We know that calorie restriction CR greatly improves insulin sensitivity - which seems to be one of the ways in which it increases life span - just as eating too much and getting fat tends to lead to insulin resistance and the diabetes that follows. Here, researchers are making slow inroads into understanding why CR does this: "Caloric restriction CR has been shown to retard aging processes, extend maximal life span, and consistently increase insulin action in experimental animals.

The mechanism by which CR enhances insulin action, specifically in higher species, is not precisely known. We sought to examine insulin receptor signaling and transcriptional alterations in skeletal muscle of nonhuman primates subjected to caloric restriction over a 4 year period.

CR increases insulin sensitivity on a whole body level and enhances insulin receptor signaling in this higher species. CR in cynomolgus monkeys may alter insulin signaling in vivo by modulating protein content of insulin receptor signaling proteins.

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Redman , Bradley R. Newcomer , Madlyn I. Frisard , Steve Anton , Steven R. Smith , Anthony Alfonso , Eric Ravussin , the Pennington CALERIE Team; Effect of Calorie Restriction With or Without Exercise on Insulin Sensitivity, β-Cell Function, Fat Cell Size, and Ectopic Lipid in Overweight Subjects.

Diabetes Care 1 June ; 29 6 : — OBJECTIVE —The purpose of this article was to determine the relationships among total body fat, visceral adipose tissue VAT , fat cell size FCS , ectopic fat deposition in liver intrahepatic lipid [IHL] and muscle intramyocellular lipid [IMCL] , and insulin sensitivity index S i in healthy overweight, glucose-tolerant subjects and the effects of calorie restriction by diet alone or in conjunction with exercise on these variables.

Weight, percent body fat, VAT, IMCL, IHL, FCS, and S i were assessed at baseline and month 6. Together the improvements in S i were related to loss in weight, fat mass, and VAT, but not IHL, IMCL, or FCS. Calorie restriction by diet alone or with exercise reverses this trend.

Insulin resistance is an early metabolic abnormality that precedes the development of hyperglycemia, hyperlipidemia, and overt type 2 diabetes. Both insulin resistance and β-cell dysfunction are associated with obesity 1 — 3.

Although total fat mass and subcutaneous abdominal adipose tissue SAT are associated with insulin resistance, visceral adipose tissue VAT is generally considered to be the most significant determinant 4.

The causal link between visceral fat accumulation and insulin resistance, however, remains unclear. A commonly accepted view is that fatty acids released from visceral fat into the portal vein have direct effects on hepatic metabolism. Another hypothesis, however, is that visceral fat may simply covary with other causal factors that affect insulin sensitivity, namely, fat cell size FCS and ectopic fat in muscle and liver.

Previous studies have shown that increased FCS, a marker of impaired adipogenesis, is related to insulin resistance and predicts the development of type 2 diabetes 5. Whether increased FCS affects insulin sensitivity by increased spillover of triglyceride into visceral fat or into muscle, liver, or other nonadipose tissues is unclear.

Calorie restriction reduces fat mass, delays the development of age-associated diseases such as type 2 diabetes, and increases lifespan in rodents. In obese humans, it is well established that calorie restriction, weight loss, and exercise improve insulin sensitivity 6 — 11 , although the additional benefits of increased exercise on insulin sensitivity are debated.

Moreover, the extent that these interventions alter ectopic fat accumulation in muscle and liver has not been explored. Moderate weight loss, by diet alone or in combination with exercise, however, does not alter muscle lipid depots, despite significant improvements in insulin sensitivity 6 , 14 , Hepatic fat, on the other hand, was significantly lowered by moderate weight reduction in obese women and in type 2 diabetes 16 , To our knowledge no study has yet determined the effect of caloric restriction with or without exercise on ectopic fat in nonobese individuals.

The goal of this study was, therefore, to determine in healthy nonobese, glucose tolerant subjects 1 the relationships among total body fat, visceral fat, FCS, intramyocellular lipid IMCL , intrahepatic lipid IHL , and insulin sensitivity index S i and 2 the effects of a calorie-restricted diet alone or in conjunction with exercise on ectopic fat, visceral fat, FCS, insulin sensitivity, and β-cell function.

Details of this study are reported elsewhere The study was approved by the Pennington Biomedical Research Center Institutional Review Board and the CALERIE Data Safety Monitoring Board, and all subjects provided written informed consent.

To carefully determine individual energy requirements, total daily energy expenditure was measured by two day measures of doubly labeled water, once while participants followed their usual diet at home and once while they were being provided with a weight maintenance diet by the metabolic kitchen.

Patients were then admitted to the ward for 5 days of metabolic testing. Participants were provided with all food for the first 12 weeks and for weeks 22— For weeks 13—22, participants self-selected their diet based on their calorie targets. CREX participants increased their energy expenditure by Participants were required to conduct three sessions per week under supervision.

For unsupervised sessions, participants wore a portable heart rate monitor Polar S, Polar Beat, Port Washington, NY with heart rate and exercise duration recorded. For support, all participants attended weekly group meetings that were led by clinical psychology professionals.

All metabolic tests were performed during inpatient stays at baseline and month 6 following a h overnight fast and at least 48 h after the last bout of exercise. Body fat was measured by dual-energy X-ray absorptiometry QDA A; Hologics, Bedford, MA and multislice computed tomography scanning of the abdominal region GE High Speed Plus; General Electric, Fairfield, CT was performed to quantify abdominal fat compartments Muscle and liver lipid stores were determined by proton magnetic resonance spectroscopy using point-resolved spectroscopy Subcutaneous abdominal needle biopsies were performed, and FCS was determined by the Multisizer-3 counter Beckman Coulter, Fullerton, CA as previously described Insulin sensitivity was determined by the insulin-modified frequently sampled intravenous glucose tolerance test 22 , At 20 min, a bolus injection of insulin 0.

The S i and acute insulin response to glucose AIR g were calculated by the minimal model Because of illness or problems with intravenous lines, four tests could not be analyzed at month 6.

Glucose was analyzed using a Synchron CX7 Beckman-Coulter, Brea, CA and insulin was analyzed via immunoassay on the DPC Diagnostic Product Corporation, Los Angeles, CA. SAS version 9. Pearson or Spearman rank order correlations were used where appropriate, and general linear regression was used to identify any interactions of the changes with sex.

To assess the effect of the intervention among the four groups, the change from baseline to month 6 was computed, and an ANCOVA was performed with baseline values included in the model as covariates and adjusted with respect to Tukey-Kramer. Two subjects withdrew during the study; one was a control subject who withdrew for personal reasons and the other subject, who was following the LCD diet, was lost to follow-up.

Data are therefore presented on 46 subjects. Characteristics of the subjects at baseline are reported in Table 1. Subjects were generally in good health with fasting glucose, insulin, and blood pressure within recommended ranges; 30 Caucasians, 15 African Americans, and 1 Asian were examined.

IMCL in the soleus was not correlated with FCS, VAT, or IHL. All of the above correlations were also statistically significant at month 6 data not shown.

The impact of the intervention can be seen in Table 1 by comparing results at month 6 versus baseline. The changes in body composition and abdominal fat were not dependent on whether the caloric deficit was achieved by exercise and diet CREX or diet alone CR and LCD.

The improvement in S i was not different among the three intervention groups. The changes in IMCL, IHL, FCS, and the other abdominal fat depots were not additional independent determinants.

These correlation analyses were repeated with the control group removed. The significance of the relationship between the changes in FCS and VAT and the changes in IHL and percent fat was lost, but no other relationships were affected.

In this study we examined the relationships between S i and various indexes of body fat in overweight, glucose-tolerant subjects before and after calorie restriction. At baseline, we found that 1 fat deposition in liver was related to the accumulation of fat in the abdominal visceral area and to enlarged subcutaneous abdominal adipocytes and 2 increased FCS but not ectopic fat deposition in muscle and liver was independently associated with reduced insulin sensitivity.

In response to 6 months of calorie restriction, we found that 1 weight, visceral fat, and FCS are reduced with improvements in S i and reduced AIR g and 2 fat deposition in liver but not muscle was reduced by the intervention, but the changes were not associated with improvements in S i.

Several studies have suggested that ectopic fat accumulation is independent of whole-body adiposity 16 , 24 — However, other studies have noted that lipid accumulation in both muscle 27 , 31 — 33 and liver 34 — 37 increases as a function of obesity, providing that subjects with a wide range of adiposity are studied.

In this study, we observed that lipid deposition in liver but not muscle was related to both total and abdominal adiposity.

Specifically, our findings indicate that ectopic fat in the liver may be related to visceral fat stores. This relationship between liver lipid and visceral adiposity has been noted in some 34 , 38 but not all 29 , 30 studies.

Most interestingly, we observed that liver lipid infiltration tended to be greater in overweight individuals who had enlarged adipocytes and increased visceral abdominal adiposity.

Furthermore, visceral fat was related to FCS. These findings support the hypothesis that inadequate subcutaneous adipose stores result in lipid overflow into visceral fat and other nonadipose tissues In this regard, visceral fat could be considered as a marker of ectopic fat.

At baseline and at month 6, large fat cells were also the strongest determinant of insulin resistance in these nondiabetic subjects. This finding prompts speculation that impaired adipogenesis may be the primary defect in insulin resistance, and the hypothesis is supported by findings that humans with partial or complete loss of adipose tissue are extremely insulin resistant 40 , that surgical replacement of adipose stores in the fatless mouse restores insulin sensitivity 41 , and that expression of Wnt signaling genes and adipogenic transcription factors are reduced in nondiabetic subjects with a family history of type 2 diabetes Large fat cells have also been shown to have a different pattern of adipocytokine secretion than smaller fat cells 43 , which may contribute to the strong association between large FCS and insulin sensitivity.

In contrast to previous studies 24 , 26 — 28 , 31 , 44 , 45 , we observed that IMCL was not related to insulin sensitivity. Furthermore, IMCL was not related to adipocyte size. Our results are consistent with the hypothesis that IMCL stores alone are not sufficient to account for impaired insulin action 46 — Liver lipid, on the other hand, was inversely related to insulin sensitivity.

Liver lipid content has previously been reported to correlate with measures of whole-body insulin sensitivity in individuals with and without diabetes 30 , 34 , 35 , 38 , 49 , but this relationship is difficult to explain mechanistically because most ingested or infused glucose is taken up by muscle.

Theoretically, IHL is expected to correlate with reduced hepatic insulin sensitivity impaired insulin suppression of glucose rate of appearance and not necessarily with whole-body insulin action. However, the accumulation of hepatic triglyceride has been hypothesized to reduce insulin clearance and lead to peripheral insulin resistance via a downregulation of insulin receptors 34 , Clearly, prospective human studies that define whether lipid accumulation in liver precedes insulin resistance would be of interest.

Contrary to some previous studies 51 , 52 , we observed that diet alone or with exercise produced identical reductions in weight, fat mass, and abdominal fat mass. These conflicting results may be due to inaccurate calculations of the energy costs of the prescribed activity in those studies, which would lead to differences in energy deficits among groups.

We also observed that FCS was reduced in response to an energy deficit, but we could not detect an additional effect of exercise. Our study was underpowered to detect differences in FCS among groups and our results contrast with the reports of You et al.

The current study is also the first to simultaneously measure ectopic fat stores in both muscle and liver in response to a calorie restriction intervention.

We found that the calorie restriction alone or with exercise did not affect IMCL in the soleus. These results are consistent with previous studies 6 , 14 , 15 and together with the findings that IMCL was not independently related to S i suggest that IMCL accumulation alone is not likely to be a causal factor leading to acquired insulin-signaling defects in muscle.

Many other factors, including lipid droplet size, location of lipid droplets relative to mitochondria, and muscle oxidative capacity, are all potential determinants of insulin resistance 15 , 48 , An alternate hypothesis is that the capacity for lipid metabolism is an important mediator in the association between IMCL and insulin resistance.

Caution must be exercised when interpreting these results because the study may have been underpowered to detect small differences in IHL among groups. The reduction in liver lipid levels is consistent with results of Tiikkainen et al.

In addition, we also observed parallel reductions in IHL and abdominal visceral fat. In summary, calorie restriction by diet alone or in conjunction with exercise leads to similar improvements in insulin sensitivity and reductions in β-cell sensitivity in overweight, glucose-tolerant subjects.

The study also provides support for the hypothesis that the underlying pathologic cause of insulin resistance is related to abnormal partitioning of fat among adipose, hepatic, muscle, and pancreatic tissues, probably as a result of an inability to make new fat cells.

However, the finding that IMCL was not responsive to weight loss despite improvements in insulin sensitivity suggests that intracellular fat accumulation is not a causal factor in insulin resistance in muscle.

Overall, this study provides new evidence to suggest that impaired adipogenesis and increased liver lipid infiltration occur early in the pathogenesis of insulin resistance.

In healthy overweight men and women at baseline, there was a strong positive correlation between abdominal subcutaneous FCS and VAT A and abdominal subcutaneous FCS and IHL B.

Groups were pooled for analysis. The improvement in insulin sensitivity with 6 months of calorie restriction was significantly associated with the loss of fat mass A and abdominal VAT depots B but not to the change in subcutaneous abdominal FCS C and IHL D.

Analyses are reported with and without the control group included. Physical characteristics of the subject groups at baseline and following 6 months of calorie restriction. Differences between treatment groups for the change scores using an ANCOVA with the absolute change as the dependent variable and the baseline score as a covariate.

This work was supported by grants U01 AG to E. and K01 DK to D. is supported by a Neil Hamilton-Fairley Training Fellowship awarded by the National Health and Medical Research Council of Australia ID The authors thank the remaining members of the Pennington CALERIE Research Team: James DeLany, Corby Martin, Julia Volaufova, Marlene Most, Lilian de Jonge, Tuong Nguyen, Frank Greenway, Emily York-Crow, Catherine Champagne, Brenda Dahmer, Andy Deutsch, Paula Geiselman, Jennifer Howard, Jana Ihrig, Michael Lefevre, Darlene Marquis, Connie Murla, Sabrina Yang, Robbie Durand, Sean Owens, Aimee Stewart, and Vanessa Tarver.

Our gratitude is extended to the excellent staffs of the Inpatient Clinic and Metabolic Kitchen. Our thanks also go to Health and Nutrition Technology Carmel, CA for providing us with all of the HealthOne formula used in the study and to Edward J. Robarge for technical assistance with collection of the magnetic resonance spectroscopy data.

Finally, our profound gratitude goes to all the volunteers who spent so much time participating in this very demanding research study. is currently affiliated with the Department of Family and Consumer Sciences, University of Wyoming, Laramie, Wyoming.

is currently affiliated with the Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia. A table elsewhere in this issue shows conventional and Système International SI units and conversion factors for many substances.

The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. Sign In or Create an Account.

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Smith, MD ; Steven R. Smith, MD. Anthony Alfonso, MAPLSTAT ; Anthony Alfonso, MAPLSTAT. Eric Ravussin, PHD ; Eric Ravussin, PHD. the Pennington CALERIE Team the Pennington CALERIE Team.

Address correspondence and reprint requests to Eric Ravussin, PhD, Pennington Biomedical Research Center, Perkins Rd.

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Figure 1—. View large Download slide. Figure 2—. Table 1— Physical characteristics of the subject groups at baseline and following 6 months of calorie restriction. Month 6. View Large. Forsey RJ, Thompson JM, Ernerudh J, Hurst TL, Strindhall J, Johansson B, Nilsson BO, Wikby A: Plasma cytokine profiles in elderly humans.

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In Proceeedings of the International Society for Magnetic Resonance in Medicine, Honolulu, Hawaii. Bjorntorp P: Liver triglycerides and metabolism. Following a 1-week acclimation period, animals were killed after anesthetization.

Livers were rapidly collected and washed briefly in PBS in preparation for tissue extraction. All care and treatment of animals were in accordance with the guidelines of the National Institutes of Health.

The protocols were subjected to prior approval by the Institutional Animal Care and Use Committee of the University of Chicago. The rats were injected i. with 1. Liver tissue extracts TEs were prepared as described previously Qiao et al.

Glutathione-S-transferase GST —IRS1 2— , GST—IRS1 — , and GST—IRS1 — were prepared as described previously Qiao et al. The DNAs encoding these regions of rat IRS1 were synthesized by PCR. PCR products were isolated, digested with appropriate restriction enzymes, subcloned into pGEX-2T, and transformed into Escherichia coli DH5.

GST fusion proteins were purified by a glutathione—sepharose column Amersham Biosciences and eluted with glutathione. Reactions were stopped by adding 6× Laemmli buffer containing 0. After washing off excess secondary antibody, blots were developed with enhanced chemiluminescence ECL, Amersham Biosciences, Pittsburgh, PA, USA and exposed using X-ray film.

ANOVA was performed to compare differences among three or more groups. Student's t -test was used to analyze the differences between two groups. They are obese and display hyperinsulinemia and hyperlipidemia but normo- or mild hyperglycemia Pederson et al.

Both food intake and body weight were higher in obese animals 1. At the end of the 20 weeks, the body weight of obese rats fed AL was 1. As expected, CR Fig. Interestingly, the growth curve of the CR obese rats could almost be superimposed onto that of the L-AL animals Fig.

Insulin sensitivity was evaluated by an ITT Fig. Consistent with body weight, the curve of the ITT in restricted obese rats was nearly superimposable upon that of L-AL animals.

The t -tests revealed significant differences between O-AL and O-CR groups as well as between O-AL and L-AL groups Fig. These data confirm that moderate CR over a period of 20 weeks improves insulin sensitivity even in animals genetically predisposed to obesity and insulin resistance.

Average daily food intake and body weight of obese and lean Zucker rats. A Ad libitum AL animals were allowed unlimited access to food; food intake was measured every day.

B Body masses were recorded daily. Data are means± s. O-CR, obese rats-calorie restriction; O-AL, obese rats- ad libitum ; L-CR, lean rats-calorie restriction; L-AL, lean rats- ad libitum.

Citation: Journal of Endocrinology , 3; Insulin tolerance test ITT in the four experimental groups following 20 weeks of ad libitum or restricted CR feeding.

Blood glucose was assayed from the tail vein at indicated times. Unpaired Student's t -test with one-tailed distribution was used to compare the two different animal groups at each time point.

CR-induced improvements in insulin sensitivity in obese Zucker rats provided a good animal model for investigating the association between abnormal activation of IRS1 serine kinases and phosphorylation of IRS1 with respect to insulin sensitivity.

To this end, we applied a well-established in vitro kinase assay using GST—IRS1 fragments as substrates and liver TEs as kinase sources Qiao et al. All these phosphorylation sites are well conserved between mice, rats, and human.

More interestingly, the phosphorylation of these sites returned to the same levels as seen in lean controls after 20 weeks of CR Fig.

This suggests that the effect of CR on the activity of the IRS1 kinases that phosphorylate the aforementioned sites is specific for those kinases whose activity is influenced by obesity. Immunoblot analysis of serine phosphorylation in IRS1 by in vitro kinase assay.

Specific phospho-IRS1 antibodies were used to detect serine phosphorylation of GST—IRS1 by western blot analysis. B—H Density of phosphoproteins in A was quantified and analyzed with Image J. P values were calculated using two-tailed Student's t -test.

Although evaluation by t -test showed a significant decrease of kinase activities toward S and S of IRS1 in lean Zucker rats, CR had little effect on kinase activity toward S, S, S, S, or S in obese Zucker rats Fig.

CR had little effect on the phosphorylation of p70 S6K and ERK in lean rats Fig. The quantity of tested kinases i. kinase protein levels was not affected by dietary treatment.

Immunoblot analysis of protein kinases in the liver of the four experimental groups. Phospho-MAPK antibody recognizes endogenous levels of p44 and p42 MAP kinase Erk1 and Erk2 when phosphorylated either individually or dually at T and Y of ERK1 T and Y of ERK2. Phospho-GSK3B antibody detects endogenous levels of GSK3B phosphorylated at S9.

Phospho-AMPKα antibody detects endogenous AMPKα phosphorylated at T B—G Density of phosphoproteins in A was quantified and analyzed with Image J. Interestingly, phosphorylation of PKCθ was significantly lower and the phosphorylation of AMPK was slightly higher in obese rats when compared with lean controls Fig.

However, CR had no effect on their phosphorylation levels in obese Zucker rats, suggesting that their activity was not associated with the improved insulin sensitivity observed following CR. JNK was given special attention based upon extensive data relating its activation to insulin resistance, presumably due to phosphorylation of IRS1 Aguirre et al.

However, neither the protein level nor the phosphorylation status of JNK showed any change in obese rats when compared with lean littermates Fig. This is consistent with the fact that we were unable to detect significant changes in IRS1 S phosphorylation Fig.

To determine the involvement of one kinase or both kinases in the changes in the phosphorylation of IRS1 as shown in Fig. The ERK inhibitor II is a relatively specific inhibitor for ERK Kelemen et al. With respect to MTOR, while rapamycin is a potent inhibitor in vivo , it does not inhibit MTOR activity in vitro Oshiro et al.

A Liver extracts were made from rats after 20 weeks of ad libitum AL or calorie-restricted CR feeding. B Inhibition of MTOR activity by LY in vitro. The reaction without ATP was used as a negative control.

Density of p70 S6K bands was quantified with Image J. Data are representative of at least two separate experiments. There is compelling evidence that CR and the consequent weight loss greatly improve glucose metabolism by augmenting insulin's action.

However, the molecular mechanism underlying the enhancement of insulin sensitivity by CR is unclear. In the current study, we systematically examined the IRS protein kinase profiles in insulin-resistant animals obese Zucker rats subjected to CR. Our data imply that ERK is likely the key modulator that is involved in the development of insulin resistance, whereas the activation of other IRS protein kinases may be a subsequent event that contributes to the worsening of insulin resistance.

The critical role of liver insulin's action in maintaining glucose homeostasis was demonstrated in mice lacking the insulin receptor gene in the liver.

These mice exhibit dramatic insulin resistance, which is evidenced by a failure of insulin to regulate hepatic glucose production and gene expression Michael et al. Thus, defects in hepatic insulin signaling may precipitate systematic insulin resistance.

Identification of key factors associated with insulin sensitivity in the liver may help us to understand the molecular mechanism of insulin resistance.

Our data provide compelling evidence that the effect of CR on the activity of IRS1 kinases is specific to ERK and p70 S6K ; our results further highlight that not all serine kinases are equally important in phosphorylating IRS1 and suppressing insulin signaling.

In fact, based on in vitro kinase assay data, we observed that S was actually the strongest phosphorylation site in IRS1 for recombinant ERK when compared with S and S unpublished data.

Others have shown that JNK can phosphorylate IRS1 at S, leading to attenuation of insulin signaling Aguirre et al. Consistent with these results, neither JNK protein level nor phosphorylation was associated with the degree of insulin sensitivity.

It is possible that TEs may contain inhibitors that suppressed JNK kinase activity. However, we have fractionated lean and obese TEs using FPLC via anion-exchanged chromatography and tested each fraction for phosphorylation of IRS1 fusion proteins.

The sum of these findings leads us to conclude that JNK, for yet unknown reasons, did not play a significant role in modulating insulin's action in our experimental system. Nevertheless, we could not exclude the possibility that our in vitro kinase assay failed to detect the phosphorylation of S by GST fusion protein of IRS1.

S in IRS1 is another interesting site, the phosphorylation of which can lead to either enhancement or attenuation of insulin's action Giraud et al. The kinases that phosphorylate S are unknown; thus far, JNK and IKK have been reported to phosphorylate this site.

CR caused a significant reduction in kinase activity toward S in lean rats; however, this was not seen in obese Zucker rats. The answer for this phenomenon is unknown. We speculate that the combination of opposing kinase activities may lead to this unusual activity pattern.

GSK3B is a serine kinase that is negatively regulated by insulin Richter et al. GSK3B is also thought to be involved in insulin resistance. In type 2 diabetics, GSK3B activity is elevated twofold in both basal muscle and insulin-stimulated muscle Nikoulina et al.

Additionally, inhibition of GSK3B improves insulin's action and glucose metabolism in human smooth muscle Nikoulina et al. In our system, we detected slightly reduced phosphorylation of GSK3B in obese rats, indicating mild activation; these data, however, did not reach statistical significance.

Consistent with GSK3B activity, obese rats showed mild increased phosphorylation of S, but again, this did not attain statistical significance. However, CR had no effect on phosphorylation of GSK3B and IRS1 at S, suggesting a lack of association of GSK3B activity with insulin resistance based on our two-criterion approach.

Thus, this enzyme may not have a critical role in controlling insulin sensitivity in obese Zucker rats or in modulating insulin signaling during CR.

Although our in vitro kinase assay provides an easy and reproducible way to examine the IRS protein kinases in TEs because of a well-controlled condition, it does have its limitations.

MC was supported in part by doctoral fellowship UC MEXUS-CONACYT and by National Institute on Minority Health and Health Disparities grant 9TMD The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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