Publications

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

OBJECTIVE: Previous studies have demonstrated that the VAMP8 protein plays a complex role in the control of granule secretion, transport vesicle trafficking, phagocytosis, and endocytosis. The present study was aimed to investigate the role of VAMP8 in mediating GLUT4 trafficking and therefore insulin action in mice.

RESEARCH DESIGN AND METHODS: Physiological parameters were measured using Oxymax indirect calorimetry system in 12-week-old VAMP8 null mice. Dynamic analysis of glucose homeostasis was assessed using euglycemic-hyperinsulinemic clamp coupled with tracer radioactively labeled 2-deoxyglucose. Insulin stimulated GLUT4 protein expressions on muscle cell surface were examined by immunofluorescence microscopy.

RESULTS: VAMP8 null mice display reduced adiposity with increased energy expenditure despite normal food intake and reduced spontaneous locomotor activity. In parallel, the VAMP8 null mice also had fasting hypoglycemia (84 ± 11 vs. 115 ± 4) and enhanced glucose tolerance with increased insulin sensitivity due to increases in both basal and insulin-stimulated glucose uptake in skeletal muscle (0.19 ± 0.04 vs. 0.09 ± 0.01 mmol/kg/min during basal, 0.6 ± 0.04 vs. 0.31 ± 0.06 mmol/kg/min during clamp in red-gastrocnemius muscle, P < 0.05). Consistent with a role for VAMP8 in the endocytosis of the insulin-responsive GLUT4, sarcolemma GLUT4 protein levels were increased in both the basal and insulin-stimulated states without any significant change in the total amount of GLUT4 protein or related facilitative glucose transporters present in skeletal muscle, GLUT1, GLUT3, and GLUT11.

CONCLUSIONS: These data demonstrate that, in the absence of VAMP8, the relative subcellular distribution of GLUT4 is altered, resulting in increased sarcolemma levels that can account for increased glucose clearance and insulin sensitivity.

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

AIMS/HYPOTHESIS: Recent work has shown that there can be significant differences when glucose disposal is assessed for high-fat induced insulin resistance by static clamp methods vs dynamic assessment during a stable isotope i.p. glucose tolerance test. MKR mice, though lean, have severe insulin resistance and decreased muscle fatty acid oxidation. Our goal was to assess dynamic vs static glucose disposal in MKR mice, and to correlate glucose disposal and muscle-adipose-liver flux interactions with metabolic flexibility (indirect calorimetry) and muscle characteristics.

METHODS: Stable isotope flux phenotyping was performed using [6,6-(2)H(2)]glucose, [U-(13)C(6)]glucose and [2-(13)C]glycerol. Muscle triacylglycerol (TAG) and diacylglycerol (DAG) content was assessed by thin layer chromatography, and histological determination of fibre type and cytochrome c activity performed. Metabolic flexibility was assessed by indirect calorimetry.

RESULTS: Indirect calorimetry showed that MKR mice used more glucose than FVB/N mice during fasting (respiratory exchange ratio [RER] 0.88 vs 0.77, respectively). Compared with FVB/N mice, MKR mice had faster dynamic glucose disposal, despite increased whole-muscle DAG and TAG, and similar hepatic glucose production with higher fasting insulin and unchanged basal glucose. Fed MKR muscle had more glycogen, and increased levels of GLUT1 and GLUT4 than FVB/N muscle. Histology indicated that MKR soleus had mildly decreased cytochrome c activity overall and more type II (glycolytic) fibres compared with that in FVB/N mice.

CONCLUSIONS/INTERPRETATION: MKR muscle adapts to using glucose, with more type II fibres present in red muscle. Fasting RER is elevated and glucose disposal during an i.p. glucose tolerance test is accelerated despite increased muscle DAG and TAG. Metabolic inflexibility may result from the compensatory use of fuel that can be best utilised for energy requirements; static vs dynamic glucose disposal assessments may measure complementary aspects of metabolic flexibility and insulin sensitivity.

In vivo insulin sensitivity can be assessed using "open loop" clamp or "closed loop" methods. Open loop clamp methods are static, and fix plasma glucose independently from plasma insulin. Closed loop methods are dynamic, and assess glucose disposal in response to a stable isotope labeled glucose tolerance test. Using PPARalpha(-/-) mice, open and closed loop assessments of insulin sensitivity/glucose disposal were compared. Indirect calorimetry done for the assessment of diurnal substrate utilization/metabolic flexibility showed that chow fed PPARalpha(-/-) mice had increased glucose utilization during the light (starved) cycle. Euglycemic clamps showed no differences in insulin stimulated glucose disposal, whether for chow or high fat diets, but did show differences in basal glucose clearance for chow fed PPARalpha(-/-) versus SV129J-wt mice. In contrast, the dynamic stable isotope labeled glucose tolerance tests reveal enhanced glucose disposal for PPARalpha(-/-) versus SV129J-wt, for chow and high fat diets. Area under the curve for plasma labeled and unlabeled glucose for PPARalpha(-/-) was approximately 1.7-fold lower, P < 0.01 during the stable isotope labeled glucose tolerance test for both diets. Area under the curve for plasma insulin was 5-fold less for the chow fed SV129J-wt (P < 0.01) but showed no difference on a high fat diet (0.30 +/- 0.1 for SV129J-wt vs. 0.13 +/- 0.10 for PPARalpha(-/-), P = 0.28). This study demonstrates that dynamic stable isotope labeled glucose tolerance test can assess "silent" metabolic phenotypes, not detectable by the static, "open loop", euglycemic or hyperglycemic clamps. Both open loop and closed loop methods may describe different aspects of metabolic inflexibility and insulin sensitivity.

Fyn null mice display reduced adiposity associated with increased fatty acid oxidation, energy expenditure, and activation of the AMP-dependent protein kinase (AMPK) in skeletal muscle and adipose tissue. The acute pharmacological inhibition of Fyn kinase activity with SU6656 in wild-type mice reproduces these metabolic effects and induced a specific reduction in fat mass with no change in lean mass. LKB1, the main upstream AMPK kinase (AMPKK) in peripheral tissues, was redistributed from the nucleus into the cytoplasm of cells treated with SU6656 and in cells expressing a kinase-deficient, but not a constitutively kinase-active, Fyn mutant. Moreover, Fyn kinase directly phosphorylated LKB1 on tyrosine 261 and 365 residues, and mutations of these sites resulted in LKB1 export into the cytoplasm and increased AMPK phosphorylation. These data demonstrate a crosstalk between Fyn tyrosine kinase and the AMPK energy-sensing pathway, through Fyn-dependent regulation of the AMPK upstream activator LKB1.

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin beta1 subunit in striated muscle results in a near complete loss of integrin beta1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin beta1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR.Rictor.LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin beta1 signaling events in mediating cross-talk to that of insulin action.

Mice null for Fyn (a member of the Src family of nonreceptor tyrosine kinases) display a reduced percentage of adipose mass associated with decreased adipocyte cell size. In parallel, there is a substantial reduction in fasting plasma glucose, insulin, triglycerides, and free fatty acids concomitant with decreased intrahepatocellular and intramyocellular lipid accumulation. Importantly, the Fyn null mice exhibit improved glucose tolerance resulting from increased peripheral tissue (adipose and skeletal muscle) insulin sensitivity with a very small effect in the liver. Moreover, whole-body, adipose, and skeletal muscle fatty acid uptake and oxidation are increased along with AMP kinase activation and acetyl-CoA carboxylase inhibition. Together, these data demonstrate crosstalk between Src-family kinase activity and fatty acid oxidation and show that the loss of Fyn markedly improves peripheral tissue insulin sensitivity by relieving a selective negative modulation of AMP kinase activity in adipose tissue and skeletal muscle.

Evidence indicates that endosomal insulin receptor (IR) trafficking plays a role in regulating insulin signal transduction. To evaluate its importance, we developed a series of biokinetic models for quantifying activated surface and endosomal IR dynamics from published experimental data. Starting with a published two-compartment Fao hepatoma model, a four-pool model was formulated that depicts IR autophosphorylation after receptor binding, IR endosomal internalization/trafficking, insulin dissociation from and dephosphorylation of internalized IR, and recycling of unliganded, dephosphorylated IR to the plasma membrane. Quantification required three additional data sets, two measured, but unmodeled by the same group. A five-pool model created to include endosomal trafficking of the nonphosphorylated insulin-IR complex was fitted using the same data sets, augmented with another published data set. Creation of a six-pool model added the physiologically relevant dissociation of insulin ligand from the activated endosomal IR. More importantly, all three models, validated against additional data not used in model fitting, predict that, mechanistically, internalization of activated IR is a rate-limiting step, at least under the receptor saturating conditions of the fitting data. This rate includes the transit time to a site where insulin dissociation from and/or dephosphorylation of the IR occurs by docking with protein-tyrosine phosphatases (PTPases), or where a sufficient conformational change occurs in the IR, perhaps due to insulin-IR dissociation, where associated PTPases may complete IR dephosphorylation. Our new models indicate that key events in endosomal IR trafficking have significance in mediating IR activity, possibly serving to regulate insulin signal transduction.

Hepatic glucose production is crucial for glucose homeostasis, and its dysregulation contributes to the pathogenesis of diabetes. Here, we show that members of the NR4A family of ligand-independent orphan nuclear receptors are downstream mediators of cAMP action in the hormonal control of gluconeogenesis. Hepatic expression of Nur77, Nurr1 and NOR1 is induced by the cAMP axis in response to glucagon and fasting in vivo and is increased in diabetic mice that exhibit elevated gluconeogenesis. Adenoviral expression of Nur77 induces genes involved in gluconeogenesis, stimulates glucose production both in vitro and in vivo, and raises blood glucose levels. Conversely, expression of an inhibitory mutant Nur77 receptor antagonizes gluconeogenic gene expression and lowers blood glucose levels in db/db mice. These results outline a previously unrecognized role for orphan nuclear receptors in the transcriptional control of glucose homeostasis.