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1.  Metabolic characteristics of long-lived mice 
Frontiers in Genetics  2012;3:288.
Genetic suppression of insulin/insulin-like growth factor signaling (IIS) can extend longevity in worms, insects, and mammals. In laboratory mice, mutations with the greatest, most consistent, and best documented positive impact on lifespan are those that disrupt growth hormone (GH) release or actions. These mutations lead to major alterations in IIS but also have a variety of effects that are not directly related to the actions of insulin or insulin-like growth factor I. Long-lived GH-resistant GHR-KO mice with targeted disruption of the GH receptor gene, as well as Ames dwarf (Prop1df) and Snell dwarf (Pit1dw) mice lacking GH (along with prolactin and TSH), are diminutive in size and have major alterations in body composition and metabolic parameters including increased subcutaneous adiposity, increased relative brain weight, small liver, hypoinsulinemia, mild hypoglycemia, increased adiponectin levels and insulin sensitivity, and reduced serum lipids. Body temperature is reduced in Ames, Snell, and female GHR-KO mice. Indirect calorimetry revealed that both Ames dwarf and GHR-KO mice utilize more oxygen per gram (g) of body weight than sex- and age-matched normal animals from the same strain. They also have reduced respiratory quotient, implying greater reliance on fats, as opposed to carbohydrates, as an energy source. Differences in oxygen consumption (VO2) were seen in animals fed or fasted during the measurements as well as in animals that had been exposed to 30% calorie restriction or every-other-day feeding. However, at the thermoneutral temperature of 30°C, VO2 did not differ between GHR-KO and normal mice. Thus, the increased metabolic rate of the GHR-KO mice, at a standard animal room temperature of 23°C, is apparently related to increased energy demands for thermoregulation in these diminutive animals. We suspect that increased oxidative metabolism combined with enhanced fatty acid oxidation contribute to the extended longevity of GHR-KO mice.
doi:10.3389/fgene.2012.00288
PMCID: PMC3521393  PMID: 23248643
growth hormone; aging; calorie restriction; dwarf mice; metabolism
2.  Targeted Loss of GHR Signaling in Mouse Skeletal Muscle Protects Against High-Fat Diet–Induced Metabolic Deterioration 
Diabetes  2011;61(1):94-103.
Growth hormone (GH) exerts diverse tissue-specific metabolic effects that are not revealed by global alteration of GH action. To study the direct metabolic effects of GH in the muscle, we specifically inactivated the growth hormone receptor (ghr) gene in postnatal mouse skeletal muscle using the Cre/loxP system (mGHRKO model). The metabolic state of the mGHRKO mice was characterized under lean and obese states. High-fat diet feeding in the mGHRKO mice was associated with reduced adiposity, improved insulin sensitivity, lower systemic inflammation, decreased muscle and hepatic triglyceride content, and greater energy expenditure compared with control mice. The obese mGHRKO mice also had an increased respiratory exchange ratio, suggesting increased carbohydrate utilization. GH-regulated suppressor of cytokine signaling-2 (socs2) expression was decreased in obese mGHRKO mice. Interestingly, muscles of both lean and obese mGHRKO mice demonstrated a higher interleukin-15 and lower myostatin expression relative to controls, indicating a possible mechanism whereby GHR signaling in muscle could affect liver and adipose tissue function. Thus, our study implicates skeletal muscle GHR signaling in mediating insulin resistance in obesity and, more importantly, reveals a novel role of muscle GHR signaling in facilitating cross-talk between muscle and other metabolic tissues.
doi:10.2337/db11-0814
PMCID: PMC3237669  PMID: 22187377
3.  Skeletal Muscle Growth Hormone Receptor Signaling Regulates Basal, but Not Fasting-Induced, Lipid Oxidation 
PLoS ONE  2012;7(9):e44777.
Background
Growth hormone (GH) stimulates whole-body lipid oxidation, but its regulation of muscle lipid oxidation is not clearly defined. Mice with a skeletal muscle-specific knockout of the GH receptor (mGHRKO model) are protected from high fat diet (HFD)–induced insulin resistance and display increased whole-body carbohydrate utilization. In this study we used the mGRHKO mice to investigate the role of muscle GHR signaling on lipid oxidation under regular chow (RC)- and HFD- fed conditions, and in response to fasting.
Methodology/Principal Findings
Expression of lipid oxidation genes was analyzed by real-time PCR in the muscles of RC- and HFD- fed mice, and after 24 h fasting in the HFD-fed mice. Expression of lipid oxidation genes was lower in the muscles of the mGHRKO mice relative to the controls, irrespective of diet. However, in response to 24 h fasting, the HFD-fed mGHRKO mice displayed up-regulation of lipid oxidation genes similar to the fasted controls. When subjected to treadmill running challenge, the HFD-fed mGHRKO mice demonstrated increased whole-body lipid utilization. Additionally, under fasted conditions, the adipose tissue of the mGHRKO mice displayed increased lipolysis as compared to both the fed mGHRKO as well as the fasted control mice.
Conclusions/Significance
Our data show that muscle GHR signaling regulates basal lipid oxidation, but not the induction of lipid oxidation in response to fasting. We further demonstrate that muscle GHR signaling is involved in muscle-adipose tissue cross-talk; however the mechanisms mediating this remain to be elucidated.
doi:10.1371/journal.pone.0044777
PMCID: PMC3443095  PMID: 23024761
4.  Leucine Supplementation Protects from Insulin Resistance by Regulating Adiposity Levels 
PLoS ONE  2013;8(9):e74705.
Background
Leucine supplementation might have therapeutic potential in preventing diet-induced obesity and improving insulin sensitivity. However, the underlying mechanisms are at present unclear. Additionally, it is unclear whether leucine supplementation might be equally efficacious once obesity has developed.
Methodology/Principal Findings
Male C57BL/6J mice were fed chow or a high-fat diet (HFD), supplemented or not with leucine for 17 weeks. Another group of HFD-fed mice (HFD-pairfat group) was food restricted in order to reach an adiposity level comparable to that of HFD-Leu mice. Finally, a third group of mice was exposed to HFD for 12 weeks before being chronically supplemented with leucine. Leucine supplementation in HFD-fed mice decreased body weight and fat mass by increasing energy expenditure, fatty acid oxidation and locomotor activity in vivo. The decreased adiposity in HFD-Leu mice was associated with increased expression of uncoupling protein 3 (UCP-3) in the brown adipose tissue, better insulin sensitivity, increased intestinal gluconeogenesis and preservation of islets of Langerhans histomorphology and function. HFD-pairfat mice had a comparable improvement in insulin sensitivity, without changes in islets physiology or intestinal gluconeogenesis. Remarkably, both HFD-Leu and HFD-pairfat mice had decreased hepatic lipid content, which likely helped improve insulin sensitivity. In contrast, when leucine was supplemented to already obese animals, no changes in body weight, body composition or glucose metabolism were observed.
Conclusions/Significance
These findings suggest that leucine improves insulin sensitivity in HFD-fed mice by primarily decreasing adiposity, rather than directly acting on peripheral target organs. However, beneficial effects of leucine on intestinal gluconeogenesis and islets of Langerhans's physiology might help prevent type 2 diabetes development. Differently, metabolic benefit of leucine supplementation is lacking in already obese animals, a phenomenon possibly related to the extent of the obesity before starting the supplementation.
doi:10.1371/journal.pone.0074705
PMCID: PMC3783457  PMID: 24086364
5.  Daily energy balance in growth hormone receptor/binding protein (GHR−/−) gene-disrupted mice is achieved through an increase in dark-phase energy efficiency 
The goal of this study was to examine factors that contribute to energy balance in female GHR −/− mice. We measured energy intake, energy expenditure (EE), fuel utilization, body mass (Mb) changes and physical activity in 17 month-old female GHR −/− mice and their age-matched wild type littermates. The GHR −/− mice were smaller, consumed more food per unit Mb, had greater EE per unit Mb and had an increase in 24-h EE/Mb that was similar to the increase in their surface-area-to-volume ratio. Locomotor activity (LMA) was reduced in the GHR −/− mice, but the energetic cost associated with their LMA was greater than in wild type controls. Furthermore, Mb and LMA were independent explanatory covariates of most of the variance in EE, and when adjusted for Mb and LMA, the GHR −/− mice had higher EE during both the light and dark phases of the daily cycle. Respiratory quotient was lower in GHR −/− mice during the light phase, which indicated a greater utilization of lipid relative to carbohydrate in these mice. Additionally, GHR −/− mice had higher ratios of caloric intake to EE at several intervals during the dark phase, and this effect was greater and more sustained in the final three hours of the dark phase. Therefore, we conclude that GHR −/− mice are able to overcome the substantial energetic challenges of dwarfism through several mechanisms that promote stable Mb. Relative to wild type mice, the GHR −/− mice consumed more calories per unit Mb, which offset the disproportionate increase in their daily energy expenditure. While GHR −/− mice oxidized a greater proportion of lipid during the light phase in order to meet their energy requirements, they achieved greater energy efficiency and storage during the dark phase through a combination of higher energy consumption and lower LMA.
doi:10.1016/j.ghir.2009.08.002
PMCID: PMC2814926  PMID: 19747867
6.  Distinct growth hormone receptor signaling modes regulate skeletal muscle development and insulin sensitivity in mice 
The Journal of Clinical Investigation  2010;120(11):4007-4020.
Skeletal muscle development, nutrient uptake, and nutrient utilization is largely coordinated by growth hormone (GH) and its downstream effectors, in particular, IGF-1. However, it is not clear which effects of GH on skeletal muscle are direct and which are secondary to GH-induced IGF-1 expression. Thus, we generated mice lacking either GH receptor (GHR) or IGF-1 receptor (IGF-1R) specifically in skeletal muscle. Both exhibited impaired skeletal muscle development characterized by reductions in myofiber number and area as well as accompanying deficiencies in functional performance. Defective skeletal muscle development, in both GHR and IGF-1R mutants, was attributable to diminished myoblast fusion and associated with compromised nuclear factor of activated T cells import and activity. Strikingly, mice lacking GHR developed metabolic features that were not observed in the IGF-1R mutants, including marked peripheral adiposity, insulin resistance, and glucose intolerance. Insulin resistance in GHR-deficient myotubes derived from reduced IR protein abundance and increased inhibitory phosphorylation of IRS-1 on Ser 1101. These results identify distinct signaling pathways through which GHR regulates skeletal muscle development and modulates nutrient metabolism.
doi:10.1172/JCI42447
PMCID: PMC2964973  PMID: 20921627
7.  Loss-of-Function Mutation in Myostatin Reduces Tumor Necrosis Factor α Production and Protects Liver Against Obesity-Induced Insulin Resistance 
Diabetes  2009;58(5):1133-1143.
OBJECTIVE
Insulin resistance develops in tandem with obesity. Ablating myostatin (Mstn) prevents obesity, so we investigated if Mstn deficiency could improve insulin sensitivity. A loss-of-function mutation (MstnLn) in either one or both alleles of the Mstn gene shows how Mstn deficiency protects whole-body insulin sensitivity.
RESEARCH DESIGN AND METHODS
MstnLn/Ln mice were weaned onto a high-fat diet (HFD) or standard diet. HFD-fed MstnLn/Ln mice exhibited high lean, low-fat body compositions compared with wild types. Wild-type and heterozygous and homozygous mutant mice were bled to determine basal levels of insulin, glucose, and homeostasis model assessment of insulin resistance. To evaluate postprandial insulin sensitivity between animals of a similar size, glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamp studies were performed with heterozygous and homozygous mutant mice. Quantitative RT-PCR quantified TNF∝, IL-6, IL-1β, F4/80, GPR43, and CD36 expression in muscle, fat, and liver. Histological analysis measured hepatosteatosis.
RESULTS
Homozygous mutants were glucose tolerant and protected against overall insulin resistance compared with heterozygous mice. Hyperinsulinemic-euglycemic clamp studies revealed a dramatically improved glucose infusion rate, glucose disposal rate, and hepatic glucose production in 11-month-old MstnLn/Ln mice on an HFD. Improvements to muscle and liver insulin sensitivity (∼200–400%) correlated with 50–75% decreased tumor necrosis factor (TNF)α production and coincided with severe Mstn deficiency. Hepatosteatosis appeared to be ameliorated. Short-term treatment of MstnLn/Ln mice with recombinant Mstn led to increased plasma TNFα and insulin resistance.
CONCLUSIONS
We find that severe Mstn deficiency caused by Ln (lean) mutations in HFD-fed mice protects muscle and liver against obesity-induced insulin resistance.
doi:10.2337/db08-0245
PMCID: PMC2671051  PMID: 19208906
8.  Age-Related and Depot-Specific Changes in White Adipose Tissue of Growth Hormone Receptor-Null Mice 
Growth hormone receptor-null (GHR−/−) mice are dwarf, insulin sensitive, and long-lived in spite of increased adiposity. However, their adiposity is not uniform, with select white adipose tissue (WAT) depots enlarged. To study WAT depot–specific effects on insulin sensitivity and life span, we analyzed individual WAT depots of 12- and 24-month-old GHR− /− and wild-type (WT) mice, as well as their plasma levels of selected hormones. Adipocyte sizes and plasma insulin, leptin, and adiponectin levels decreased with age in both GHR− /− and WT mice. Two-dimensional gel electrophoresis proteomes of WAT depots were similar among groups, but several proteins involved in endocytosis and/or cytoskeletal organization (Ehd2, S100A10, actin), anticoagulation (S100A10, annexin A5), and age-related conditions (alpha2-macroglobulin, apolipoprotein A-I, transthyretin) showed significant differences between genotypes. Because Ehd2 may regulate endocytosis of Glut4, we measured Glut4 levels in the WAT depots of GHR− /− and WT mice. Inguinal WAT of 12-month-old GHR− /− mice displayed lower levels of Glut4 than WT. Overall, the protein changes detected in this study offer new insights into possible mechanisms contributing to enhanced insulin sensitivity and extended life span in GHR− /− mice.
doi:10.1093/gerona/glt110
PMCID: PMC3859361  PMID: 23873966
Aging; Growth hormone receptor; Adipose tissue depots; Endocytosis; Glut4.
9.  Metabolic Actions of Estrogen Receptor Beta (ERβ) are Mediated by a Negative Cross-Talk with PPARγ 
PLoS Genetics  2008;4(6):e1000108.
Estrogen receptors (ER) are important regulators of metabolic diseases such as obesity and insulin resistance (IR). While ERα seems to have a protective role in such diseases, the function of ERβ is not clear. To characterize the metabolic function of ERβ, we investigated its molecular interaction with a master regulator of insulin signaling/glucose metabolism, the PPARγ, in vitro and in high-fat diet (HFD)-fed ERβ -/- mice (βERKO) mice. Our in vitro experiments showed that ERβ inhibits ligand-mediated PPARγ-transcriptional activity. That resulted in a blockade of PPARγ-induced adipocytic gene expression and in decreased adipogenesis. Overexpression of nuclear coactivators such as SRC1 and TIF2 prevented the ERβ-mediated inhibition of PPARγ activity. Consistent with the in vitro data, we observed increased PPARγ activity in gonadal fat from HFD-fed βERKO mice. In consonance with enhanced PPARγ activation, HFD-fed βERKO mice showed increased body weight gain and fat mass in the presence of improved insulin sensitivity. To directly demonstrate the role of PPARγ in HFD-fed βERKO mice, PPARγ signaling was disrupted by PPARγ antisense oligonucleotide (ASO). Blockade of adipose PPARγ by ASO reversed the phenotype of βERKO mice with an impairment of insulin sensitization and glucose tolerance. Finally, binding of SRC1 and TIF2 to the PPARγ-regulated adiponectin promoter was enhanced in gonadal fat from βERKO mice indicating that the absence of ERβ in adipose tissue results in exaggerated coactivator binding to a PPARγ target promoter. Collectively, our data provide the first evidence that ERβ-deficiency protects against diet-induced IR and glucose intolerance which involves an augmented PPARγ signaling in adipose tissue. Moreover, our data suggest that the coactivators SRC1 and TIF2 are involved in this interaction. Impairment of insulin and glucose metabolism by ERβ may have significant implications for our understanding of hormone receptor-dependent pathophysiology of metabolic diseases, and may be essential for the development of new ERβ-selective agonists.
Author Summary
In the present study, we demonstrate for the first time a pro-diabetogenic function of the ERβ. Our experiments indicate that ERβ impairs insulin sensitivity and glucose tolerance in mice challenged with a high fat diet (HFD). Loss of ERβ, studied in ERβ -/- mice (βERKO mice), results in increased body weight gain and fat deposition under HFD-treatment. Conversely, absence of ERβ averted accumulation of triglycerides and preserved regular insulin signaling in liver and skeletal muscle. This observation was associated with improved whole-body insulin sensitivity and glucose tolerance. Increased adipose tissue mass in the presence of improved insulin sensitivity and glucose tolerance is usually observed under chronic stimulation of the nuclear hormone receptor PPARγ. In consonance, we show that activation of PPARγ was markedly induced in gonadal fat from βERKO mice and blockade of adipose PPARγ signaling by antisense oligonucleotide injection reversed the metabolic phenotype. Moreover, our cell culture experiments indicate that ERβ is a negative regulator of ligand-induced PPARγ activity in vitro. Finally, we identify SRC1 and TIF2 as key players in the ERβ-PPARγ interaction. In summary, the present study demonstrates that ERβ impairs insulin and glucose metabolism, which may, at least in part, result from a negative cross-talk with adipose PPARγ.
doi:10.1371/journal.pgen.1000108
PMCID: PMC2432036  PMID: 18584035
10.  12/15-Lipoxygenase Is Required for the Early Onset of High Fat Diet-Induced Adipose Tissue Inflammation and Insulin Resistance in Mice 
PLoS ONE  2009;4(9):e7250.
Background
Recent understanding that insulin resistance is an inflammatory condition necessitates searching for genes that regulate inflammation in insulin sensitive tissues. 12/15-lipoxygenase (12/15LO) regulates the expression of proinflammatory cytokines and chemokines and is implicated in the early development of diet-induced atherosclerosis. Thus, we tested the hypothesis that 12/15LO is involved in the onset of high fat diet (HFD)-induced insulin resistance.
Methodology/Principal Findings
Cells over-expressing 12/15LO secreted two potent chemokines, MCP-1 and osteopontin, implicated in the development of insulin resistance. We assessed adipose tissue inflammation and whole body insulin resistance in wild type (WT) and 12/15LO knockout (KO) mice after 2–4 weeks on HFD. In adipose tissue from WT mice, HFD resulted in recruitment of CD11b+, F4/80+ macrophages and elevated protein levels of the inflammatory markers IL-1β, IL-6, IL-10, IL-12, IFNγ, Cxcl1 and TNFα. Remarkably, adipose tissue from HFD-fed 12/15LO KO mice was not infiltrated by macrophages and did not display any increase in the inflammatory markers compared to adipose tissue from normal chow-fed mice. WT mice developed severe whole body (hepatic and skeletal muscle) insulin resistance after HFD, as measured by hyperinsulinemic euglycemic clamp. In contrast, 12/15LO KO mice exhibited no HFD-induced change in insulin-stimulated glucose disposal rate or hepatic glucose output during clamp studies. Insulin-stimulated Akt phosphorylation in muscle tissue from HFD-fed mice was significantly greater in 12/15LO KO mice than in WT mice.
Conclusions
These results demonstrate that 12/15LO mediates early stages of adipose tissue inflammation and whole body insulin resistance induced by high fat feeding.
doi:10.1371/journal.pone.0007250
PMCID: PMC2746280  PMID: 19787041
11.  Hepatocyte-Specific Ptpn6 Deletion Protects From Obesity-Linked Hepatic Insulin Resistance 
Diabetes  2012;61(8):1949-1958.
The protein-tyrosine phosphatase Shp1 negatively regulates insulin action on glucose homeostasis in liver and muscle, but its potential role in obesity-linked insulin resistance has not been examined. To investigate the role of Shp1 in hepatic insulin resistance, we generated hepatocyte-specific Shp1 knockout mice (Ptpn6H-KO), which were subjected to extensive metabolic monitoring throughout an 8-week standard chow diet (SD) or high-fat diet (HFD) feeding. We report for the first time that Shp1 expression is upregulated in metabolic tissues of HFD-fed obese mice. When compared with their Shp1-expressing Ptpn6f/f littermates, Ptpn6H-KO mice exhibited significantly lowered fasting glycemia and heightened hepatic insulin sensitivity. After HFD feeding, Ptpn6H-KO mice developed comparable levels of obesity as Ptpn6f/f mice, but they were remarkably protected from liver insulin resistance, as revealed by euglycemic clamps and hepatic insulin signaling determinations. Although Ptpn6H-KO mice still acquired diet-induced peripheral insulin resistance, they were less hyperinsulinemic during a glucose tolerance test because of reduced insulin secretion. Ptpn6H-KO mice also exhibited increased insulin clearance in line with enhanced CC1 tyrosine phosphorylation in liver. These results show that hepatocyte Shp1 plays a critical role in the development of hepatic insulin resistance and represents a novel therapeutic target for obesity-linked diabetes.
doi:10.2337/db11-1502
PMCID: PMC3402325  PMID: 22698917
12.  Age- and Sex-Associated Plasma Proteomic Changes in Growth Hormone Receptor Gene–Disrupted Mice 
Growth hormone receptor gene–disrupted (GHR−/−) mice are dwarf, insulin sensitive, and long lived despite being obese. In order to identify characteristics associated with their increased longevity, we studied age-related plasma proteomic changes in these mice. Male and female GHR−/− mice and their littermate controls were followed longitudinally at 8, 16, and 24 months of ages for plasma proteomic analysis. Relative to control littermates, GHR−/− mice had increased levels of apolipoprotein A-4 and retinol-binding protein-4 and decreased levels of apolipoprotein E, haptoglobin, and mannose-binding protein-C. Female GHR−/− mice showed decreased inflammatory cytokines including interleukin-1β and monocyte chemotactic protein-1. Additionally, sex differences were found in specific isoforms of apolipoprotein E, RBP-4, haptoglobin, albumin, and hemoglobin subunit beta. In conclusion, we find plasma proteomic changes in GHR−/− mice that favor a longer life span as well as sex differences indicative of an improved health span in female mice.
doi:10.1093/gerona/glr212
PMCID: PMC3403865  PMID: 22156438
Growth hormone receptor; Plasma; Proteomics; Sex; Aging
13.  Liraglutide Increases FGF-21 Activity and Insulin Sensitivity in High Fat Diet and Adiponectin Knockdown Induced Insulin Resistance 
PLoS ONE  2012;7(11):e48392.
Background
Liraglutide is a glucagon-like peptide-1 analogue that stimulates insulin secretion and improves β-cell function. However, it is not clear whether liraglutide achieves its glucose lowering effect only by its known effects or whether other as yet unknown mechanisms are involved. The aim of this study was to examine the effects of liraglutide on Fibroblast growth factor-21 (FGF-21) activity in High-fat diet (HFD) fed ApoE−/− mice with adiponectin (Acrp30) knockdown.
Method
HFD-fed ApoE−/− mice were treated with adenovirus vectors expressing shAcrp30 to produce insulin resistance. Hyperinsulinemic-euglycemic clamp studies were performed to evaluate insulin sensitivity of the mouse model. QRT-PCR and Western blot were used to measure the mRNA and protein expression of the target genes.
Results
The combination of HFD, ApoE deficiency, and hypoadiponectinemia resulted in an additive effect on insulin resistance. FGF-21 mRNA expressions in both liver and adipose tissues were significantly increased while FGF-21 receptor 1 (FGFR-1) and β-Klotho mRNA levels in adipose tissue, as well as FGFR-1-3 and β-Klotho mRNA levels in liver were significantly decreased in this model. Liraglutide treatment markedly improved insulin resistance and increased FGF-21 expression in liver and FGFR-3 in adipose tissue, restored β-Klotho mRNA expression in adipose tissue as well as FGFR-1-3, β-Klotho levels and phosphorylation of FGFR1 up to the levels observed in control mice in liver. Liraglutide treatment also further increased FGF-21 proteins in liver and plasma. In addition, as shown by hyperinsulinemic-euglycemic clamp, liraglutide treatment also markedly improved glucose metabolism and insulin sensitivity in these animals.
Conclusion
These findings demonstrate an additive effect of HFD, ApoE deficiency, and adiponectin knockdown on insulin resistance and unveil that the regulation of glucose metabolism and insulin sensitivity by liraglutide may be partly mediated via increased FGF-21 and its receptors action.
doi:10.1371/journal.pone.0048392
PMCID: PMC3495944  PMID: 23152772
14.  Attenuation of Cocaine Induced Locomotor Sensitization in Rats Sustaining Genetic or Pharmacologic Antagonism of Ghrelin Receptors 
Addiction biology  2011;17(6):956-963.
Systemic infusions of the orexigenic peptide ghrelin (GHR) increase dopamine levels within the nucleus accumbens and augment cocaine stimulated locomotion and conditioned place preference in rats; observations that suggest an important role for ghrelin and GHR receptors (GHR-Rs) in drug reinforcement. In the present studies, we examined the development of cocaine locomotor sensitization in rats sustaining either pharmacologic antagonism or genetic ablation of GHR-Rs. In a pharmacologic study, adult male rats were injected (i.p.) with either 0, 3 or 6 mg/kg JMV 2959 (a GHR-R1 receptor antagonist) and 20 minutes later with either vehicle or 10 mg/kg cocaine HCl on each of 7 consecutive days. Rats pretreated with JMV 2959 showed significantly attenuated cocaine-induced hyperlocomotion. In a second study, adult wild type (WT) or mutant rats sustaining ENU-induced knockout of GHR-R (GHR-R (−/−)) received daily injections (i.p) of vehicle (0.9% saline) or 10.0 mg/kg cocaine HCl for 14 successive days. GHR-R null rats treated repeatedly with cocaine showed diminished development of cocaine locomotor sensitization relative to WT rats treated with cocaine. To verify the lack of GHR-R function in the GHR-R (−/−) rats, a separate feeding experiment was conducted in which WT rats, but not GHR-R (−/−) rats, were noted to eat more after a systemic injection of 15 nmol ghrelin than after vehicle. These results suggest that GHR-R activity is required for the induction of locomotor sensitization to cocaine and complement an emerging literature implicating central GHR systems in drug reward. Ghrelin (GHR) is an orexigenic gut peptide that is transported across the blood brain barrier and interacts with GHR receptors (GHR-R) located on ventral tegmental dopamine neurons.
doi:10.1111/j.1369-1600.2011.00339.x
PMCID: PMC3204336  PMID: 21790898
ghrelin; ghrelin receptors; drug abuse; dopamine; locomotion; sensitization; ENU mutagenesis; GHR-R receptor antagonists
15.  Serum IGF-1 is insufficient to restore skeletal size in the total absence of the growth hormone receptor 
States of growth hormone (GH) resistance, such those observed in Laron’s dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout, GHRKO) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKOHIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1 allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and from poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron’s syndrome does not achieve full skeletal growth.
doi:10.1002/jbmr.1920
PMCID: PMC3843230  PMID: 23456957
IGF-1; growth hormone receptor; bone; micro-computed tomography; betaislet; glucose tolerance
16.  High levels of dietary stearate promote adiposity and deteriorate hepatic insulin sensitivity 
Background
Relatively little is known about the role of specific saturated fatty acids in the development of high fat diet induced obesity and insulin resistance. Here, we have studied the effect of stearate in high fat diets (45% energy as fat) on whole body energy metabolism and tissue specific insulin sensitivity.
Methods
C57Bl/6 mice were fed a low stearate diet based on palm oil or one of two stearate rich diets, one diet based on lard and one diet based on palm oil supplemented with tristearin (to the stearate level of the lard based diet), for a period of 5 weeks. Ad libitum fed Oxidative metabolism was assessed by indirect calorimetry at week 5. Changes in body mass and composition was assessed by DEXA scan analysis. Tissue specific insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp analysis and Western blot at the end of week 5.
Results
Indirect calorimetry analysis revealed that high levels of dietary stearate resulted in lower caloric energy expenditure characterized by lower oxidation of fatty acids. In agreement with this metabolic phenotype, mice on the stearate rich diets gained more adipose tissue mass. Whole body and tissue specific insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp and analysis of insulin induced PKBser473 phosphorylation. Whole body insulin sensitivity was decreased by all high fat diets. However, while insulin-stimulated glucose uptake by peripheral tissues was impaired by all high fat diets, hepatic insulin sensitivity was affected only by the stearate rich diets. This tissue-specific pattern of reduced insulin sensitivity was confirmed by similar impairment in insulin-induced phosphorylation of PKBser473 in both liver and skeletal muscle.
Conclusion
In C57Bl/6 mice, 5 weeks of a high fat diet rich in stearate induces a metabolic state favoring low oxidative metabolism, increased adiposity and whole body insulin resistance characterized by severe hepatic insulin resistance. These results indicate that dietary fatty acid composition per sé rather than dietary fat content determines insulin sensitivity in liver of high fat fed C57Bl/6 mice.
doi:10.1186/1743-7075-7-24
PMCID: PMC2852377  PMID: 20346174
17.  Opposing Roles of Leptin and Ghrelin in the Equine Corpus Luteum Regulation: An In Vitro Study 
Mediators of Inflammation  2014;2014:682193.
Metabolic hormones have been associated with reproductive function modulation. Thus, the aim of this study was: (i) to characterize the immunolocalization, mRNA and protein levels of leptin (LEP), Ghrelin (GHR) and respective receptors LEPR and Ghr-R1A, throughout luteal phase; and (ii) to evaluate the role of LEP and GHR on progesterone (P4), prostaglandin (PG) E2 and PGF2α, nitric oxide (nitrite), tumor necrosis factor-α (TNF); macrophage migration inhibitory factor (MIF) secretion, and on angiogenic activity (BAEC proliferation), in equine corpus luteum (CL) from early and mid-luteal stages. LEPR expression was decreased in late CL, while GHR/Ghr-R1A system was increased in the same stage. Regarding secretory activity, GHR decreased P4 in early CL, but increased PGF2α, nitrite and TNF in mid CL. Conversely, LEP increased P4, PGE2, angiogenic activity, MIF, TNF and nitrite during early CL, in a dose-dependent manner. The in vitro effect of LEP on secretory activity was reverted by GHR, when both factors acted together. The present results evidence the presence of LEP and GHR systems in the equine CL. Moreover, we suggest that LEP and GHR play opposing roles in equine CL regulation, with LEP supporting luteal establishment and GHR promoting luteal regression. Finally, a dose-dependent luteotrophic effect of LEP was demonstrated.
doi:10.1155/2014/682193
PMCID: PMC4122068  PMID: 25125800
18.  The SOCS2 Ubiquitin Ligase Complex Regulates Growth Hormone Receptor Levels 
PLoS ONE  2011;6(9):e25358.
Growth Hormone is essential for the regulation of growth and the homeostatic control of intermediary metabolism. GH actions are mediated by the Growth Hormone Receptor; a member of the cytokine receptor super family that signals chiefly through the JAK2/STAT5 pathway. Target tissue responsiveness to GH is under regulatory control to avoid excessive and off-target effects upon GHR activation. The suppressor of cytokine signalling 2 (SOCS) is a key regulator of GHR sensitivity. This is clearly shown in mice where the SOCS2 gene has been inactivated, which show 30–40% increase in body length, a phenotype that is dependent on endogenous GH secretion. SOCS2 is a GH-stimulated, STAT5b-regulated gene that acts in a negative feedback loop to downregulate GHR signalling. Since the biochemical basis for these actions is poorly understood, we studied the molecular function of SOCS2. We demonstrated that SOCS2 is part of a multimeric complex with intrinsic ubiquitin ligase activity. Mutational analysis shows that the interaction with Elongin B/C controls SOCS2 protein turnover and affects its molecular activity. Increased GHR levels were observed in livers from SOCS2−/− mice and in the absence of SOCS2 in in vitro experiments. We showed that SOCS2 regulates cellular GHR levels through direct ubiquitination and in a proteasomally dependent manner. We also confirmed the importance of the SOCS-box for the proper function of SOCS2. Finally, we identified two phosphotyrosine residues in the GHR to be responsible for the interaction with SOCS2, but only Y487 to account for the effects of SOCS2. The demonstration that SOCS2 is an ubiquitin ligase for the GHR unveils the molecular basis for its physiological actions.
doi:10.1371/journal.pone.0025358
PMCID: PMC3183054  PMID: 21980433
19.  Grp78 Heterozygosity Promotes Adaptive Unfolded Protein Response and Attenuates Diet-Induced Obesity and Insulin Resistance 
Diabetes  2009;59(1):6-16.
OBJECTIVE
To investigate the role of the endoplasmic reticulum (ER) chaperone glucose-regulated protein (GRP) 78/BiP in the pathogenesis of obesity, insulin resistance, and type 2 diabetes.
RESEARCH DESIGN AND METHODS
Male Grp78+/− mice and their wild-type littermates were subjected to a high-fat diet (HFD) regimen. Pathogenesis of obesity and type 2 diabetes was examined by multiple approaches of metabolic phenotyping. Tissue-specific insulin sensitivity was analyzed by hyperinsulinemic-euglycemic clamps. Molecular mechanism was explored via immunoblotting and tissue culture manipulation.
RESULTS
Grp78 heterozygosity increases energy expenditure and attenuates HFD-induced obesity. Grp78+/− mice are resistant to diet-induced hyperinsulinemia, liver steatosis, white adipose tissue (WAT) inflammation, and hyperglycemia. Hyperinsulinemic-euglycemic clamp studies revealed that Grp78 heterozygosity improves glucose metabolism independent of adiposity and following an HFD increases insulin sensitivity predominantly in WAT. As mechanistic explanations, Grp78 heterozygosity in WAT under HFD stress promotes adaptive unfolded protein response (UPR), attenuates translational block, and upregulates ER degradation-enhancing α-mannosidase–like protein (EDEM) and ER chaperones, thus improving ER quality control and folding capacity. Further, overexpression of the active form of ATF6 induces protective UPR and improves insulin signaling upon ER stress.
CONCLUSIONS
HFD-induced obesity and type 2 diabetes are improved in Grp78+/− mice. Adaptive UPR in WAT could contribute to this improvement, linking ER homeostasis to energy balance and glucose metabolism.
doi:10.2337/db09-0755
PMCID: PMC2797945  PMID: 19808896
20.  Estrogen Treatment After Ovariectomy Protects Against Fatty Liver and May Improve Pathway-Selective Insulin Resistance 
Diabetes  2013;62(2):424-434.
Pathway-selective insulin resistance where insulin fails to suppress hepatic glucose production but promotes liver fat storage may underlie glucose and lipid abnormalities after menopause. We tested the mechanisms by which estrogen treatment may alter the impact of a high-fat diet (HFD) when given at the time of ovariectomy (OVX) in mice. Female C57BL/6J mice underwent sham operation, OVX, or OVX with estradiol (E2) treatment and were fed an HFD. Hyperinsulinemic-euglycemic clamps were used to assess insulin sensitivity, tracer incorporation into hepatic lipids, and liver triglyceride export. OVX mice had increased adiposity that was prevented with E2 at the time of OVX. E2 treatment increased insulin sensitivity with OVX and HFD. In sham and OVX mice, HFD feeding induced fatty liver, and insulin reduced hepatic apoB100 and liver triglyceride export. E2 treatment reduced liver lipid deposition and prevented the decrease in liver triglyceride export during hyperinsulinemia. In mice lacking the liver estrogen receptor α, E2 after OVX limited adiposity but failed to improve insulin sensitivity, to limit liver lipid deposition, and to prevent insulin suppression of liver triglyceride export. In conclusion, estrogen treatment may reverse aspects of pathway-selective insulin resistance by promoting insulin action on glucose metabolism but limiting hepatic lipid deposition.
doi:10.2337/db11-1718
PMCID: PMC3554377  PMID: 22966069
21.  Nature of fatty acids in high fat diets differentially delineates obesity-linked metabolic syndrome components in male and female C57BL/6J mice 
Background
Adverse effects of high-fat diets (HFD) on metabolic homeostasis are linked to adipose tissue dysfunction. The goal of this study was to examine the effect of the HFD nature on adipose tissue activity, metabolic disturbances and glucose homeostasis alterations in male mice compared with female mice.
Methods
C57BL/6J mice were fed either a chow diet or HFD including vegetal (VD) or animal (AD) fat. Body weight, plasmatic parameters and adipose tissue mRNA expression levels of key genes were evaluated after 20 weeks of HFD feeding.
Results
HFD-fed mice were significantly heavier than control at the end of the protocol. Greater abdominal visceral fat accumulation was observed in mice fed with AD compared to those fed a chow diet or VD. Correlated with weight gain, leptin levels in systemic circulation were increased in HFD-fed mice in both sexes with a significant higher level in AD group compared to VD group. Circulating adiponectin levels as well as adipose tissue mRNA expression levels were significantly decreased in HFD-fed male mice. Although its plasma levels remained unchanged in females, adiponectin mRNA levels were significantly reduced in adipose tissue of both HFD-fed groups with a more marked decrease in AD group compared to VD group. Only HFD-fed male mice were diabetic with increased fasting glycaemia. On the other hand, insulin levels were only increased in AD-fed group in both sexes associated with increased resistin levels. VD did not induce any apparent metabolic alteration in females despite the increased weight gain. Peroxisome Proliferator-Activated Receptors gamma-2 (PPARγ2) and estrogen receptor alpha (ERα) mRNA expression levels in adipose tissue were decreased up to 70% in HFD-fed mice but were more markedly reduced in male mice as compared with female mice.
Conclusions
The nature of dietary fat determines the extent of metabolic alterations reflected in adipocytes through modifications in the pattern of adipokines secretion and modulation of key genes mRNA expression. Compared with males, female mice demonstrate higher capacity in controlling glucose homeostasis in response to 20 weeks HFD feeding. Our data suggest gender specific interactions between the diet's fatty acid source, the adipocyte-secreted proteins and metabolic disorders.
doi:10.1186/1758-5996-3-34
PMCID: PMC3277487  PMID: 22166251
Adipocyte; Adipokines; Diabetes; High Fat Diet; Metabolic disorders; Obesity; Type 2 Diabetes
22.  Enhanced Lipid Oxidation and Maintenance of Muscle Insulin Sensitivity Despite Glucose Intolerance in a Diet-Induced Obesity Mouse Model 
PLoS ONE  2013;8(8):e71747.
Background
Diet-induced obesity is a rising health concern which can lead to the development of glucose intolerance and muscle insulin resistance and, ultimately, type II diabetes mellitus. This research investigates the associations between glucose intolerance or muscle insulin resistance and tissue specific changes during the progression of diet-induced obesity.
Methodology
C57BL/6J mice were fed a normal or high-fat diet (HFD; 60% kcal fat) for 3 or 8 weeks. Disease progression was monitored by measurements of body/tissue mass changes, glucose and insulin tolerance tests, and ex vivo glucose uptake in intact muscles. Lipid metabolism was analyzed using metabolic chambers and ex vivo palmitate assays in intact muscles. Skeletal muscle, liver and adipose tissues were analyzed for changes in inflammatory gene expression. Plasma was analyzed for insulin levels and inflammatory proteins. Histological techniques were used on muscle and liver cryosections to assess metabolic and morphological changes.
Principal Findings/Conclusions
A rapid shift in whole body metabolism towards lipids was observed with HFD. Following 3 weeks of HFD, elevated total lipid oxidation and an oxidative fiber type shift had occurred in the skeletal muscle, which we propose was responsible for delaying intramyocellular lipid accumulation and maintaining muscle’s insulin sensitivity. Glucose intolerance was present after three weeks of HFD and was associated with an enlarged adipose tissue depot, adipose tissue inflammation and excess hepatic lipids, but not hepatic inflammation. Furthermore, HFD did not significantly increase systemic or muscle inflammation after 3 or 8 weeks of HFD suggesting that early diet-induced obesity does not cause inflammation throughout the whole body. Overall these findings indicate skeletal muscle did not contribute to the development of HFD-induced impairments in whole-body glucose tolerance following 3 weeks of HFD.
doi:10.1371/journal.pone.0071747
PMCID: PMC3741110  PMID: 23951235
23.  Specific suppression of insulin sensitivity in growth hormone receptor gene-disrupted (GHR-KO) mice attenuates phenotypic features of slow aging 
Aging Cell  2014;13(6):981-1000.
In addition to their extended lifespans, slow-aging growth hormone receptor/binding protein gene-disrupted (knockout) (GHR-KO) mice are hypoinsulinemic and highly sensitive to the action of insulin. It has been proposed that this insulin sensitivity is important for their longevity and increased healthspan. We tested whether this insulin sensitivity of the GHR-KO mouse is necessary for its retarded aging by abrogating that sensitivity with a transgenic alteration that improves development and secretory function of pancreatic β-cells by expressing Igf-1 under the rat insulin promoter 1 (RIP::IGF-1). The RIP::IGF-1 transgene increased circulating insulin content in GHR-KO mice, and thusly fully normalized their insulin sensitivity, without affecting the proliferation of any non-β-cell cell types. Multiple (nonsurvivorship) longevity-associated physiological and endocrinological characteristics of these mice (namely beneficial blood glucose regulatory control, altered metabolism, and preservation of memory capabilities) were partially or completely normalized, thus supporting the causal role of insulin sensitivity for the decelerated senescence of GHR-KO mice. We conclude that a delayed onset and/or decreased pace of aging can be hormonally regulated.
doi:10.1111/acel.12262
PMCID: PMC4326932  PMID: 25244225
endocrinology and metabolism; growth hormone hormonal signaling; insulin sensitivity; longevity regulation; (neuro)endocrinology of senescence
24.  NOD1 Activators Link Innate Immunity to Insulin Resistance 
Diabetes  2011;60(9):2206-2215.
OBJECTIVE
Insulin resistance associates with chronic inflammation, and participatory elements of the immune system are emerging. We hypothesized that bacterial elements acting on distinct intracellular pattern recognition receptors of the innate immune system, such as bacterial peptidoglycan (PGN) acting on nucleotide oligomerization domain (NOD) proteins, contribute to insulin resistance.
RESEARCH DESIGN AND METHODS
Metabolic and inflammatory properties were assessed in wild-type (WT) and NOD1/2−/− double knockout mice fed a high-fat diet (HFD) for 16 weeks. Insulin resistance was measured by hyperinsulinemic euglycemic clamps in mice injected with mimetics of meso-diaminopimelic acid–containing PGN or the minimal bioactive PGN motif, which activate NOD1 and NOD2, respectively. Systemic and tissue-specific inflammation was assessed using enzyme-linked immunosorbent assays in NOD ligand–injected mice. Cytokine secretion, glucose uptake, and insulin signaling were assessed in adipocytes and primary hepatocytes exposed to NOD ligands in vitro.
RESULTS
NOD1/2−/− mice were protected from HFD-induced inflammation, lipid accumulation, and peripheral insulin intolerance. Conversely, direct activation of NOD1 protein caused insulin resistance. NOD1 ligands induced peripheral and hepatic insulin resistance within 6 h in WT, but not NOD1−/−, mice. NOD2 ligands only modestly reduced peripheral glucose disposal. NOD1 ligand elicited minor changes in circulating proinflammatory mediators, yet caused adipose tissue inflammation and insulin resistance of muscle AS160 and liver FOXO1. Ex vivo, NOD1 ligand caused proinflammatory cytokine secretion and impaired insulin-stimulated glucose uptake directly in adipocytes. NOD1 ligand also caused inflammation and insulin resistance directly in primary hepatocytes from WT, but not NOD1−/−, mice.
CONCLUSIONS
We identify NOD proteins as innate immune components that are involved in diet-induced inflammation and insulin intolerance. Acute activation of NOD proteins by mimetics of bacterial PGNs causes whole-body insulin resistance, bolstering the concept that innate immune responses to distinctive bacterial cues directly lead to insulin resistance. Hence, NOD1 is a plausible, new link between innate immunity and metabolism.
doi:10.2337/db11-0004
PMCID: PMC3161332  PMID: 21715553
25.  Long term effects of high fat or high carbohydrate diets on glucose tolerance in mice with heterozygous carnitine palmitoyltransferase-1a (CPT-1a) deficiency 
Nutrition & diabetes  2011;1:e14.
Background
Abnormal fatty acid metabolism is an important feature in the mechanisms of insulin resistance and beta-cell dysfunction. Carnitine palmitoyltransferase-1a (CPT-1a, liver isoform) plays a pivotal role in the regulation of mitochondrial fatty acid oxidation. We investigated the role of CPT-1a in the development of impaired glucose tolerance using a mouse model for CPT-1a deficiency when challenged by either a high-carbohydrate (HCD) or a high-fat diet (HFD) for a total duration of up to 46 weeks.
Methods
Insulin sensitivity and glucose tolerance were assessed in heterozygous CPT-1a deficient (CPT-1a+/−) male mice after being fed either a HCD or a HFD for durations of 28 weeks and 46 weeks. Both glucose and insulin tolerance tests were used to investigate beta-cell function and insulin sensitivity. Differences in islet insulin content and hepatic steatosis were evaluated by morphological analysis.
Results
CPT-1a+/− mice were more insulin sensitive than CPT-1a+/+ mice when fed either HCD or HFD. The increased insulin sensitivity was associated with an increased expression of Cpt-1b (muscle isoform) in liver, as well as increased microvesicular hepatic steatosis compared to CPT-1a+/+ mice. CPT-1a+/− mice were more glucose tolerant than CPT-1a+/+ mice when fed the HCD, but there was no significant difference when fed HFD. Moreover, CPT-1a+/− mice fed HFD or HCD had fewer and smaller pancreatic islets than CPT-1a+/+ mice.
Conclusions
CPT-1a deficiency preserved insulin sensitivity when challenged by long term feeding of either diet. Furthermore, CPT-1a deficient mice had distinct phenotypes dependent on the diet fed demonstrating that both diet and genetics collectively play a role in the development of impaired glucose tolerance.
doi:10.1038/nutd.2011.11
PMCID: PMC3251273  PMID: 22229081
carnitine palmitoyltransterase-1a deficiency; mouse model; glucose tolerance; long term diet challenge; high carbohydrate diet; high fat diet; fatty acid oxidation disorders

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