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University of Tuebingen, Department of Internal Medicine IV, Tuebingen, Germany
1Correspondence: University of Tuebingen, Department of Internal Medicine IV, Tuebingen D-72076, Germany. Email: Hans-Ulrich.Haering{at}med.uni-tuebingen.de
SPECIFIC AIMS
Obesity is the most common endocrine disorder that causes insulin resistance and finally contributes to the development of type 2 diabetes. Extensive studies were made to unveil the cause of peripheral insulin resistance, but the precise mechanisms are largely unknown. Nevertheless, there is increasing evidence that adipose-derived cytokines and hormones like tumor necrosis factor-
, interleukins, and leptin are relevant for impaired insulin action in liver, fat and skeletal muscle. Recent data provide evidence that high leptin levels lead to the hallmarks of insulin resistance including inhibition of insulin-stimulated glucose (Glc) uptake, and elevated plasma leptin levels correlate positively with total body fat mass, and negatively with insulin sensitivity. These data suggest that obesity, insulin resistance and type 2 diabetes are tightly linked by a crosstalk of the leptin and insulin signaling cascade. In the present study, we focused on the impact of the adipose hormone leptin on the insulin signaling cascade at the concentration of Ser-318 phosphorylation in cell lines, as well as in mouse and human tissues.
PRINCIPAL FINDINGS
1. Leptin stimulates Ser-318 phosphorylation in insulin receptor substrate 1
Our data reveal that leptin phosphorylates Ser-318 in Irs1 in HEK 293 cells, L6 myoblasts, and mouse skeletal muscle determined by Western blot analysis. The extent of Ser-318 phosphorylation was strongly dependent on the amount of Irs2 protein present in the cell, suggesting that Irs2 is involved in this activation loop.
2. Specificity of protein kinase C isoforms to phosphorylate Ser-318
To identify the signaling molecules that are involved in leptin-induced Ser-318 phosphorylation, we used an in vitro phosphorylation approach to examine the specificity of different protein kinase C (PKC) classes to phosphorylate an Irs1 protein fragment covering amino acid residues 265–522. The Irs1 wild-type protein fragment was incubated with a member of the "classical" PKCs (PKC-ßbeta;1) or with PKC-
as a member of the "novel" PKCs and the "atypical" PKC-
, respectively, and incubation of PKC-ßbeta;1 with this fragment resulted in a 32P-incorporation of 39 pmolATP/80 µg substrate, whereas for PKC- the incorporation rate was elevated to 63 pmolATP/80 µg substrate (P<0.005, ßbeta;1 vs. , n=6), and 81 pmolATP/80 µg substrate for PKC- (P<0.001, ßbeta;1 vs. , n=6). Using RP-HPLC, we confirmed that all three PKCs, PKC-ßbeta;1, PKC-, and PKC- are a substrate of this Irs1 fragment.
3. Ser-318 phosphorylation is downstream of Jak-2 and PKC-
To determine which of these PKC isoforms are involved in leptin-induced Ser-318 phosphorylation, we treated L6 myoblasts with insulin, leptin, or leptin and bisindolylmaleimide to inhibit classical (ßbeta;) and novel () but not atypical () PKC activity. Bisindolylmaleimide was able to prevent Ser-318 phosphorylation, suggesting a PKC-ßbeta;/-dependent mechanism. Moreover, leptin-induced Ser-318 phosphorylation was disrupted by the use of the Jak-2 inhibitor tyrphostin. As our in vitro studies revealed that PKC- is one of the major PKCs to phosphorylate Ser-318 in Irs1, we further established the signaling cascade in C2C12 cells that are known to express tiny amounts of PKC-. In this cell line, the stimulatory effect of leptin was not detectable, suggesting a PKC- dependent mechanism. To finally validate the importance of PKC- on Ser-318 phosphorylation, we overexpressed constitutively active PKC- in C2C12 cells. The results obtained revealed that Ser-318 phosphorylation is dependent on the presence of PKC-. It is noteworthy that insulin-induced Ser-318 phosphorylation was preserved, due to the presence of PKC- in C2C12 cells.
4. Ser-318 phosphorylation down-regulates insulin action and 2-deoxyglucose uptake
To test the hypothesis whether leptin-induced Ser-318 phosphorylation also results in impaired insulin signaling, we transfected L6 cells with either Irs1 or a Ser-318 to Ala (318) point-mutated-Irs1 and performed Western blot analysis on Irs1 tyrosine phosphorylation. The data reveal that insulin stimulates tyrosine phosphorylation of Irs1 while leptin had no effect. However, leptin pretreatment leads to a greatly diminished insulin-induced tyrosine phosphorylation of Irs1, suggesting an inhibitory effect of leptin on the insulin signaling cascade. In the presence of the Ala318 mutant, the inhibitory effect of leptin pretreatment on Irs1 tyrosine phosphorylation was absent, indicating that Ser-318 is required for leptin-induced inhibition of the insulin signal. To validate the importance of this serine-site in Glc metabolism, we determined Glc uptake in L6 myoblasts transfected with Glut-4 and either Irs1-wt or the Ala318 mutant. Compared to Irs1-wt, where leptin pretreatment leads to a diminished Glc uptake rate (Fig. 1A
, right panel), leptin was unable to decrease Glc uptake in the presence of the Ala318 mutant. This strongly suggests that Ser-318 is the key serine-phosphorylation site involved in leptin-induced peripheral insulin resistance.
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5. Ser-318 phosphorylation is elevated in muscle tissue of obese mice
To determine whether Ser-318 phosphorylation is indeed present and regulated in vivo, we injected C57Bl/6 mice with leptin and insulin and determined the phosphorylation levels of Ser-318 in muscle tissue. Leptin and insulin were able to activate Ser-318 phosphorylation as determined by the phospho-specific antibody in muscle tissue of lean animals (Fig. 1
A, left panel). To proof the fact that Ser-318 phosphorylation is regulated in mouse physiology, we investigated the extent of Ser-318 phosphorylation in the presence of obesity. In muscle tissue of high fat diet fed mice, where insulin and adipose-derived cytokines and hormones are elevated, basal Ser-318 phosphorylation was greatly enhanced compared to low-fat diet fed mice. Moreover, in obese mice, insulin and leptin are not able to substantially increase Ser-318 phosphorylation in vivo compared to lean animals (Fig. 1A
, right panel).
6. Ser-318 phosphorylation is regulated in human lymphocytes
As another system to investigate the role of Ser-318 phosphorylation in the presence of metabolic alterations in humans, we made use of lymphocytes isolated from human blood. Therefore, Ser-318 phosphorylation was investigated in lymphocytes isolated from nondiabetic human subjects before and at the end of an euglycemic hyperinsulinemic clamp. In analogy to the data obtained from mouse muscle, Western blot analysis in human lymphocytes of lean revealed an increase in Ser-318 phosphorylation by the end of the clamp (Fig. 1B
, left panel), while in obese the phosphorylation levels were already up-regulated in the basal state. Moreover, further stimulation during the clamp is missing in obese subjects (Fig. 1B
, right panel). Looking at data of 47 individuals, basal Ser-318 phosphorylation is negatively associated with age (Fig. 1C
) and positively with body fat mass (Fig. 1D
), suggesting that the previously described decrease in cytosolic PKCs during aging is relevant for the extent of Ser-318 phosphorylation in vivo.
After the mouse data, we further analyzed the impact of obesity on the potential to increase Ser-318 phosphorylation during hyperinsulinemia in humans. Therefore, subjects were stratified in quartiles of percentage of body fat. Western blot analysis revealed that the basal Ser-318 phosphorylation in the presence of elevated leptin levels in obese (4th quartile) was already up-regulated and further stimulation of Ser-318 during the clamp was absent in these subjects, reflecting the data obtained from muscle of overweight mice (Fig. 1F
). By contrast, Ser-318 phosphorylation was inducible in lymphocytes of lean human subjects (1st quartile, Fig. 1E
) and correlated to the data obtained from mice fed a low fat diet.
CONCLUSIONS AND SIGNIFICANCE
Adipose tissue is known to be an endocrine organ that communicates with the brain and peripheral tissues by secreting hormones to regulate food intake and Glc metabolism. Among a large number of hormones secreted from adipocytes, leptin is supposed to signal the nutrient status to the central nervous system, especially the hypothalamus where it decreases food intake. There is a bulk of evidence supporting the idea that leptin acts as a insulin-sensitizing factor as leptin administration either centrally or peripherally leads to a reduction in body wt, and the loss of the leptin gene results in massive obesity, reversed by leptin administration.
On the other hand, leptin levels are elevated in the presence of obesity, and circulating levels are proportional to body adiposity, consistent with the idea that obesity causes leptin resistance in peripheral tissues and the brain. As this phenomenon is mechanistically related to insulin resistance, and insulin and leptin share downstream signaling elements, studies are focusing on the crosstalk of the insulin and leptin signaling cascade to alter its downstream action. In L6 muscle cells, elevated leptin levels are able to diminish MAP kinase phosphorylation and inhibit insulin-stimulated Glc uptake. Moreover, leptin pretreatment impairs insulin receptor downstream action in liver cells and adipocytes. This dual effect of leptin action on Glc metabolism is supposed to be regulated by the nutritional status, as in obese rats, leptin administration leads to a reduced insulin signal in liver, whereas in lean rats, leptin was not able to diminish the insulin response. Therefore, the insulin-antagonizing effects of leptin on peripheral tissues such as muscle, liver or fat may become prominent in the presence of obesity that leads to central leptin resistance (scheme 1).
To uncouple obesity from insulin resistance in peripheral tissue, more data about the mechanism involved are needed to design pharmacological tools that abolish obesity-related inhibition of the insulin signaling cascade.
In this study, we defined a potential mechanism for the inhibition of the insulin signal in the face of obesity at the concentration of insulin receptor substrate proteins measured by Ser-318 phosphorylation. This serine-site is known to reduce the coupling of Irs1 to the insulin receptor and therefore reduces the pool of Irs1 molecules available for adequate insulin signaling in peripheral tissues and/or creating binding sites for inhibitory molecules.
In summary, our data reveal that leptin phosphorylates Ser-318 in Irs1 in various cell models through a Jak-2, Irs2, and PKC dependent pathway, and therefore down-regulates the insulin signal in the presence of obesity. As this effect is increased in muscle tissue of obese high fat diet fed mice as well as in lymphocytes isolated from obese humans, we propose that Ser-318 in Irs1 is a common site for integrating the inhibitory signaling of leptin on the insulin signaling cascade in vitro and in vivo.
Therefore, inhibition of leptin-dependent phosphorylation of Ser-318 in Irs1 could be a rational treatment to improve peripheral insulin signaling in overweight subjects.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4635fje
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