost completely suppressed during anesthesia such that hepatic insulin sensitivity was decreased by 75.5% and; 116.2% in lean and fat-fed groups, respectively. CONCLUSION: Inhaled anesthesia induces severe hepatic insulin resistance in a canine model. Countermeasures that preserve hepatic insulin sensitivity may represent a therapeutic target that could improve surgical outcomes in both diabetic and healthy patients.

Insulin access to skeletal muscle is impaired during the early stages of diet-induced obesity.

OBJECTIVE: Insulin must move from the blood to the interstitium to initiate signaling, yet access to the interstitium may be impaired in cases of insulin resistance, such as obesity. This study investigated whether consuming a short- and long-term high-fat diet (HFD) impairs insulin access to skeletal muscle, the major site of insulin-mediated glucose uptake. METHODS: Male mongrel dogs were divided into three groups consisting of control diet (n = 16), short-term (n = 8), and long-term HFD (n = 8). Insulin sensitivity was measured with intravenous glucose tolerance tests. A hyperinsulinemic euglycemic clamp was performed in each animal at the conclusion of the study. During the clamp, lymph fluid was measured as a representation of the interstitial space to assess insulin access to muscle. RESULTS: Short- and long-term HFD induced obesity and reduced insulin sensitivity. Lymph insulin concentrations were approximately 50% of plasma insulin concentrations under control conditions. Long-term HFD caused fasting plasma hyperinsulinemia; however, interstitial insulin concentrations were not increased, suggesting impaired insulin access to muscle. CONCLUSIONS: A HFD rapidly induces insulin resistance at the muscle and impairs insulin access under basal insulin concentrations. Hyperinsulinemia induced by a long-term HFD may be a compensatory mechanism necessary to maintain healthy insulin levels in muscle interstitium.

Exenatide Treatment Alone Improves ß-Cell Function in a Canine Model of Pre-Diabetes.

BACKGROUND: Exenatide's effects on glucose metabolism have been studied extensively in diabetes but not in pre-diabetes. OBJECTIVE: We examined the chronic effects of exenatide alone on glucose metabolism in pre-diabetic canines. DESIGN AND METHODS: After 10 weeks of high-fat diet (HFD), adult dogs received one injection of streptozotocin (STZ, 18.5 mg/kg). After induction of pre-diabetes, while maintained on HFD, animals were randomized to receive either exenatide (n = 7) or placebo (n = 7) for 12 weeks. ß-Cell function was calculated from the intravenous glucose tolerance test (IVGTT, expressed as the acute insulin response, AIRG), the oral glucose tolerance test (OGTT, insulinogenic index) and the graded-hyperglycemic clamp (clamp insulinogenic index). Whole-body insulin sensitivity was assessed by the IVGTT. At the end of the study, pancreatic islets were isolated to assess ß-cell function in vitro. RESULTS: OGTT: STZ caused an increase in glycemia at 120 min by 22.0% (interquartile range, IQR, 31.5%) (P = 0.011). IVGTT: This protocol also showed a reduction in glucose tolerance by 48.8% (IQR, 36.9%) (P = 0.002). AIRG decreased by 54.0% (IQR, 40.7%) (P = 0.010), leading to mild fasting hyperglycemia (P = 0.039). Exenatide, compared with placebo, decreased body weight (P<0.001) without altering food intake, fasting glycemia, insulinemia, glycated hemoglobin A1c, or glucose tolerance. Exenatide, compared with placebo, increased both OGTT- (P = 0.040) and clamp-based insulinogenic indexes (P = 0.016), improved insulin secretion in vitro (P = 0.041), but had no noticeable effect on insulin sensitivity (P = 0.405). CONCLUSIONS: In pre-diabetic canines, 12-week exenatide treatment improved ß-cell function but not glucose tolerance or insulin sensitivity. These findings demonstrate partial beneficial metabolic effects of exenatide alone on an animal model of pre-diabetes.

Rapid development of cardiac dysfunction in a canine model of insulin resistance and moderate obesity.

AIMS/HYPOTHESIS: The worldwide incidence of obesity and diabetes continues to rise at an alarming rate. A major cause of the morbidity and mortality associated with obesity and diabetes is heart disease, yet the mechanisms that lead to cardiovascular complications remain unclear. METHODS: We performed cardiac MRI to assess left ventricular morphology and function during the development of moderate obesity and insulin resistance in a well-established canine model (n = 26). To assess the influence of dietary fat composition, we randomised animals to a traditional lard diet (rich in saturated and monounsaturated fat; n = 12), a salmon oil diet (rich in polyunsaturated fat; n = 8) or a control diet (n = 6). RESULTS: High-fat feeding with lard increased body weight and fasting insulin and markedly reduced insulin sensitivity. Lard feeding also significantly reduced left ventricular function, evidenced by a worsening of circumferential strain and impairment in left ventricular torsion. High-fat feeding with salmon oil increased body weight; however, salmon oil feeding did not impair insulin sensitivity or cardiac function. CONCLUSIONS/INTERPRETATION: These data emphasise the importance of dietary fat composition on both metabolic and cardiac function, and have important implications for the relationship between diet and health.

CB1R antagonist increases hepatic insulin clearance in fat-fed dogs likely via upregulation of liver adiponectin receptors.

The improvement of hepatic insulin sensitivity by the cannabinoid receptor 1 (CB1R) antagonist rimonabant (RIM) has been recently been reported to be due to upregulation of adiponectin. Several studies demonstrated that improvement in insulin clearance accompanies the enhancement of hepatic insulin sensitivity. However, the effects of RIM on hepatic insulin clearance (HIC) have not been fully explored. The aim of this study was to explore the molecular mechanism(s) by which RIM affects HIC, specifically to determine whether upregulation of liver adiponectin receptors (ADRs) and other key genes regulated by adiponectin mediate the effects. To induce insulin resistance in skeletal muscle and liver, dogs were fed a hypercaloric high-fat diet (HFD) for 6 wk. Thereafter, while still maintained on a HFD, animals received RIM (HFD+RIM; n = 11) or placebo (HFD+PL; n = 9) for an additional 16 wk. HIC, calculated as the metabolic clearance rate (MCR), was estimated from the euglycemic-hyperinsulinemic clamp. The HFD+PL group showed a decrease in MCR; in contrast, the HFD+RIM group increased MCR. Consistently, the expression of genes involved in HIC, CEACAM-1 and IDE, as well as gene expression of liver ADRs, were increased in the HFD+RIM group, but not in the HFD+PL group. We also found a positive correlation between CEACAM-1 and the insulin-degrading enzyme IDE with ADRs. Interestingly, expression of liver genes regulated by adiponectin and involved in lipid oxidation were increased in the HFD+RIM group. We conclude that in fat-fed dogs RIM enhances HIC, which appears to be linked to an upregulation of the adiponectin pathway.

Elevated nocturnal NEFA are an early signal for hyperinsulinaemic compensation during diet-induced insulin resistance in dogs.

AIMS/HYPOTHESIS: A normal consequence of increased energy intake and insulin resistance is compensatory hyperinsulinaemia through increased insulin secretion and/or reduced insulin clearance. Failure of compensatory mechanisms plays a central role in the pathogenesis of type 2 diabetes mellitus; consequently, it is critical to identify in vivo signal(s) involved in hyperinsulinaemic compensation. We have previously reported that high-fat feeding leads to an increase in nocturnal NEFA concentration. We therefore designed this study to test the hypothesis that elevated nocturnal NEFA are an early signal for hyperinsulinaemic compensation for insulin resistance. METHODS: Blood sampling was conducted in male dogs to determine 24 h profiles of NEFA at baseline and during high-fat feeding with and without acute nocturnal NEFA suppression using a partial A1 adenosine receptor agonist. RESULTS: High-fat feeding increased nocturnal NEFA and reduced insulin sensitivity, effects countered by an increase in acute insulin response to glucose (AIR(g)). Pharmacological NEFA inhibition after 8 weeks of high-fat feeding lowered NEFA to baseline levels and reduced AIR(g) with no effect on insulin sensitivity. A significant relationship emerged between nocturnal NEFA levels and AIR(g). This relationship indicates that the hyperinsulinaemic compensation induced in response to high-fat feeding was prevented when the nocturnal NEFA pattern was returned to baseline. CONCLUSIONS/INTERPRETATION: Elevated nocturnal NEFA are an important signal for hyperinsulinaemic compensation during diet-induced insulin resistance.

Lipid-induced insulin resistance does not impair insulin access to skeletal muscle.

Elevated plasma free fatty acids (FFA) induce insulin resistance in skeletal muscle. Previously, we have shown that experimental insulin resistance induced by lipid infusion prevents the dispersion of insulin through the muscle, and we hypothesized that this would lead to an impairment of insulin moving from the plasma to the muscle interstitium. Thus, we infused lipid into our anesthetized canine model and measured the appearance of insulin in the lymph as a means to sample muscle interstitium under hyperinsulinemic euglycemic clamp conditions. Although lipid infusion lowered the glucose infusion rate and induced both peripheral and hepatic insulin resistance, we were unable to detect an impairment of insulin access to the lymph. Interestingly, despite a significant, 10-fold increase in plasma FFA, we detected little to no increase in free fatty acids or triglycerides in the lymph after lipid infusion. Thus, we conclude that experimental insulin resistance induced by lipid infusion does not reduce insulin access to skeletal muscle under clamp conditions. This would suggest that the peripheral insulin resistance is likely due to reduced cellular sensitivity to insulin in this model, and yet we did not detect a change in the tissue microenvironment that could contribute to cellular insulin resistance.