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1.  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
2.  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
3.  Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets 
Diabetes  2009;58(11):2607-2615.
OBJECTIVE
The G-protein–coupled receptor GPR40 mediates fatty acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets.
RESEARCH DESIGN AND METHODS
Insulin secretion and sensitivity were assessed in GPR40 knockout mice and their wild-type littermates by hyperglycemic clamps and hyperinsulinemic euglycemic clamps, respectively. Transcriptomic analysis, metabolic studies, and lipid profiling were used to ascertain whether GPR40 modulates intracellular fuel metabolism in islets.
RESULTS
Both glucose- and arginine-stimulated insulin secretion in vivo were decreased by ∼60% in GPR40 knockout fasted and fed mice, without changes in insulin sensitivity. Neither gene expression profiles nor intracellular metabolism of glucose and palmitate in isolated islets were affected by GPR40 deletion. Lipid profiling of isolated islets revealed that the increase in triglyceride and decrease in lyso-phosphatidylethanolamine species in response to palmitate in vitro was similar in wild-type and knockout islets. In contrast, the increase in intracellular inositol phosphate levels observed in wild-type islets in response to fatty acids in vitro was absent in knockout islets.
CONCLUSIONS
These results indicate that deletion of GPR40 impairs insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism in islets, via a mechanism that may involve the generation of inositol phosphates downstream of GPR40 activation.
doi:10.2337/db09-0362
PMCID: PMC2768167  PMID: 19720802
4.  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
5.  Pharmacologic Antagonism of Ghrelin Receptors Attenuates Development of Nicotine Induced Locomotor Sensitization in Rats 
Regulatory peptides  2011;172(1-3):77-80.
Aims
Ghrelin (GHR) is an orexigenic gut peptide that interacts with ghrelin receptors (GHR-Rs) to modulate brain reinforcement circuits. Systemic GHR infusions augment cocaine stimulated locomotion and conditioned place preference (CPP) in rats, whereas genetic or pharmacological ablation of GHR-Rs has been shown to attenuate the acute locomotor-enhancing effects of nicotine, cocaine, amphetamine and alcohol and to blunt the CPP induced by food, alcohol, amphetamine and cocaine in mice. The stimulant nicotine can induce CPP and like amphetamine and cocaine, repeated administration of nicotine induces locomotor sensitization in rats. A key issue is whether pharmacological antagonism of GHR-Rs would similarly attenuate nicotine-induced locomotor sensitization.
Method
To examine the role of GHR-Rs in the behavioral sensitizing effects of nicotine, adult male rats were injected with either 0, 3 or 6 mg/kg of the GHR-R receptor antagonist JMV 2959 (i.p.) and 20 minutes later with either vehicle or 0.4 mg/kg nicotine hydrogen tartrate (s.c.) on each of 7 consecutive days.
Results
Rats treated with nicotine alone showed robust locomotor sensitization, whereas rats pretreated with JMV 2959 showed significantly attenuated nicotine-induced hyperlocomotion.
Conclusions
These results suggest that GHR-R activity is required for the induction of locomotor sensitization to nicotine and complement an emerging literature implicating central GHR systems in drug reward/reinforcement.
doi:10.1016/j.regpep.2011.08.014
PMCID: PMC3197247  PMID: 21903141
ghrelin; ghrelin receptors; JMV 2959; locomotion; sensitization
6.  Quantifying Insulin Sensitivity and Entero-Insular Responsiveness to Hyper- and Hypoglycemia in Ferrets 
PLoS ONE  2014;9(3):e90519.
Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5–6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes.
doi:10.1371/journal.pone.0090519
PMCID: PMC3940889  PMID: 24594704
7.  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
8.  Brain PPARγ Promotes Obesity and is Required for the Insulin–Sensitizing Effect of Thiazolidinediones 
Nature Medicine  2011;17(5):618-622.
In adipose, muscle, liver and macrophages, signaling by the nuclear receptor PPARγ is a determinant of insulin sensitivity and this receptor mediates the insulin–sensitizing effects of thioazolidinediones (TZDs)1-4. Since PPARγ is also expressed in neurons5, we generated mice with neuron–specific Pparγ knockout (Pparγ BKO) to determine whether neuronal PPARγ signaling contributes to either weight gain or insulin resistance. During high fat diet (HFD) feeding, food intake was reduced and energy expenditure increased in Pparγ BKO mice, resulting in reduced weight gain. When treated with the TZD rosiglitazone, Pparγ BKO mice were resistant to rosiglitazone–induced hyperphagia and weight gain and, relative to rosiglitazone–treated controls, experienced only a marginal improvement in glucose metabolism. Hyperinsulinemic euglycemic clamp studies showed that the effect of rosiglitazone treatment to increase hepatic insulin sensitivity during HFD feeding was completely abolished in Pparγ BKO mice, an effect associated with the failure of rosiglitazone to improve liver insulin receptor signal transduction. We conclude that excess weight gain induced by HFD feeding depends in part on the effect of neuronal PPARγ signaling to limit thermogenesis and increase food intake. Neuronal PPARγ signaling is also required for the hepatic insulin sensitizing effects of TZDs.
doi:10.1038/nm.2332
PMCID: PMC3380629  PMID: 21532596
PPARγ; Central Nervous System; rosiglitazone; Insulin Sensitivity; Hyperphagia
9.  Liraglutide Prevents Hypoadiponectinemia-Induced Insulin Resistance and Alterations of Gene Expression Involved in Glucose and Lipid Metabolism 
Molecular Medicine  2011;17(11-12):1168-1178.
Liraglutide is a glucagonlike peptide (GLP)-1 analog that reduces blood glucose levels, increases insulin secretion and improves insulin sensitivity through mechanisms that are not completely understood. Therefore, we aimed to evaluate the metabolic impact and underlying mechanisms of liraglutide in a hypoadiponectinemia and high-fat diet (HFD)-induced insulin resistance (IR) model. Adiponectin gene targeting was achieved using adenovirus-transduced RNAi and was used to lower plasma adiponectin levels. Liraglutide (1 mg/kg) was given twice daily for 8 wks to HFD-fed apolipoprotein (Apo)E−/− mice. Insulin sensitivity was examined by a hyperinsulinemic-euglycemic clamp. Gene mRNA and protein expressions were measured by quantitative real-time polymerase chain reaction (PCR) and Western blot, respectively. Administration of liraglutide prevented hypoadiponectinemia-induced increases in plasma insulin, free fatty acids, triglycerides and total cholesterol. Liraglutide also attenuated hypoadiponectinemia-induced deterioration in peripheral and hepatic insulin sensitivity and alterations in key regulatory factors implicated in glucose and lipid metabolism. These findings demonstrated for the first time that liraglutide could be used to rescue IR induced by hypoadiponectinemia and HFD via regulating gene and protein expression involved in glucose and lipid metabolism.
doi:10.2119/molmed.2011.00051
PMCID: PMC3321815  PMID: 21785811
10.  Nesfatin-1 Action in the Brain Increases Insulin Sensitivity Through Akt/AMPK/TORC2 Pathway in Diet-Induced Insulin Resistance 
Diabetes  2012;61(8):1959-1968.
Nesfatin-1, derived from nucleobindin 2, was recently identified as an anorexigenic signal peptide. However, its neural role in glucose homeostasis and insulin sensitivity is unknown. To evaluate the metabolic impact and underlying mechanisms of central nesfatin-1 signaling, we infused nesfatin-1 in the third cerebral ventricle of high-fat diet (HFD)–fed rats. The effects of central nesfatin-1 on glucose metabolism and changes in transcription factors and signaling pathways were assessed during euglycemic-hyperinsulinemic clamping. The infusion of nesfatin-1 into the third cerebral ventricle markedly inhibited hepatic glucose production (HGP), promoted muscle glucose uptake, and was accompanied by decreases in hepatic mRNA and protein expression and enzymatic activity of PEPCK in both standard diet- and HFD-fed rats. In addition, central nesfatin-1 increased insulin receptor (InsR)/insulin receptor substrate-1 (IRS-1)/AMP-dependent protein kinase (AMPK)/Akt kinase (Akt)/target of rapamycin complex (TORC) 2 phosphorylation and resulted in an increase in Fos immunoreactivity in the hypothalamic nuclei that mediate glucose homeostasis. Taken together, these results reveal what we believe to be a novel site of action of nesfatin-1 on HGP and the PEPCK/InsR/IRS-1/AMPK/Akt/TORC2 pathway and suggest that hypothalamic nesfatin-1 action through a neural-mediated pathway can contribute to increased peripheral and hepatic insulin sensitivity by decreasing gluconeogenesis and promoting peripheral glucose uptake in vivo.
doi:10.2337/db11-1755
PMCID: PMC3402309  PMID: 22688332
11.  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
12.  Interleukin-10 Prevents Diet-Induced Insulin Resistance by Attenuating Macrophage and Cytokine Response in Skeletal Muscle 
Diabetes  2009;58(11):2525-2535.
OBJECTIVE
Insulin resistance is a major characteristic of type 2 diabetes and is causally associated with obesity. Inflammation plays an important role in obesity-associated insulin resistance, but the underlying mechanism remains unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine with lower circulating levels in obese subjects, and acute treatment with IL-10 prevents lipid-induced insulin resistance. We examined the role of IL-10 in glucose homeostasis using transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10).
RESEARCH DESIGN AND METHODS
MCK-IL10 and wild-type mice were fed a high-fat diet (HFD) for 3 weeks, and insulin sensitivity was determined using hyperinsulinemic-euglycemic clamps in conscious mice. Biochemical and molecular analyses were performed in muscle to assess glucose metabolism, insulin signaling, and inflammatory responses.
RESULTS
MCK-IL10 mice developed with no obvious anomaly and showed increased whole-body insulin sensitivity. After 3 weeks of HFD, MCK-IL10 mice developed comparable obesity to wild-type littermates but remained insulin sensitive in skeletal muscle. This was mostly due to significant increases in glucose metabolism, insulin receptor substrate-1, and Akt activity in muscle. HFD increased macrophage-specific CD68 and F4/80 levels in wild-type muscle that was associated with marked increases in tumor necrosis factor-α, IL-6, and C-C motif chemokine receptor-2 levels. In contrast, MCK-IL10 mice were protected from diet-induced inflammatory response in muscle.
CONCLUSIONS
These results demonstrate that IL-10 increases insulin sensitivity and protects skeletal muscle from obesity-associated macrophage infiltration, increases in inflammatory cytokines, and their deleterious effects on insulin signaling and glucose metabolism. Our findings provide novel insights into the role of anti-inflammatory cytokine in the treatment of type 2 diabetes.
doi:10.2337/db08-1261
PMCID: PMC2768157  PMID: 19690064
13.  Glucose Metabolism In Vivo in Four Commonly Used Inbred Mouse Strains 
Diabetes  2008;57(7):1790-1799.
OBJECTIVE—To characterize differences in whole-body glucose metabolism between commonly used inbred mouse strains.
RESEARCH DESIGN AND METHODS—Hyperinsulinemic-euglycemic (∼8.5 mmol/l) and -hypoglycemic (∼3.0 mmol/l) clamps were done in catheterized, 5-h-fasted mice to assess insulin action and hypoglycemic counter-regulatory responsiveness. Hyperglycemic clamps (∼15 mmol/l) were done to assess insulin secretion and compared with results in perifused islets.
RESULTS—Insulin action and hypoglycemic counter-regulatory and insulin secretory phenotypes varied considerably in four inbred mouse strains. In vivo insulin secretion was greatest in 129X1/Sv mice, but the counter-regulatory response to hypoglycemia was blunted. FVB/N mice in vivo showed no increase in glucose-stimulated insulin secretion, relative hepatic insulin resistance, and the highest counter-regulatory response to hypoglycemia. In DBA/2 mice, insulin action was lowest among the strains, and islets isolated had the greatest glucose-stimulated insulin secretion in vitro. In C57BL/6 mice, in vivo physiological responses to hyperinsulinemia at euglycemia and hypoglycemia were intermediate relative to other strains. Insulin secretion by C57BL/6 mice was similar to that in other strains in contrast to the blunted glucose-stimulated insulin secretion from isolated islets.
CONCLUSIONS—Strain-dependent differences exist in four inbred mouse strains frequently used for genetic manipulation and study of glucose metabolism. These results are important for selecting inbred mice to study glucose metabolism and for interpreting and designing experiments.
doi:10.2337/db07-1615
PMCID: PMC2453626  PMID: 18398139
14.  SGLT2 Deletion Improves Glucose Homeostasis and Preserves Pancreatic β-Cell Function 
Diabetes  2011;60(3):890-898.
OBJECTIVE
Inhibition of the Na+-glucose cotransporter type 2 (SGLT2) is currently being pursued as an insulin-independent treatment for diabetes; however, the behavioral and metabolic consequences of SGLT2 deletion are unknown. Here, we used a SGLT2 knockout mouse to investigate the effect of increased renal glucose excretion on glucose homeostasis, insulin sensitivity, and pancreatic β-cell function.
RESEARCH DESIGN AND METHODS
SGLT2 knockout mice were fed regular chow or a high-fat diet (HFD) for 4 weeks, or backcrossed onto the db/db background. The analysis used metabolic cages, glucose tolerance tests, euglycemic and hyperglycemic clamps, as well as isolated islet and perifusion studies.
RESULTS
SGLT2 deletion resulted in a threefold increase in urine output and a 500-fold increase in glucosuria, as well as compensatory increases in feeding, drinking, and activity. SGLT2 knockout mice were protected from HFD-induced hyperglycemia and glucose intolerance and had reduced plasma insulin concentrations compared with controls. On the db/db background, SGLT2 deletion prevented fasting hyperglycemia, and plasma insulin levels were also dramatically improved. Strikingly, prevention of hyperglycemia by SGLT2 knockout in db/db mice preserved pancreatic β-cell function in vivo, which was associated with a 60% increase in β-cell mass and reduced incidence of β-cell death.
CONCLUSIONS
Prevention of renal glucose reabsorption by SGLT2 deletion reduced HFD- and obesity-associated hyperglycemia, improved glucose intolerance, and increased glucose-stimulated insulin secretion in vivo. Taken together, these data support SGLT2 inhibition as a viable insulin-independent treatment of type 2 diabetes.
doi:10.2337/db10-1328
PMCID: PMC3046850  PMID: 21357472
15.  Impaired Insulin Secretion and Enhanced Insulin Sensitivity in Cholecystokinin-Deficient Mice 
Diabetes  2011;60(7):2000-2007.
OBJECTIVE
Cholecystokinin (CCK) is released in response to lipid intake and stimulates insulin secretion. We hypothesized that CCK deficiency would alter the regulation of insulin secretion and glucose homeostasis.
RESEARCH DESIGN AND METHODS
We used quantitative magnetic resonance imaging to determine body composition and studied plasma glucose and insulin secretion of CCK gene knockout (CCK-KO) mice and their wild-type controls using intraperitoneal glucose and arginine infusions. The area of anti-insulin staining in pancreatic islets was measured by immunohistochemistry. Insulin sensitivity was assessed with euglycemic-hyperinsulemic clamps.
RESULTS
CCK-KO mice fed a low-fat diet had a reduced acute insulin response to glucose but a normal response to arginine and normal glucose tolerance, associated with a trend toward greater insulin sensitivity. However, when fed a high-fat diet (HFD) for 10 weeks, CCK-KO mice developed glucose intolerance despite increased insulin sensitivity that was associated with low insulin secretion in response to both glucose and arginine. The deficiency of insulin secretion in CCK-KO mice was not associated with changes in β-cell or islet size.
CONCLUSIONS
CCK is involved in regulating insulin secretion and glucose tolerance in mice eating an HFD. The impaired insulin response to intraperitoneal stimuli that do not typically elicit CCK release suggests that this hormone has chronic effects on β-cell adaptation to diet in addition to acute incretin actions.
doi:10.2337/db10-0789
PMCID: PMC3121422  PMID: 21602512
16.  Overexpression of the Adiponectin Receptor AdipoR1 in Rat Skeletal Muscle Amplifies Local Insulin Sensitivity 
Endocrinology  2012;153(11):5231-5246.
Adiponectin is an adipokine whose plasma levels are inversely related to degrees of insulin resistance (IR) or obesity. It enhances glucose disposal and mitochondrial substrate oxidation in skeletal muscle and its actions are mediated through binding to receptors, especially adiponectin receptor 1 (AdipoR1). However, the in vivo significance of adiponectin sensitivity and the molecular mechanisms of muscle insulin sensitization by adiponectin have not been fully established. We used in vivo electrotransfer to overexpress AdipoR1 in single muscles of rats, some of which were fed for 6 wk with chow or high-fat diet (HFD) and then subjected to hyperinsulinemic-euglycemic clamp. After 1 wk, the effects on glucose disposal, signaling, and sphingolipid metabolism were investigated in test vs. contralateral control muscles. AdipoR1 overexpression (OE) increased glucose uptake and glycogen accumulation in the basal and insulin-treated rat muscle and also in the HFD-fed rats, locally ameliorating muscle IR. These effects were associated with increased phosphorylation of insulin receptor substrate-1, Akt, and glycogen synthase kinase-3β. AdipoR1 OE also caused increased phosphorylation of p70S6 kinase, AMP-activated protein kinase, and acetyl-coA carboxylase as well as increased protein levels of adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif-1 and adiponectin, peroxisome proliferator activated receptor-γ coactivator-1α, and uncoupling protein-3, indicative of increased mitochondrial biogenesis. Although neither HFD feeding nor AdipoR1 OE caused generalized changes in sphingolipids, AdipoR1 OE did reduce levels of sphingosine 1-phosphate, ceramide 18:1, ceramide 20:2, and dihydroceramide 20:0, plus mRNA levels of the ceramide synthetic enzymes serine palmitoyl transferase and sphingolipid Δ-4 desaturase, changes that are associated with increased insulin sensitivity. These data demonstrate that enhancement of local adiponectin sensitivity is sufficient to improve skeletal muscle IR.
doi:10.1210/en.2012-1368
PMCID: PMC3498583  PMID: 22989629
17.  The Growth Hormone Receptor Gene-Disrupted (GHR-KO) Mouse Fails to Respond to an Intermittent Fasting (IF) Diet 
Aging cell  2009;8(6):756-760.
SUMMARY
The interaction of longevity-conferring genes with longevity-conferring diets is poorly understood. The growth hormone receptor gene-disrupted (GHR-KO) mouse is long-lived; and this longevity is not responsive to 30% caloric restriction (CR), in contrast to wild-type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction (DR), we subjected GHR-KO mice to a different dietary restriction regimen, an intermittent fasting (IF) diet.
The IF diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR-KO mice.
From the results of two paradigms of dietary restriction we postulate that GHR-KO mice would be resistant to any manner of DR; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan-extending potential of an intervention.
doi:10.1111/j.1474-9726.2009.00520.x
PMCID: PMC2783987  PMID: 19747233
aging; longevity; caloric restriction; intermittent fasting; growth hormone; insulin sensitivity
18.  Hepatic Cannabinoid Receptor-1 Mediates Diet-Induced Insulin Resistance via Inhibition of Insulin Signaling and Clearance in Mice 
Gastroenterology  2012;142(5):1218-1228.e1.
BACKGROUND & AIMS
Obesity-related insulin resistance contributes to cardiovascular disease. Cannabinoid receptor-1 (CB1) blockade improves insulin sensitivity in obese animals and people, suggesting endocannabinoid involvement. We explored the role of hepatic CB1 in insulin resistance and inhibition of insulin signaling pathways.
METHODS
Wild-type mice and mice with disruption of CB1 (CB1−/− mice) or with hepatocyte-specific deletion or transgenic overexpression of CB1 were maintained on regular chow or a high-fat diet (HFD) to induce obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp analysis was used to analyze the role of the liver and hepatic CB1 in HFD-induced insulin resistance. The cellular mechanisms of insulin resistance were analyzed in mouse and human isolated hepatocytes using small interfering or short hairpin RNAs and lentiviral knockdown of gene expression.
RESULTS
The HFD induced hepatic insulin resistance in wild-type mice, but not in CB1−/− mice or mice with hepatocyte-specific deletion of CB1. CB1−/− mice that overexpressed CB1 specifically in hepatocytes became hyperinsulinemic as a result of reduced insulin clearance due to down-regulation of the insulin-degrading enzyme. However, they had increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance; this was further increased by the HFD. In mice with hepatocytes that express CB1, the HFD or CB1 activation induced the endoplasmic reticulum stress response via activation of the Bip-PERK-eIF2α protein translation pathway. In hepatocytes isolated from human or mouse liver, CB1 activation caused endoplasmic reticulum stress-dependent suppression of insulin-induced phosphorylation of akt-2 via phosphorylation of IRS1 at serine-307 and by inducing the expression of the serine and threonine phosphatase Phlpp1. Expression of CB1 was up-regulated in samples from patients with nonalcoholic fatty liver disease.
CONCLUSIONS
Endocannabinoids contribute to diet-induced insulin resistance in mice via hepatic CB1-mediated inhibition of insulin signaling and clearance.
doi:10.1053/j.gastro.2012.01.032
PMCID: PMC3482511  PMID: 22307032
NASH; Signal Transduction; Mouse Model; Liver Disease
19.  Hyperinsulinemia in African-American Adolescents Compared With Their American White Peers Despite Similar Insulin Sensitivity 
Diabetes Care  2008;31(7):1445-1447.
OBJECTIVE—African-American (AA) children are hyperinsulinemic and insulin resistant compared with American white (AW) children. Previously, we demonstrated that insulin secretion relative to insulin sensitivity was ∼75% higher in AA compared with AW children, suggesting that hyperinsulinemia in AA children is not merely a compensatory response to lower insulin sensitivity. The aim of the present investigation was to assess whether glucose-stimulated insulin response is higher in AA versus AW adolescents who have comparable in vivo insulin sensitivity.
RESEARCH DESIGN AND METHODS—The hyperinsulinemic-euglycemic and hyperglycemic clamp techniques were utilized to assess first- and second-phase insulin secretion. Insulin secretion relative to insulin sensitivity was calculated as the glucose disposition index.
RESULTS—AA adolescents compared with their AW peers with comparable insulin sensitivity and body composition had higher first-phase insulin concentrations.
CONCLUSIONS—The quantitative relationship between insulin sensitivity and first-phase insulin appears to differ among AA and AW adolescents.
doi:10.2337/dc08-0116
PMCID: PMC2453672  PMID: 18417751
20.  A Novel EPO Receptor Agonist Improves Glucose Tolerance via Glucose Uptake in Skeletal Muscle in a Mouse Model of Diabetes 
Experimental Diabetes Research  2011;2011:910159.
Patients treated with recombinant human Epo demonstrate an improvement in insulin sensitivity. We aimed to investigate whether CNTO 530, a novel Epo receptor agonist, could affect glucose tolerance and insulin sensitivity. A single administration of CNTO 530 significantly and dose-dependently reduced the area under the curve in a glucose tolerance test in diet-induced obese and diabetic mice after 14, 21, and 28 days. HOMA analysis suggested an improvement in insulin sensitivity, and this effect was confirmed by a hyperinsulinemic-euglycemic clamp. Uptake of 14C-2-deoxy-D-glucose indicated that animals dosed with CNTO 530 transported more glucose into skeletal muscle and heart relative to control animals. In conclusion, CNTO530 has a profound effect on glucose tolerance in insulin-resistant rodents likely because of improving peripheral insulin sensitivity. This effect was observed with epoetin-α and darbepoetin-α, suggesting this is a class effect, but the effect with these compounds relative to CNTO530 was decreased in duration and magnitude.
doi:10.1155/2011/910159
PMCID: PMC3132469  PMID: 21754921
21.  Upregulation of the Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Receptor Axis in the Heart and the Kidney of Growth Hormone Receptor knock-out Mice 
Objective
Growth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR−/−) mouse.
Design
RAS components were evaluated in the heart and the kidney of GHR−/− and control mice by immunohistochemistry and western blotting (n=12 for both groups).
Results
The immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR−/− mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR−/− mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western Blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues.
Conclusions
The shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR−/− mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.
doi:10.1016/j.ghir.2012.08.003
PMCID: PMC3698955  PMID: 22947377
Angiotensin-(1-7); AT1 receptor; Mas receptor; Growth hormone; Renin-angiotensin system
22.  Advantages of dynamic “closed loop” stable isotope flux phenotyping over static “open loop” clamps in detecting silent genetic and dietary phenotypes 
Metabolomics  2009;6(2):180-190.
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 PPARα−/− 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 PPARα−/− 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 PPARα−/− versus SV129J-wt mice. In contrast, the dynamic stable isotope labeled glucose tolerance tests reveal enhanced glucose disposal for PPARα−/− versus SV129J-wt, for chow and high fat diets. Area under the curve for plasma labeled and unlabeled glucose for PPARα−/− was ≈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 PPARα−/−, 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.
doi:10.1007/s11306-009-0190-2
PMCID: PMC2862950  PMID: 20445758
Orphan nuclear receptor action; Stable isotope flux phenotyping; Metabolic flexibility
23.  High-Fat Diet with Acyl-Ghrelin Treatment Leads to Weight Gain with Low Inflammation, High Oxidative Capacity and Normal Triglycerides in Rat Muscle 
PLoS ONE  2011;6(10):e26224.
Obesity is associated with muscle lipid accumulation. Experimental models suggest that inflammatory cytokines, low mitochondrial oxidative capacity and paradoxically high insulin signaling activation favor this alteration. The gastric orexigenic hormone acylated ghrelin (A-Ghr) has antiinflammatory effects in vitro and it lowers muscle triglycerides while modulating mitochondrial oxidative capacity in lean rodents. We tested the hypothesis that A-Ghr treatment in high-fat feeding results in a model of weight gain characterized by low muscle inflammation and triglycerides with high muscle mitochondrial oxidative capacity. A-Ghr at a non-orexigenic dose (HFG: twice-daily 200-µg s.c.) or saline (HF) were administered for 4 days to rats fed a high-fat diet for one month. Compared to lean control (C) HF had higher body weight and plasma free fatty acids (FFA), and HFG partially prevented FFA elevation (P<0.05). HFG also had the lowest muscle inflammation (nuclear NFkB, tissue TNF-alpha) with mitochondrial enzyme activities higher than C (P<0.05 vs C, P = NS vs HF). Under these conditions HFG prevented the HF-associated muscle triglyceride accumulation (P<0.05). The above effects were independent of changes in redox state (total-oxidized glutathione, glutathione peroxidase activity) and were not associated with changes in phosphorylation of AKT and selected AKT targets. Ghrelin administration following high-fat feeding results in a novel model of weight gain with low inflammation, high mitochondrial enzyme activities and normalized triglycerides in skeletal muscle. These effects are independent of changes in tissue redox state and insulin signaling, and they suggest a potential positive metabolic impact of ghrelin in fat-induced obesity.
doi:10.1371/journal.pone.0026224
PMCID: PMC3198460  PMID: 22039445
24.  Myostatin Inhibition in Muscle, but Not Adipose Tissue, Decreases Fat Mass and Improves Insulin Sensitivity 
PLoS ONE  2009;4(3):e4937.
Myostatin (Mstn) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Mstn−/− mice have a dramatic increase in muscle mass, reduction in fat mass, and resistance to diet-induced and genetic obesity. To determine how Mstn deletion causes reduced adiposity and resistance to obesity, we analyzed substrate utilization and insulin sensitivity in Mstn−/− mice fed a standard chow. Despite reduced lipid oxidation in skeletal muscle, Mstn−/− mice had no change in the rate of whole body lipid oxidation. In contrast, Mstn−/− mice had increased glucose utilization and insulin sensitivity as measured by indirect calorimetry, glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamp. To determine whether these metabolic effects were due primarily to the loss of myostatin signaling in muscle or adipose tissue, we compared two transgenic mouse lines carrying a dominant negative activin IIB receptor expressed specifically in adipocytes or skeletal muscle. We found that inhibition of myostatin signaling in adipose tissue had no effect on body composition, weight gain, or glucose and insulin tolerance in mice fed a standard diet or a high-fat diet. In contrast, inhibition of myostatin signaling in skeletal muscle, like Mstn deletion, resulted in increased lean mass, decreased fat mass, improved glucose metabolism on standard and high-fat diets, and resistance to diet-induced obesity. Our results demonstrate that Mstn−/− mice have an increase in insulin sensitivity and glucose uptake, and that the reduction in adipose tissue mass in Mstn−/− mice is an indirect result of metabolic changes in skeletal muscle. These data suggest that increasing muscle mass by administration of myostatin antagonists may be a promising therapeutic target for treating patients with obesity or diabetes.
doi:10.1371/journal.pone.0004937
PMCID: PMC2654157  PMID: 19295913
25.  Stimulatory Effect of Insulin on Glucose Uptake by Muscle Involves the Central Nervous System in Insulin-Sensitive Mice 
Diabetes  2011;60(12):3132-3140.
OBJECTIVE
Insulin inhibits endogenous glucose production (EGP) and stimulates glucose uptake in peripheral tissues. Hypothalamic insulin signaling is required for the inhibitory effects of insulin on EGP. We examined the contribution of central insulin signaling on circulating insulin–stimulated tissue-specific glucose uptake.
RESEARCH DESIGN AND METHODS
Tolbutamide, an inhibitor of ATP-sensitive K+ channels (KATP channels), or vehicle was infused into the lateral ventricle in the basal state and during hyperinsulinemic-euglycemic conditions in postabsorptive, chow-fed C57Bl/6J mice and in postabsorptive C57Bl/6J mice with diet-induced obesity. Whole-body glucose uptake was measured by d-[14C]glucose kinetics and tissue-specific glucose uptake by 2-deoxy-d-[3H]glucose uptake.
RESULTS
During clamp conditions, intracerebroventricular administration of tolbutamide impaired the ability of insulin to inhibit EGP by ∼20%. In addition, intracerebroventricular tolbutamide diminished insulin-stimulated glucose uptake in muscle (by ∼59%) but not in heart or adipose tissue. In contrast, in insulin-resistant mice with diet-induced obesity, intracerebroventricular tolbutamide did not alter the effects of insulin during clamp conditions on EGP or glucose uptake by muscle.
CONCLUSIONS
Insulin stimulates glucose uptake in muscle in part through effects via KATP channels in the central nervous system, in analogy with the inhibitory effects of insulin on EGP. High-fat diet–induced obesity abolished the central effects of insulin on liver and muscle. These observations stress the role of central insulin resistance in the pathophysiology of diet-induced insulin resistance.
doi:10.2337/db10-1100
PMCID: PMC3219951  PMID: 22028182

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