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Fishberg Center for Neurobiology, Neurobiology of Aging Laboratories and Department of Geriatrics, Mount Sinai School of Medicine, New York, NY 10029 and * Department of Physiology, Yokohama City University School of Medicine, Yokohama, 236-0004, Japan
3To whom correspondence should be addressed. E-mail: Charles.Mobbs{at}mssm.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • insulin resistance • jun-B • c-fos • leptin
The importance of individual tissues in determining whole-body insulin resistance has not yet been fully resolved. Impaired responses to insulin in muscle, fat, and liver can be demonstrated in association with whole-body insulin resistance (1), and insulin resistance in these tissues has classically been considered to contribute to whole-body insulin resistance. On the other hand, recent studies suggested that insulin resistance in muscle and fat may not play a major role in causing whole-body insulin resistance (2). Furthermore, because plasma insulin levels increase to compensate for insulin resistance, plasma glucose levels are often normal in the presence of whole-body insulin resistance. Although insulin resistance may be exactly compensated by hyperinsulinemia at the whole-body level, all responses to insulin may not be equally impaired. If some biosynthetic responses continue to be sensitive to insulin, the presence of hyperinsulinemia may actually produce a state of hyperstimulation. For example, it was proposed that obesity can lead to hypertension because the vasodilatory response to insulin is impaired, but the sodium-retaining effect of insulin is not impaired, thus leading to sodium retention due to hyperinsulinemia (3). Similarly, the maintenance of leptin sensitivity to insulin despite whole-body insulin resistance could explain the elevated expression of leptin (normalized to wet weight or per microgram RNA) observed in obesity (4) because insulin stimulates leptin and insulin is elevated in insulin resistance (4). However, it is unclear whether leptin sensitivity to insulin is normal in the presence of insulin resistance because insulin-induced phosphorylation of signaling proteins is impaired in the fat tissue of insulin-resistant animals (1). On the other hand, it is not clear whether impaired phosphorylation of signaling proteins necessarily implies impaired synthetic responses to insulin.
To address the hypothesis that liver and adipose tissue might exhibit differential impairments in synthetic responses to insulin in the presence of whole-body insulin resistance, it was necessary to assess a synthetic response to insulin exhibited by both tissues. The immediate-early genes c-fos and jun-B have been used as markers of insulin and glucose stimulation in a variety of cell types in vivo and in vitro (5–12). Furthermore, these immediate-early genes can interact to form the activator protein (AP)-1 transcription factor, and the leptin promoter sequence contains a potential AP-1 binding site (13,14). We therefore assessed expression of c-fos, jun-B, and leptin in liver and fat after insulin or glucose stimulation in insulin-sensitive and insulin-resistant mice. The present studies suggest that some biosynthetic responses to insulin stimulation are impaired in liver before adipose tissue, and that the elevated expression of leptin in insulin-resistant mice may be due to elevated expression of immediate-early genes secondary to hyperinsulinemia acting on insulin-sensitive adipose tissue.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In all studies, upon killing, liver and adipose tissues from the epididymal or gonadal fat pad were removed, frozen on dry ice, and stored at –70°C until use. Blood glucose was measured by a Lifescan One-Touch ll glucose meter (Johnson & Johnson), and serum insulin was measured by a species-nonspecific RIA using rat insulin as a standard (15).
RNA analysis. Total RNA was extracted in TRIzol (GIBCO BRL); 7 µg of total RNA, estimated by spectrophotometer, was subjected to Northern blot analysis to detect jun-B and c-fos mRNA in the liver and adipose tissue and leptin mRNA in adipose tissue. Northern blot analysis was performed as described previously (4). Templates for a single-stranded cDNA probe for jun-B and c-fos were generated from a source DNA fragments by using PCR. The template for the leptin probe was generated as described previously (4). Primers to generate and label probes were as follows: jun-B forward primer: 5'-CACGACGACTCTTACGCAGC-3', jun-B reverse primer: 5'-GATGCGCCTGTGTCTGATCC-3', c-fos forward primer: 5'-AGCCGACTCCTTCTCGAGCA-3', c-fos reverse primer: 5'-CCTCCTGACAGGCTCTTCAC-3', leptin forward primer: 5'-CTGCAAGGTGCAAGAAGAAG-3', and leptin reverse primer: 5'-TCGGAGATTCTCCAGGTCAT-3'. Single-stranded internally labeled DNA probes were produced as described previously (4). Membranes were reprobed and hybridized with 32P-labeled probe encoding 18S ribosomal RNA. The total integrated densities of hybridization signals were determined by computerized densitometric scanning (MCID System) or phosphoimager (to quantify Northern blots: STORM 860, Molecular Dynamics).
Statistical analysis. Gene expression data were expressed as a percentage relative to the saline-injected lean control group in each experiment. Data represent means ± SEM. Initially, statistical analysis was performed by a two-way ANOVA followed by a Tukey-Kramer post-hoc test; comparisons between 2 groups was by unpaired Student’s t test. In the time course study after glucose injection, effects of time were analyzed by a two-way ANOVA followed by Dunnett’s post-hoc test to compare each time point with time 0 within each genotype, or a Tukey-Kramer post-hoc test to compare values at same time point between genotypes. When the variances across groups were not equal, the Wilcoxon (nonparametric) test was used to assess statistical significance. Then the relative contributions of factors, such as glucose, to jun-B or leptin mRNA were assessed using the general linear model (GLM) as described previously (16). The statistical analysis was performed using the JMP statistical package (Version 5.0.1a, SAS Institute) implemented on a Macintosh operating system. Part of the data for leptin mRNA was reported previously (4).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Insulin-induced hepatic immediate-early gene expression in obese insulin-resistant (male) Ay/a mice. As expected, male Ay/a mice were heavier than wild-type a/a mice (47.8 ± 0.5 g vs. 32.1 ± 0.6 g, P < 0.05, unpaired Student’s t test). In contrast to female Ay/a mice, male Ay/a exhibited clear evidence of insulin resistance because serum insulin was significantly elevated in male Ay/a mice compared with a/a mice (P < 0.05, Tukey-Kramer test, Table 1); insulin injection decreased blood glucose in male a/a mice (from 232.6 ± 14.4 mmol/L to 99.5 ± 26.5 mmol/L, P < 0.001, Tukey-Kramer test), but did not do so in male Ay/a mice (from 200.6 ± 40.5 mmol/L to 158.5 ± 50.7 mmol/L, P = 0.48, Tukey-Kramer test).
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There was a significant main effect of genotype (P < 0.0001, ANOVA) on adipose jun-B mRNA without a significant main effect of time course (P = 0.09, ANOVA) or a significant interaction (genotype x time course, P = 0.63, ANOVA). Adipose jun-B mRNA was increased 30 min after glucose injection (454% of time 0, P < 0.05 compared with 0 min, Dunnett’s test, Fig. 4A) and remained elevated 75 min after glucose injection (408% of time 0, P < 0.005, Dunnett’s test) in a/a mice. In Ay/a mice, adipose jun-B mRNA was increased by 15 min after glucose injection (P < 0.05, Dunnett’s test, Fig. 4A). There was a significant main effect of genotype (P < 0.0001, ANOVA) and injection (P < 0.005, ANOVA) and a significant interaction (genotype x injection, P < 0.0001, ANOVA) on leptin mRNA. Basal expression of leptin mRNA was elevated in male insulin-resistant Ay/a mice (532% of a/a mice) compared with a/a mice (P < 0.0001, Tukey-Kramer test, Fig. 4B). In a/a mice, leptin mRNA peaked at 30 min after glucose injection (329% of time 0, P < 0.01 compared with 0 min, Dunnett’s test) and returned to basal level at 75 min. In contrast, although leptin mRNA was not changed by 30 min after glucose injection, it was significantly elevated at 75 min in Ay/a mice (173% of time 0, P < 0.005 compared with 0 min, Dunnett’s test, Fig. 4B). Furthermore, leptin mRNA was positively correlated with adipose jun-B mRNA (r = 0.61, P < 0.005, Fig. 4C), blood glucose (r = 0.33, P < 0.05), serum insulin (r = 0.39, P < 0.05), and body weight (r = 0.73, P < 0.0001). The correlation between jun-B mRNA and leptin mRNA was significant even after removing the effect of blood glucose (P < 0.05 by GLM). In contrast, the correlation between blood glucose and leptin mRNA was no longer significant after the effect of jun-B mRNA was removed. In contrast to a/a mice, leptin mRNA in Ay/a mice was not correlated with adipose jun-B mRNA (r = 0.27, P = 0.24), blood glucose (r = 0.29, P = 0.21), serum insulin (r = –0.15, P = 0.53), or body weight (r = –0.07, P = 0.76). However, the correlation between blood glucose and jun-B mRNA was significant in Ay/a mice (r = 0.66, P < 0.005).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The present study demonstrated that both insulin and glucose injection stimulated expression of jun-B and c-fos mRNA in the liver and adipose tissue in lean insulin-sensitive a/a mice. These results are consistent with earlier studies demonstrating that expression of immediate early genes in hepatoma cells and adipose tissue is induced by insulin treatment both in vitro and in vivo (5–8). In the present study, we also found that induction of jun-B and c-fos mRNA by insulin injection was remarkably attenuated in the liver of obese insulin-resistant Ay/a mice. The impaired induction of liver immediate-early gene expression appeared to be due to insulin resistance, rather than obesity or the presence of the Ay agouti allele, because obese female Ay/a mice that were not insulin resistant did not exhibit impaired insulin-induced immediate-early gene expression.
In contrast to the impairment of immediate-early gene responses to insulin in liver, these responses to insulin were preserved in adipose tissue from insulin-resistant mice. These results are consistent with the observation that insulin inhibited the epinephrine-stimulated lipolysis in adipose tissue in Ay/a mice as well as in a/a mice (21). In fact, even basal expression of immediate-early gene expression was elevated in adipose tissue from insulin-resistant mice. Because insulin-resistant mice exhibited hyperinsulinemia, this latter result is consistent with the hypothesis that fat cells in insulin-resistant mice respond normally to insulin, leading to hyperinsulinemia-induced hyperstimulation of insulin-induced biosynthetic responses to insulin in adipose tissue. To support this hypothesis, adipose jun-B mRNA correlated with blood glucose in Ay/a mice as well as in a/a mice in the present study. However, leptin mRNA was not correlated with either blood glucose or jun-B mRNA in Ay/a mice, suggesting that insulin-resistance might develop downstream of jun-B induction in adipose tissue. It should be noted that at least 1 major cause of insulin resistance, a high-fat diet, leads to impaired insulin-induced phosphorylation of IRS-1 and IRS-2 in adipose and muscle tissue but not in liver (1). The same study demonstrated that inhibition of hepatic glucose output by insulin was impaired in rats fed the high-fat diet (1). The differences in results among these studies may be due to the different variables to be examined (i.e., gene expression vs. phosphorylation of signaling protein) because phosphorylation of signaling proteins may not correlate with functional responses to insulin. In addition, a high-fat diet causes insulin resistance through mechanisms other than those mediating obesity in our study.
Basal (unstimulated) leptin mRNA, like jun-B mRNA, was also elevated in insulin-resistant Ay/a mice, as previously reported (4). Furthermore, leptin mRNA correlated with insulin and glucose, as previously reported (4), and leptin mRNA also correlated with jun-B mRNA. Interestingly, multiple regression analysis indicated that after removal of the effect of glucose, jun-B mRNA continued to predict leptin mRNA, whereas after removal of the effect of jun-B mRNA, glucose no longer predicted leptin mRNA. This observation could be interpreted as indicating that induction of jun-B mRNA is a more proximal factor than glucose in determining leptin mRNA, consistent with the observation that the leptin gene contains a potential AP-1 binding site in the promoter sequence (13,14). It was shown that glucose alone affects the expression of several genes in cultured adipocytes with an additive or synergic effect of insulin (22–24). Insulin is also known to exert its effects on gene expression through glucose-dependent and -independent mechanisms (25). Furthermore, leptin promoter activity is increased in the presence of glucose and insulin compared with glucose alone (26); insulin-induced increases in leptin mRNA, secretion of leptin in cultured adipocytes, and leptin promoter activity were closely related to glucose uptake and were attenuated by inhibition of glucose transport or uptake (27,28). In fact, insulin stimulates the translocation of glucose transporter-containing vesicles from intracellular storage sites to the plasma membrane, leading to the stimulation of glucose uptake into peripheral tissue including adipose tissue (29–31). Taken together, these results are consistent with previous reports that at least part of the effect of insulin to stimulate leptin production is mediated through enhanced glucose metabolism (32–34).
In conclusion, immediate-early genes constitute potentially useful molecular markers for the activation of nutrition-sensitive tissues such as liver and adipose tissue by metabolic factors such as glucose and insulin, and they are also useful molecular markers for hepatic insulin resistance. The present data suggest that biosynthetic responses to insulin may be impaired in the liver before similar biosynthetic responses are impaired in fat cells. Because insulin resistance in the liver may lead to whole-body insulin resistance and hyperinsulinemia, the maintenance of insulin sensitivity in fat cells in the presence of hyperinsulinemia may explain why the elevated expression of leptin and other insulin-stimulated genes is associated with whole-body insulin resistance.
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FOOTNOTES |
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2 Present affiliation: Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA.
Manuscript received 29 October 2003. Initial review completed 16 December 2003. Revision accepted 17 February 2004.
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Different from a/a mice at same time point, P < 0.05, Tukey-Kramer test. Correlations were determined by pooling all time points after glucose injection in panels A and B. Part of the data for leptin mRNA was reported previously (