An insulin-regulated arrestin domain protein controls hepatic glucagon action.

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the in vivo functions of the arrestin domain-containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver.

Physiological and Pathophysiological Roles of Thioredoxin Interacting Protein: A Perspective on Redox Inflammation and Metabolism.

Significance: Thioredoxin interacting protein (TXNIP) is a member of the arrestin fold superfamily with important cellular functions, including cellular transport, mitochondrial energy generation, and protein cycling. It is the only arrestin-domain protein known to covalently bind to thioredoxin and plays roles in glucose metabolism, inflammation, apoptosis, and cancer. Recent Advances: The crystal structure of the TXNIP-thioredoxin complex provided details about this fascinating interaction. Recent studies showed that TXNIP is induced by endoplasmic reticulum (ER) stress, activates NLR family pyrin domain containing 3 (NLRP3) inflammasomes, and can regulate glucose transport into cells. The tumor suppressor role of TXNIP in various cancer types and the role of TXNIP in fructose absorption are now described. Critical Issues: The influence of TXNIP on redox state is more complex than its interaction with thioredoxin. Future Directions: It is incompletely understood which functions of TXNIP are thioredoxin-dependent. It is also unclear whether TXNIP binding can inhibit glucose transporters without endocytosis. TXNIP-regulated control of ER stress should also be investigated further. Antioxid. Redox Signal. 38, 442-460.

SATB2 preserves colon stem cell identity and mediates ileum-colon conversion via enhancer remodeling.

Adult stem cells maintain regenerative tissue structure and function by producing tissue-specific progeny, but the factors that preserve their tissue identities are not well understood. The small and large intestines differ markedly in cell composition and function, reflecting their distinct stem cell populations. Here we show that SATB2, a colon-restricted chromatin factor, singularly preserves LGR5+ adult colonic stem cell and epithelial identity in mice and humans. Satb2 loss in adult mice leads to stable conversion of colonic stem cells into small intestine ileal-like stem cells and replacement of the colonic mucosa with one that resembles the ileum. Conversely, SATB2 confers colonic properties on the mouse ileum. Human colonic organoids also adopt ileal characteristics upon SATB2 loss. SATB2 regulates colonic identity in part by modulating enhancer binding of the intestinal transcription factors CDX2 and HNF4A. Our study uncovers a conserved core regulator of colonic stem cells able to mediate cross-tissue plasticity in mature intestines.

Thioredoxin Interacting Protein Is Required for a Chronic Energy-Rich Diet to Promote Intestinal Fructose Absorption.

Increased consumption of fats and added sugars has been associated with an increase in metabolic syndromes. Here we show that mice chronically fed an energy-rich diet (ERD) with high fat and moderate sucrose have enhanced the absorption of a gastrointestinal fructose load, and this required expression of the arrestin domain protein Txnip in the intestinal epithelial cells. ERD feeding induced gene and protein expression of Glut5, and this required the expression of Txnip. Furthermore, Txnip interacted with Rab11a, a small GTPase that facilitates the apical localization of Glut5. We also demonstrate that ERD promoted Txnip/Glut5 complexes in the apical intestinal epithelial cell. Our findings demonstrate that ERD facilitates fructose absorption through a Txnip-dependent mechanism in the intestinal epithelial cell, suggesting that increased fructose absorption could potentially provide a mechanism for worsening of metabolic syndromes in the setting of a chronic ERD.

Arrestin domain-containing 3 (Arrdc3) modulates insulin action and glucose metabolism in liver.

Insulin action in the liver is critical for glucose homeostasis through regulation of glycogen synthesis and glucose output. Arrestin domain-containing 3 (Arrdc3) is a member of the α-arrestin family previously linked to human obesity. Here, we show that Arrdc3 is differentially regulated by insulin in vivo in mice undergoing euglycemic-hyperinsulinemic clamps, being highly up-regulated in liver and down-regulated in muscle and fat. Mice with liver-specific knockout (KO) of the insulin receptor (IR) have a 50% reduction in Arrdc3 messenger RNA, while, conversely, mice with liver-specific KO of Arrdc3 (L-Arrdc3 KO) have increased IR protein in plasma membrane. This leads to increased hepatic insulin sensitivity with increased phosphorylation of FOXO1, reduced expression of PEPCK, and increased glucokinase expression resulting in reduced hepatic glucose production and increased hepatic glycogen accumulation. These effects are due to interaction of ARRDC3 with IR resulting in phosphorylation of ARRDC3 on a conserved tyrosine (Y382) in the carboxyl-terminal domain. Thus, Arrdc3 is an insulin target gene, and ARRDC3 protein directly interacts with IR to serve as a feedback regulator of insulin action in control of liver metabolism.

Exogenous GDF11, but not GDF8, reduces body weight and improves glucose homeostasis in mice.

Insulin resistance is associated with aging in mice and humans. We have previously shown that administration of recombinant GDF11 (rGDF11) to aged mice alters aging phenotypes in the brain, skeletal muscle, and heart. While the closely related protein GDF8 has a role in metabolism, limited data are available on the potential metabolic effects of GDF11 or GDF8 in aging. To determine the metabolic effects of these two ligands, we administered rGDF11 or rGDF8 protein to young or aged mice fed a standard chow diet, short-term high-fat diet (HFD), or long-term HFD. Under nearly all of these diet conditions, administration of exogenous rGDF11 reduced body weight by 3-17% and significantly improved glucose tolerance in aged mice fed a chow (~30% vs. saline) or HF (~50% vs. saline) diet and young mice fed a HFD (~30%). On the other hand, exogenous rGDF8 showed signifcantly lesser effect or no effect at all on glucose tolerance compared to rGDF11, consistent with data demonstrating that GFD11 is a more potent signaling ligand than GDF8. Collectively, our results show that administration of exogenous rGDF11, but not rGDF8, can reduce diet-induced weight gain and improve metabolic homeostasis.

Adiposity-Independent Effects of Aging on Insulin Sensitivity and Clearance in Mice and Humans.

OBJECTIVE: Aging is associated with impaired insulin sensitivity and increased prevalence of type 2 diabetes. However, it remains unclear whether aging-associated insulin resistance is due to increased adiposity or other age-related factors. To address this question, the impact of aging on insulin sensitivity was investigated independently of changes in body composition. METHODS: Cohorts of mice aged 4 to 8 months ("young") and 18 to 27 months ("aged") exhibiting similar body composition were characterized for glucose metabolism on chow and high-fat diets. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp analyses. The relationship between aging and insulin resistance in humans was investigated in 1,250 nondiabetic Mexican Americans who underwent hyperinsulinemic-euglycemic clamps. RESULTS: In mice with similar body composition, age had no detrimental effect on plasma glucose and insulin levels. While aging did not diminish glucose tolerance, hyperinsulinemic-euglycemic clamps demonstrated impaired insulin sensitivity and reduced insulin clearance in aged mice on chow and high-fat diets. Consistent with results in the mouse, age remained an independent determinant of insulin resistance after adjustment for body composition in Mexican American males. CONCLUSIONS: This study demonstrates that in addition to altered body composition, adiposity-independent mechanisms also contribute to aging-associated insulin resistance in mice and humans.

Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78-/-) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78-/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78-/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78-/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

Liver-specific reconstitution of CEACAM1 reverses the metabolic abnormalities caused by its global deletion in male mice.

AIMS/HYPOTHESIS: The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Mice with global null mutation (Cc1 -/-) or with liver-specific inactivation (L-SACC1) of Cc1 (also known as Ceacam1) gene display hyperinsulinaemia resulting from impaired insulin clearance, insulin resistance, steatohepatitis and obesity. Because increased lipolysis contributes to the metabolic phenotype caused by transgenic inactivation of CEACAM1 in the liver, we aimed to further investigate the primary role of hepatic CEACAM1-dependent insulin clearance in insulin and lipid homeostasis. To this end, we examined whether transgenic reconstitution of CEACAM1 in the liver of global Cc1 -/- mutant mice reverses their abnormal metabolic phenotype. METHODS: Insulin response was assessed by hyperinsulinaemic-euglycaemic clamp analysis and energy balance was analysed by indirect calorimetry. Mice were overnight-fasted and refed for 7 h to assess fatty acid synthase activity in the liver and the hypothalamus in response to insulin release during refeeding. RESULTS: Liver-based rescuing of CEACAM1 restored insulin clearance, plasma insulin level, insulin sensitivity and steatohepatitis caused by global deletion of Cc1. It also reversed the gain in body weight and total fat mass observed with Cc1 deletion, in parallel to normalising energy balance. Mechanistically, reversal of hyperphagia appeared to result from reducing fatty acid synthase activity and restoring insulin signalling in the hypothalamus. CONCLUSIONS/INTERPRETATION: Despite the potential confounding effects of deleting Cc1 from extrahepatic tissues, liver-based rescuing of CEACAM1 resulted in full normalisation of the metabolic phenotype, underscoring the key role that CEACAM1-dependent hepatic insulin clearance pathways play in regulating systemic insulin sensitivity, lipid homeostasis and energy balance.

IL-10 prevents aging-associated inflammation and insulin resistance in skeletal muscle.

Altered energy balance and insulin resistance are important characteristics of aging. Skeletal muscle is a major site of glucose disposal, and the role of aging-associated inflammation in skeletal muscle insulin resistance remains unclear. To investigate, we examined glucose metabolism in 18-mo-old transgenic mice with muscle-specific overexpression of IL-10 (MIL10) and in wild-type mice during hyperinsulinemic-euglycemic clamping. Despite similar fat mass and energy balance, MIL10 mice were protected from aging-associated insulin resistance with significant increases in glucose infusion rates, whole-body glucose turnover, and skeletal muscle glucose uptake (∼60%; P < 0.05), as compared to age-matched WT mice. This protective effect was associated with decreased muscle inflammation, but no changes in adipose tissue inflammation in aging MIL10 mice. These results demonstrate the importance of skeletal muscle inflammation in aging-mediated insulin resistance, and our findings further implicate a potential therapeutic role of anti-inflammatory cytokine in the treatment of aging-mediated insulin resistance.-Dagdeviren, S., Jung, D. Y., Friedline, R. H., Noh, H. L., Kim, J. H., Patel, P. R., Tsitsilianos, N., Inashima, K., Tran, D. A., Hu, X., Loubato, M. M., Craige, S. M., Kwon, J. Y., Lee, K. W., Kim, J. K. IL-10 prevents aging-associated inflammation and insulin resistance in skeletal muscle.

Adipocyte-specific Hypoxia-inducible gene 2 promotes fat deposition and diet-induced insulin resistance.

OBJECTIVE: Adipose tissue relies on lipid droplet (LD) proteins in its role as a lipid-storing endocrine organ that controls whole body metabolism. Hypoxia-inducible Gene 2 (Hig2) is a recently identified LD-associated protein in hepatocytes that promotes hepatic lipid storage, but its role in the adipocyte had not been investigated. Here we tested the hypothesis that Hig2 localization to LDs in adipocytes promotes adipose tissue lipid deposition and systemic glucose homeostasis. METHOD: White and brown adipocyte-deficient (Hig2fl/fl × Adiponection cre+) and selective brown/beige adipocyte-deficient (Hig2fl/fl × Ucp1 cre+) mice were generated to investigate the role of Hig2 in adipose depots. Additionally, we used multiple housing temperatures to investigate the role of active brown/beige adipocytes in this process. RESULTS: Hig2 localized to LDs in SGBS cells, a human adipocyte cell strain. Mice with adipocyte-specific Hig2 deficiency in all adipose depots demonstrated reduced visceral adipose tissue weight and increased glucose tolerance. This metabolic effect could be attributed to brown/beige adipocyte-specific Hig2 deficiency since Hig2fl/fl × Ucp1 cre+ mice displayed the same phenotype. Furthermore, when adipocyte-deficient Hig2 mice were moved to thermoneutral conditions in which non-shivering thermogenesis is deactivated, these improvements were abrogated and glucose intolerance ensued. Adipocyte-specific Hig2 deficient animals displayed no detectable changes in adipocyte lipolysis or energy expenditure, suggesting that Hig2 may not mediate these metabolic effects by restraining lipolysis in adipocytes. CONCLUSIONS: We conclude that Hig2 localizes to LDs in adipocytes, promoting adipose tissue lipid deposition and that its selective deficiency in active brown/beige adipose tissue mediates improved glucose tolerance at 23 °C. Reversal of this phenotype at thermoneutrality in the absence of detectable changes in energy expenditure, adipose mass, or liver triglyceride suggests that Hig2 deficiency triggers a deleterious endocrine or neuroendocrine pathway emanating from brown/beige fat cells.

Altered Interleukin-10 Signaling in Skeletal Muscle Regulates Obesity-Mediated Inflammation and Insulin Resistance.

Skeletal muscle insulin resistance is a major characteristic of obesity and type 2 diabetes. Although obesity-mediated inflammation is causally associated with insulin resistance, the underlying mechanism is unclear. Here, we examined the effects of chronic obesity in mice with muscle-specific overexpression of interleukin-10 (MIL10). After 16 weeks of a high-fat diet (HFD), MIL10 mice became markedly obese but showed improved insulin action compared to that of wild-type mice, which was largely due to increased glucose metabolism and reduced inflammation in skeletal muscle. Since leptin regulates inflammation, the beneficial effects of interleukin-10 (IL-10) were further examined in leptin-deficient ob/ob mice. Muscle-specific overexpression of IL-10 in ob/ob mice (MCK-IL10ob/ob) did not affect spontaneous obesity, but MCK-IL10ob/ob mice showed increased glucose turnover compared to that in ob/ob mice. Last, mice with muscle-specific ablation of IL-10 receptor (M-IL10R-/-) were generated to determine whether IL-10 signaling in skeletal muscle is involved in IL-10 effects on glucose metabolism. After an HFD, M-IL10R-/- mice developed insulin resistance with reduced glucose metabolism compared to that in wild-type mice. Overall, these results demonstrate IL-10 effects to attenuate obesity-mediated inflammation and improve insulin sensitivity in skeletal muscle, and our findings implicate a potential therapeutic role of anti-inflammatory cytokines in treating insulin resistance and type 2 diabetes.

Genetic ablation of lymphocytes and cytokine signaling in nonobese diabetic mice prevents diet-induced obesity and insulin resistance.

Obesity is characterized by a dysregulated immune system, which may causally associate with insulin resistance and type 2 diabetes. Despite widespread use of nonobese diabetic (NOD) mice, NOD with severe combined immunodeficiency (scid) mutation (SCID) mice, and SCID bearing a null mutation in the IL-2 common γ chain receptor (NSG) mice as animal models of human diseases including type 1 diabetes, the underlying metabolic effects of a genetically altered immune system are poorly understood. For this, we performed a comprehensive metabolic characterization of these mice fed chow or after 6 wk of a high-fat diet. We found that NOD mice had ∼50% less fat mass and were 2-fold more insulin sensitive, as measured by hyperinsulinemic-euglycemic clamp, than C57BL/6 wild-type mice. SCID mice were also more insulin sensitive with increased muscle glucose metabolism and resistant to diet-induced obesity due to increased energy expenditure (∼10%) and physical activity (∼40%) as measured by metabolic cages. NSG mice were completely protected from diet-induced obesity and insulin resistance with significant increases in glucose metabolism in peripheral organs. Our findings demonstrate an important role of genetic background, lymphocytes, and cytokine signaling in diet-induced obesity and insulin resistance.

CD40 deficiency in mice exacerbates obesity-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance.

The pathophysiology of obesity and type 2 diabetes in rodents and humans is characterized by low-grade inflammation in adipose tissue and liver. The CD40 receptor and its ligand CD40L initiate immune cell signaling promoting inflammation, but conflicting data on CD40L-null mice confound its role in obesity-associated insulin resistance. Here, we demonstrate that CD40 receptor-deficient mice on a high-fat diet display the expected decrease in hepatic cytokine levels but paradoxically exhibit liver steatosis, insulin resistance, and glucose intolerance compared with their age-matched wild-type controls. Hyperinsulinemic-euglycemic clamp studies also demonstrated insulin resistance in glucose utilization by the CD40-null mice compared with wild-type mice. In contrast to liver, adipose tissue in CD40-deficient animals harbors elevated cytokine levels and infiltration of inflammatory cells, particularly macrophages and CD8(+) effector T cells. In addition, ex vivo explants of epididymal adipose tissue from CD40(-/-) mice display elevated basal and isoproterenol-stimulated lipolysis, suggesting a potential increase of lipid efflux from visceral fat to the liver. These findings reveal that 1) CD40-null mice represent an unusual model of hepatic steatosis with reduced hepatic inflammation, and 2) CD40 unexpectedly functions in adipose tissue to attenuate its inflammation in obesity, thereby protecting against hepatic steatosis.

Vancomycin containing PLLA/ß-TCP controls MRSA in vitro.

BACKGROUND: Osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) often requires surgery and prolonged systemic antibiotic treatment. Local antibiotic delivery systems of bioceramics or polymers have been developed to treat osteomyelitis. A disadvantage of biodegradable polymers is the initial burst of antibiotics into the environment; one advantage of bioceramics is its osteoconductivity. We therefore developed a vancomycin-containing poly-l-lactic acid/ß-tricalcium phosphate (PLLA/ß-TCP) composite to control antibiotic release and stimulate bone formation. QUESTIONS/PURPOSES: We (1) characterized these composites, (2) assessed vancomycin release in inhibitory doses, and (3) determined whether they would permit cell adhesion, proliferation, and mineralization in vitro. METHODS: We molded 250 vancomycin-containing (VC) and 125 vancomycin-free (VUC) composites using PLLA, ß-TCP, and chloroform. One hundred twenty-five VC composites were further dip-coated with PLLA (CVC) to delay antibiotic release. Composites were characterized according to their pore structure, size, volume, density, and surface area. Vancomycin release and bioactivity were determined. Adhesion, proliferation, and mineralization were assessed for two and three replicates on Days 3 and 7 with mesenchymal stem (MSC) and Saos type 2 cells. RESULTS: Pore size, volume, apparent density, and surface area of the CVC were 3.5 ± 1.9 µm, 0.005 ± 0.002 cm(3)/g, 1.18 g/cm(3) and 3.68 m(2)/g, respectively. CVC released 1.71 ± 0.13 mg (63.1%) and 2.49 ± 0.64 mg (91.9%) of its vancomycin on Day 1 and Week 6, respectively. MSC and Saos type 2 cells attached and proliferated on composites on Days 3 and 7. CONCLUSIONS: Vancomycin-containing PLLA/ß-TCP composites release antibiotics in inhibitory doses after dip coating and appeared biocompatible based on adhesion, proliferation, and mineralization. CLINICAL RELEVANCE: Vancomycin-containing PLLA/ß-TCP composites may be useful for controlling MRSA but will require in vivo confirmation.

Tumor necrosis factor-alpha antagonist administration recovers skeletal muscle dysfunction in ovariectomized rats.

Skeletal muscles deteriorate after ovariectomy. Molecular pathway of this deterioration has not been defined. Tumor necrosis factor (TNF)-alpha activation is assumed to trigger muscle atrophy and administration of its antagonist is hypothesized to recover this atrophy in rats. Slow-twitch soleus and fast-twitch extensor digitorum longus muscle functions were investigated in intact, ovariectomized (OVX), and OVX plus 10 µg/g/week TNF-alpha antagonist administered female rats. Maximum isometric twitch and tetanic contraction responses were lower in the OVX groups. Maximum isometric twitch amplitudes recovered in the extensor digitorum longus but not in the soleus muscles after TNF-alpha antagonist administration. The decrease in responses to tetanic stimulations recovered in the OVX-TNF group at frequencies higher than 20 Hz in both muscle types. OVX animals body weight was 21% higher than intact animals. Muscle weight to body weight ratios of the OVX groups were higher than the control group which recovered after TNF-alpha antagonist administration. Findings suggest that the functional loss in OVX rat muscles is TNF-alpha pathway dependent. Skeletal muscle atrophy and function after OVX recovered by TNF-alpha antagonist administration.