Alexandra Chadt

Tbc1d1 mutation in lean mouse strain confers leanness and protects from diet-induced obesity

We previously identified Nob1 as a quantitative trait locus for high-fat diet–induced obesity and diabetes in genome-wide scans of outcross populations of obese and lean mouse strains. Additional crossbreeding experiments indicated that Nob1 represents an obesity suppressor from the lean Swiss Jim Lambert (SJL) strain. Here we identify a SJL-specific mutation in the Tbc1d1 gene that results in a truncated protein lacking the TBC Rab–GTPase-activating protein domain. TBC1D1, which has been recently linked to human obesity, is related to the insulin signaling protein AS160 and is predominantly expressed in skeletal muscle. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, whereas overexpression of TBC1D1 had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation and reduced glucose uptake in isolated skeletal muscle. Our data strongly suggest that mutation of Tbc1d1 suppresses high-fat diet–induced obesity by increasing lipid use in skeletal muscle.

The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL)) and wild-type littermates were studied. Glucose and insulin tolerance, glucose utilization, hepatic glucose production, and tissue-specific insulin-mediated glucose uptake were determined. The effect of insulin, AICAR, or contraction on glucose transport was studied in isolated skeletal muscle. Glucose and insulin tolerance tests were normal in TBC1D1-deficient Nob1.10(SJL) mice, yet the 4-h-fasted insulin concentration was increased. Insulin-stimulated peripheral glucose utilization during a euglycemic hyperinsulinemic clamp was similar between genotypes, whereas the suppression of hepatic glucose production was increased in TBC1D1-deficient mice. In isolated extensor digitorum longus (EDL) but not soleus muscle, glucose transport in response to insulin, AICAR, or contraction was impaired by TBC1D1 deficiency. The reduction in glucose transport in EDL muscle from TBC1D1-deficient Nob1.10(SJL) mice may be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice. In conclusion, TBC1D1 plays a role in regulation of glucose metabolism in skeletal muscle. Moreover, functional TBC1D1 is required for AICAR- or contraction-induced metabolic responses, implicating a role in energy-sensing signals.

Conventional Knockout of Tbc1d1 in Mice Impairs Insulin- and AICAR-Stimulated Glucose Uptake in Skeletal Muscle

In the obesity-resistant SJL mouse strain, we previously identified a naturally occurring loss-of-function mutation in the gene for Tbc1d1. Characterization of recombinant inbred mice that carried the Tbc1d1(SJL) allele on a C57BL/6J background indicated that loss of TBC1D1 protects from obesity, presumably by increasing the use of fat as energy source. To provide direct functional evidence for an involvement of TBC1D1 in energy substrate metabolism, we generated and characterized conventional Tbc1d1 knockout mice. TBC1D1-deficient mice showed moderately reduced body weight, decreased respiratory quotient, and an elevated resting metabolic rate. Ex vivo analysis of intact isolated skeletal muscle revealed a severe impairment in insulin- and AICAR-stimulated glucose uptake in glycolytic extensor digitorum longus muscle and a substantially increased rate of fatty acid oxidation in oxidative soleus muscle. Our results provide direct evidence that TBC1D1 plays a major role in glucose and lipid utilization, and energy substrate preference in skeletal muscle.

Deletion of Both Rab-GTPase–Activating Proteins TBC1D1 and TBC1D4 in Mice Eliminates Insulin- and AICAR-Stimulated Glucose Transport

The Rab-GTPase–activating proteins TBC1D1 and TBC1D4 (AS160) were previously shown to regulate GLUT4 translocation in response to activation of AKT and AMP-dependent kinase. However, knockout mice lacking either Tbc1d1 or Tbc1d4 displayed only partially impaired insulin-stimulated glucose uptake in fat and muscle tissue. The aim of this study was to determine the impact of the combined inactivation of Tbc1d1 and Tbc1d4 on glucose metabolism in double-deficient (D1/4KO) mice. D1/4KO mice displayed normal fasting glucose concentrations but had reduced tolerance to intraperitoneally administered glucose, insulin, and AICAR. D1/4KO mice showed reduced respiratory quotient, indicating increased use of lipids as fuel. These mice also consistently showed elevated fatty acid oxidation in isolated skeletal muscle, whereas insulin-stimulated glucose uptake in muscle and adipose cells was almost completely abolished. In skeletal muscle and white adipose tissue, the abundance of GLUT4 protein, but not GLUT4 mRNA, was substantially reduced. Cell surface labeling of GLUTs indicated that RabGAP deficiency impairs retention of GLUT4 in intracellular vesicles in the basal state. Our results show that TBC1D1 and TBC1D4 together play essential roles in insulin-stimulated glucose uptake and substrate preference in skeletal muscle and adipose cells.

The ARF-like GTPase ARFRP1 is essential for lipid droplet growth and is involved in the regulation of lipolysis.

ABSTRACT ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1ad−/−) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1ad−/− mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1ad−/− adipocytes, suggesting that lipid droplet growth is defective in Arfrp1ad−/− mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1ad−/− mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.

Skeletal muscle uncoupling-induced longevity in mice is linked to increased substrate metabolism and induction of the endogenous antioxidant defense system.

Ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) mitochondria increases lifespan considerably in high-fat diet-fed UCP1 Tg mice compared with wild types (WT). To clarify the underlying mechanisms, we investigated substrate metabolism as well as oxidative stress damage and antioxidant defense in SM of low-fat- and high-fat-fed mice. Tg mice showed an increased protein expression of phosphorylated AMP-activated protein kinase, markers of lipid turnover (p-ACC, FAT/CD36), and an increased SM ex vivo fatty acid oxidation. Surprisingly, UCP1 Tg mice showed elevated lipid peroxidative protein modifications with no changes in glycoxidation or direct protein oxidation. This was paralleled by an induction of catalase and superoxide dismutase activity, an increased redox signaling (MAPK signaling pathway), and increased expression of stress-protective heat shock protein 25. We conclude that increased skeletal muscle mitochondrial uncoupling in vivo does not reduce the oxidative stress status in the muscle cell. Moreover, it increases lipid metabolism and reactive lipid-derived carbonyls. This stress induction in turn increases the endogenous antioxidant defense system and redox signaling. Altogether, our data argue for an adaptive role of reactive species as essential signaling molecules for health and longevity.

Re-patterning of Skeletal Muscle Energy Metabolism by Fat Storage-inducing Transmembrane Protein 2

Background: Fat storage-inducing transmembrane protein 2 (FIT2) is implicated to be important in the formation of triacylglyceride lipid droplets. Results: Mice with skeletal muscle-specific overexpression of FIT2 had increased muscle triacylglycerides, were completely protected from diet-induced weight gain, and had altered muscle energy metabolism. Conclusion: FIT2 plays an unexpected function in regulating muscle energy metabolism. Significance: This is the first study describing a function for FIT2 in energy metabolism. Triacylglyceride stored in cytosolic lipid droplets (LDs) constitutes a major energy reservoir in most eukaryotes. The regulated turnover of triacylglyceride in LDs provides fatty acids for mitochondrial β-oxidation and ATP generation in physiological states of high demand for energy. The mechanisms for the formation of LDs in conditions of energy excess are not entirely understood. Fat storage-inducing transmembrane protein 2 (FIT2/FITM2) is the anciently conserved member of the fat storage-inducing transmembrane family of proteins implicated to be important in the formation of LDs, but its role in energy metabolism has not been tested. Here, we report that expression of FIT2 in mouse skeletal muscle had profound effects on muscle energy metabolism. Mice with skeletal muscle-specific overexpression of FIT2 (CKF2) had significantly increased intramyocellular triacylglyceride and complete protection from high fat diet-induced weight gain due to increased energy expenditure. Mass spectrometry-based metabolite profiling suggested that CKF2 skeletal muscle had increased oxidation of branched chain amino acids but decreased oxidation of fatty acids. Glucose was primarily utilized in CKF2 muscle for synthesis of the glycerol backbone of triacylglyceride and not for glycogen production. CKF2 muscle was ATP-deficient and had activated AMP kinase. Together, these studies indicate that FIT2 expression in skeletal muscle plays an unexpected function in regulating muscle energy metabolism and indicates an important role for lipid droplet formation in this process.

Over-expression of PRAS40 enhances insulin sensitivity in skeletal muscle

Abstract Context: Silencing proline-rich Akt substrate of 40-kDa (PRAS40) impairs insulin signalling in skeletal muscle. Objective: This study assessed the effects of over-expressing wild type or mutant AAA-PRAS40, in which the major phosphorylation sites and mTORC1-binding site were mutated, on insulin signalling in skeletal muscle. Results: Over-expression of WT-PRAS40, but not AAA-PRAS40, impaired the insulin-mediated activation of the mTORC1-pathway in human skeletal muscle cells (hSkMC). However, insulin-mediated Akt-phosphorylation was increased upon over-expression of WT-PRAS40 both in hSkMC and mouse skeletal muscle. Also over-expression of AAA-PRAS40 had an insulin-sensitizing effect, although to a lesser extent as WT-PRAS40. The insulin-sensitizing effect associated with increased IRS1 protein abundance and inhibition of proteasome activity. Finally, over-expression of WT-PRAS40 reversed hyperinsulinemia-induced insulin resistance. Conclusion: This study identifies PRAS40 as a regulator of insulin sensitivity in hSkMC. In contrast to the mTORC1-pathway, the insulin-sensitizing action of PRAS40 occurs independent of binding of PRAS40 to the mTORC1-complex.

An Interval of the Obesity QTL Nob3.38 within a QTL Hotspot on Chromosome 1 Modulates Behavioral Phenotypes

A region on mouse distal chromosome 1 (Chr. 1) that is highly enriched in quantitative trait loci (QTLs) controlling neural and behavioral phenotypes overlaps with the peak region of a major obesity QTL (Nob3.38), which we identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6). By positional cloning we recently identified a microdeletion within this locus causing the disruption of Ifi202b that protects from adiposity by suppressing expression of 11β-Hsd1. Here we show that the Nob3.38 segment also corresponds with the QTL rich region (Qrr1) on Chr. 1 and associates with increased voluntary running wheel activity, Rota-rod performance, decreased grip strength, and anxiety-related traits. The characterization of a subcongenic line carrying 14.2 Mbp of Nob3.38 with a polymorphic region of 4.4 Mbp indicates that the microdeletion and/or other polymorphisms in its proximity alter body weight, voluntary activity, and exploration. Since 27 out of 32 QTL were identified in crosses with B6, we hypothesized that the microdeletion and or adjacent SNPs are unique for B6 mice and responsible for some of the complex Qrr1-mediated effects. Indeed, a phylogenic study of 28 mouse strains revealed a NZO-like genotype for 22 and a B6-like genotype for NZW/LacJ and 4 other C57BL strains. Thus, we suggest that a Nob3.38 interval (173.0–177.4 Mbp) does not only modify adiposity but also neurobehavioral traits by a haplotype segregating with C57BL strains.