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. 2004 Jul 2;559(Pt 2):583–591. doi: 10.1113/jphysiol.2004.067751

A novel function of insulin in rat dermis

Torbjørn Nedrebø 1, Tine V Karlsen 1, Gerd S Salvesen 1, Rolf K Reed 1
PMCID: PMC1665113  PMID: 15235083

Abstract

In this study we present a novel function of insulin in rat dermis. We investigated local effects of insulin on interstitial fluid pressure (Pif), and capillary albumin leakage and pro-inflammatory cytokine production in skin and serum after intravenous lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) challenge treated with a glucose–insulin–potassium regimen (GIK). The main objective for this study was to investigate anti-inflammatory effects of insulin. Work by others shows that insulin stimulates cell adhesion, and that this effect is dependent upon phosphatidylinositol 3-kinase (PI3K) activity. Cytokines like platelet-derived growth factor BB (PDGF-BB) attenuate lowering of Pif, possibly via PI3K. LPS and pro-inflammatory cytokines contribute to oedema development during acute inflammation by lowering the Pif. Intravenous injection of LPS, TNF-α or IL-1β to Wistar Møller rats caused a lowering of Pif, but after local injection of insulin in the paw, Pif increased back to control values. IL-1β caused a lowering in control from −0.5 ± 0.2 mmHg to −3.0 ± 0.2 mmHg after 20 min (mean ± s.e.m.) (P < 0.05). Within 50 min after insulin injection the pressure was increased to −0.6 ± 0.2 mmHg (P > 0.05 compared with control). Insulin was given together with a PI3K inhibitor (wortmannin) locally in the skin, almost abolishing the effect of insulin on Pif. A GIK regimen was given as a continuous intravenous infusion, significantly attenuating the oedema formation after LPS or TNF-α/IL-1β challenge. The same GIK regimen caused a significant reduction in pro-inflammatory cytokines in serum and in interstitial fluid in skin of endotoxaemic rats. These experiments show a possible role for insulin in the interstitium during inflammation induced by LPS and TNF-α/IL-1β. Insulin can attenuate a lowering of Pif possibly via PI3K, and it has an anti-inflammatory effect by inhibiting production of pro-inflammatory cytokines.

Hyperglycaemia and insulin resistance are common in critically ill patients, and recently it was shown that intensive insulin therapy in such critically ill patients significantly reduced mortality (van den Berghe et al. 2001). There is clearly a beneficial effect of a lowered blood glucose level, but there are also indications of insulin being anti-inflammatory (Das, 2001). Administration of insulin in a glucose–insulin–potassium regimen (GIK) at time of reoxygenation in acute myocardial infarction (AMI) reduced cardiomyocyte injury (Jonassen et al. 2001). Also, during AMI, circulating levels of tumour necrosis factor-α (TNF-α) are elevated (Cain et al. 1999), and exogenous injection of insulin inhibits this TNF-α production (Das, 2001). Sepsis is also known to induce insulin resistance (Westfall & Sayeed, 1988), but the exact mechanism is still not clear. In septicaemia and septic shock, increased levels of TNF-α play an important role, and continuous infusion of GIK regimen has improved survival, possibly by inhibiting TNF-α production (Fraker et al. 1989). Infusion of glucose alone, however, augments the TNF-α and haemodynamic response to endotoxin administration in rabbits (Losser et al. 1997).

TNF-α and interleukin-1β (IL-1β) are pro-inflammatory cytokines produced by many cells during acute and chronic inflammation. During acute inflammation a cardinal sign is oedema formation. Lowering of interstitial fluid pressure (Pif) has been shown to be an important factor in oedema generation during acute inflammatory reactions (Lund et al. 1987). There is agreement that the interstitium can be an ‘active’ tissue, meaning that cell–matrix interactions can influence Pif, transcapillary fluid exchange and interstitial volume (IFV) via Pif (Wiig et al. 2003b). The mechanisms behind this change in Pif have been investigated, and it has been shown that platelet-derived growth factor BB (PDGF-BB) reverses a lowering of Pif, and that this in vivo control of tissue fluid homeostasis is most likely via the phosphatidylinositol 3-kinase (PI3K) pathway (Heuchel et al. 1999). There is a close relation between insulin and PI3K, since after insulin stimulation of its receptor, the insulin-receptor 1 substrate associates with PI3K (White & Kahn, 1994). PI3K activity in such signalling complexes appears to be required for insulin to exert its effect on many cellular processes. Inhibition of PI3K activity by wortmannin, a well-recognized PI3K-inhibitor, ablates the effect of insulin on several of these processes. Insulin action also promotes dephosphorylation of tyrosine phosphates on proteins like focal adhesion kinase (FAK) and paxillin, thought to be involved in cell regulation by integrins. It has been shown that insulin stimulates cell adhesion, and that this effect was dependent upon PI3K activity (Guilherme et al. 1998).

In this study potential effects of insulin on Pif were investigated for a possible connection with PI3K by using a PI3K inhibitor (wortmannin). We also wanted to measure total tissue water (TTW) and extravasation of albumin (Ealb) in skin after challenge with either LPS or a TNF-α/IL-1β combination given intravenously (i.v.) and treated with a continuous infusion of a GIK regimen. Also the response of TNF-α and IL-1β was measured in interstitial fluid in skin and in serum of rats with and without treatment with a GIK regimen. Briefly, we report a novel function of insulin in skin. Insulin abolishes oedema formation by attenuating lowering of Pif, and a continuous infusion of a GIK regimen not only attenuated the increase in Ealb and TTW, but also reduced the concentrations of pro-inflammatory cytokines (TNF-α and IL-1β) in serum and interstitial fluid in skin. Thus, we have demonstrated that insulin has an anti-inflammatory function, as it can attenuate oedema formation in skin and also inhibit pro-inflammatory cytokine production in interstitial fluid induced by LPS.

Methods

Animal preparation

Female Wistar Møller rats (190–230 g) from M & B, Denmark were used. Animals were fed a standard diet and tap water ad libitum. All animals were anaesthetized with pentobarbital sodium (60 mg (kg body wt)−1) given intraperitoneally and placed in dorsal recumbence on a heating pad or under a heating lamp, maintaining a body temperature at 37.5–38°C. The right external jugular vein was cannulated with a PE-50 catheter for intravenous administration of substances used in the experiment. The glucose–insulin–potassium (GIK) was administered at 0.75 ml h−1 (glucose 1.5 g kg−1 h−1, insulin 0.3 U kg−1 h−1 and potassium 0.3 mmol kg−1 h−1). Circulatory arrest was induced with an intravenous injection of 0.5 ml saturated potassium chloride (KCl) during anaesthesia. The experiments described in this article have been carried out with the approval of and in accordance with the recommendations laid down by the National Animal Research Authority.

Test substances

Insulin actrapid (100 U ml−1) was obtained from Novo Nordisk, Denmark. Insulin was stored undiluted at +4°C, under dark conditions. LPS (Sigma, USA) was stored at below −20°C at a stock solution of 10 mg ml−1. Upon reconstitution it was diluted in phosphate-buffered saline (PBS) with 2% bovine serum albumin. TNF-α and IL-1β (R&D Systems, UK) were stored at a concentration of 10 μg ml−1 at below −70°C. Upon reconstitution they were diluted in PBS with 2% bovine serum albumin. Wortmannin (Sigma) was diluted in dimethyl sulphoxide (DMSO) to 2.3 mm, and further diluted in saline to 23 μm.

Measurements

Interstitial fluid pressure

Pif was measured by a micropuncture technique using sharpened pipettes connected to a servocontrolled counterpressure device. Pipettes were made from glass capillaries (1.00 mm o.d., 0.58 mm i.d.) (Harvard Apparatus Ltd, UK). They were pulled on a micropipette puller (P-87, Sutter Instrument Co, USA) and sharpened to achieve a tip diameter of 2–6 μm (MB3/T-PSU5 microbeveller, World Precision Instruments, USA). The pipettes were filled with 0.5 m NaCl solution coloured with Evans blue and connected to a micromanipulator (Leica, Heerbrugg, Switzerland). The method has earlier been validated and criteria for acceptance of the measurements have been defined elsewhere (Wiig et al. 1981). Punctures of the skin were performed through intact skin on the dorsal aspect of the hindlimb under visual guidance using a stereomicroscope (Wild M5, Leitz, Germany). Pif was first measured prior to injection of substances, and then for the subsequent 90 min after circulatory arrest and the measurements were grouped into the following time periods: 1–20, 21–40, 41–60 and 61–90 min. After 20 min 5 μl of either saline or insulin (20 U ml−1, based on pilot studies) was injected subdermally with a syringe. To avoid a possible underestimation of Pif, circulatory arrest was induced as part of the experimental protocol by intravenous injection of KCl under general anaesthesia. The reason for this is that inflammation will enhance microvascular filtration, thereby raising IFV. Due to accumulation of fluid in the tissue, Pif will then become more positive as a consequence of tissue compliance.

Albumin extravasation and total tissue water

Ealb was measured as the 25 min extravascular clearance of 125I-labelled-human serum albumin (125I-HSA). 125I-HSA (0.05 MBq) in 0.2 ml saline was given i.v. at the same time as the animals were given test substances. It circulated for 25 min before 131I-HSA (0.05 MBq) in 0.2 ml saline was given i.v. Five minutes after injection of 131I-HSA, blood samples were obtained, and the rats were killed by an i.v. injection of saturated KCl. Ealb was then estimated as the extravascular plasma equivalent volume of 125I-HSA at 25 min after injection, i.e. calculated as the difference between the plasma equivalent distribution volume of 125I-HSA and that of 131I-HSA. Tissue samples for determining Ealb were obtained from paw and back skin, and thereafter put in pre-weighed vials, bottled immediately, reweighed as soon as possible and then placed in a drying chamber at 50°C. The vials were weighed twice a week until stable weight was obtained (usually after 2–3 weeks). Radioactivity was determined in a gamma-counter (LKB Wallac 1285, Turku, Finland) with automatic background and spillover correction. Distribution volumes were calculated as plasma-equivalent space (counts per min per gram dry tissue weight divided by counts per min per ml plasma). Total tissue water (TTW) in the tissue samples was calculated as water content per gram of dry tissue weight ((wet weight − dry weight)/(dry weight)).

Sampling of interstitial fluid in skin

All animals received an i.v. injection of LPS or saline followed by a continuous GIK infusion. When the experimental period ended, the animals were transferred to an incubator kept at room temperature and 100% relative humidity. Skin from back and paw was carefully excised. Any superficial blood contamination was removed by flushing with saline followed by careful blotting with tissue paper. The centrifugation method has recently been described (Wiig et al. 2003a). Briefly, a skin sample was transferred to a centrifuge tube, provided with a basket of nylon mesh with pore size ∼15–20 μm, and the skin was placed with the subcutis facing the mesh. The nylon mesh will keep the skin sample up from the bottom of the tube (Aukland et al. 1997). The tube was capped and spun at 2000 r.p.m. (424 g) for 10 min in an Eppendorff 5417 R centrifuge. After centrifugation the tube was brought back into the incubator. The fluid at the bottom of the tube (0.5–10 μl) was collected with a graded micropipette. Thereafter the samples were analysed or frozen (−70°C) as soon as possible, and kept at this temperature until later analysis.

Analysis of TNF-α and IL-1β in interstitial fluid

TNF-α and IL-1β in interstitial fluid were measured with a commercial ELISA kit (R&D Systems, UK). All plates were pre-coated with either TNF-α or IL-1β antibodies, and controls, standards and experimental samples were added. The washing, the adding of antibodies, substrate solutions and stop solutions, and the analyses were performed according to the manufacturers' specifications. For each well the optical density was read using a microplate reader (Spectramax, Molecular Devices, USA) set to 450 nm, with wavelength correction. The detection limit was 5 pg ml−1.

Glucose

Glucose was measured in serum of non-fasted rats. Blood was sampled by intracardiac puncture in anaesthetized rats, and thereafter analysed with a Reflotron Plus (Roche, Germany).

Experimental protocol

Interstitial fluid pressure

The animals were randomly divided into two control groups (A and B) and eight different experimental groups (C–J) (see Table 1 for details). Immediately after recording of a control pressure in the paw the rats received either saline or a test substance i.v. The administered substance circulated 5 min before circulatory arrest was induced as part of the experimental protocol. After 20 min of recording, animals received either 5 μl saline or insulin subdermally in the paw, and Pif was measured for an additional 70 min. Doses of LPS, TNF-α and IL-1β used in the protocol are based on earlier experiments in our laboratory (Nedrebø et al. 1999, 2004) and pilot studies prior to this experiment. In group I we wanted to investigate the effect of the PI3K inhibitor wortmannin (23 μm), and in group J, animals received wortmannin together with insulin to see whether insulin acted via this signalling pathway.

Table 1.

Interstitial fluid pressure (Pif) in skin of rats. Mean ± s.e.m. (mmHg). Pressures recorded in 90 min

Substance
Group n i.v. s.c. Control 1–20 min 21–40 min 41–60 min 61–90 min
A 8 Saline Saline −0.7 ± 0.2 −0.3 ± 0.1 −0.4 ± 0.2 −0.6 ± 0.2 −0.2 ± 0.3
B 6 Saline Insulin −0.7 ± 0.2 −0.3 ± 0.1 −0.8 ± 0.1 −0.6 ± 0.2 −0.9 ± 0.2
C 6 LPS Saline −0.7 ± 0.2 −1.9 ± 0.2a −3.2 ± 0.2* −3.1 ± 0.3*† −2.9 ± 0.1*g
D 6 LPS Insulin −0.7 ± 0.2 −2.0 ± 0.2b −2.0 ± 0.3* −0.9 ± 0.2 −0.4 ± 0.1
E 6 TNF-α Saline −0.6 ± 0.1 −2.5 ± 0.2* −3.8 ± 0.3* −3.6 ± 0.4*† −4.0 ± 0.7*†
F 6 TNF-α Insulin −0.6 ± 0.1 −2.8 ± 0.2* −1.8 ± 0.3* −1.2 ± 0.2 −1.2 ± 0.2
G 6 IL-1β Saline −0.5 ± 0.2 −2.8 ± 0.2* − 4.4 ± 0.4*† −5.0 ± 0.4*† −4.4 ± 0.3*†
H 6 IL-1β Insulin −0.5 ± 0.2 −3.0 ± 0.2* −1.8 ± 0.3c −1.2 ± 0.2 −0.6 ± 0.2
I 7 Saline Wortmannin −0.6 ± 0.1 −2.3 ± 0.3* −1.8 ± 0.2d −2.1 ± 0.3* −1.9 ± 0.3e
J 6 LPS Wortmannin/insulin −0.6 ± 0.1 −2.7 ± 0.4c −3.1 ± 0.5* −2.2 ± 0.3f −4.0 ± 1.3*
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*

P = 0.001

a

P = 0.003

b

P = 0.002

c

P = 0.007

d

P = 0.006

e

P = 0.018 and

f

P = 0.047 compared to own control.

†

P ≤ 0.001 and

g

P = 0.014 compared to corresponding experimental group receiving insulin s.c.

Albumin extravasation and total tissue water

The animals were divided into five groups:

Experiment A, group 1 (n = 8): LPS (4 mg kg−1) i.v. followed by a continuous saline infusion.

Treatment A, group 2 (n = 8): LPS (4 mg kg−1) i.v. followed by a continuous GIK infusion.

Experimental B, group 3 (n = 8): TNF-α/IL-1β combination i.v. (400 and 80 ng kg−1, respectively) followed by a saline infusion.

Treatment B, group 4 (n = 7): TNF-α/IL-1β combination i.v. (400 and 80 ng kg−1, respectively), followed by a continuous GIK infusion.

Control, group 5 (n = 6): saline i.v. followed by a GIK infusion.

Interstitial fluid in skin

Interstitial fluid was obtained from two experimental groups of rats and one control; one group received only LPS i.v. (n = 6) and the second LPS followed by GIK infusion (n = 6). The control group (n = 6) received saline (0.2 ml) followed by GIK infusion. The substances were allowed to circulate for a 120 min period, based on results from previous experiment (Nedrebø et al. 2004). After 120 min circulatory arrest was induced, skin was taken from the back and undisturbed interstitial fluid was obtained by the centrifugation method.

Statistical analysis

Data are means ± s.e.m. unless otherwise stated. For all statistical analysis one-way analysis of variance (ANOVA) and subsequent Bonferroni t test was used. (Dunn's method was used if normality test failed.) A value of P < 0.05 was considered statistically significant. The exact P-values are given in the text, figures and tables when between 0.05 and 0.001.

Results

Interstitial fluid pressure (Table 1)

In all experimental animals Pif was lowered significantly within 20 min after i.v. injection of LPS, TNF-α or IL-1β in agreement with previous experiments. Within 10–20 min after subdermal injection of insulin in the paw, the pressure increased to values observed in control groups at corresponding time levels (Fig. 1). Wortmannin caused a significant fall in Pif within 20 min and Pif stayed at this level during the observation period. Wortmannin given s.c. together with insulin to rats receiving LPS i.v. (Group J) also lowered Pif significantly within 20 min, and it did not change significantly within the remaining period of pressure recordings.

Figure 1. Interstitial fluid pressure after i.v. injection of IL-1β (upper), TNF-α (middle) and LPS (lower).

Figure 1

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A control group (▾) receiving saline i.v. and s.c. is shown on all three graphs. ○, recordings done in experimental animals receiving insulin after 20 min; •, experimental animals receiving saline s.c. after 20 min. Recordings are done for 90 min. Data are presented as means ± s.e.m. *P < 0.001, aP = 0.003, bP = 0.002 and cP = 0.007 compared with own control. †P < 0.001 compared with own control and experimental group receiving insulin s.c.

Albumin extravasation and total tissue water (Fig. 2)

Figure 2.

Figure 2

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A, extravasation of albumin (Ealb) in skin of paw and back after i.v. injection of LPS, LPS + GIK, TNF-α/IL-1β, TNF-α/IL-1β + GIK or saline + GIK (control). B, total tissue water (TTW) in skin of paw and back after i.v. injection of LPS, LPS + GIK, TNF-α/IL-1β, TNF-α/IL-1β + GIK or saline + GIK (control). Data are presented as means ± s.e.m. LPS had a significantly higher Ealb compared with corresponding GIK group and control, described with † (P < 0.001). TNF-α/IL-1β had a significantly higher Ealb compared with control (P = 0.002) but not to GIK treatment (P > 0.05). LPS increased TTW compared to control P = 0.009), but not GIK treatment (P > 0.05). TNF-α/IL-1β increased TTW both compared with control and GIK treatment (P = 0.007 for both).

In the experimental groups receiving LPS or a combination of TNF-α/IL-1β followed by saline, Ealb increased significantly (P < 0.001 and P = 0.002, respectively, compared with control). In rats receiving LPS or TNF-α/IL-1β followed by a continuous GIK infusion, the Ealb increase was not significantly different from control (P > 0.05), but was significantly different from LPS-challenged rats not receiving a GIK infusion (P < 0.001). Group 3, receiving TNF-α/IL-1β, had a significant increase (P = 0.007) in TTW compared with group 5 (receiving saline) and group 4, treated with GIK (P = 0.007). The change in TTW for group 1 (LPS + saline) was significantly different from control (P = 0.009), but not from group 4 receiving GIK (P > 0.05).

TNF-α and IL-1β in the interstitial fluid and serum (Fig. 3)

Figure 3.

Figure 3

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A, concentrations of TNF-α and IL-1β in interstitial fluid in skin after i.v. injections of LPS, LPS + GIK or saline + GIK (control). B, concentrations of TNF-α and IL-1β in serum after i.v. injections of LPS, LPS + GIK or saline + GIK (control). Data are presented as means ± s.e.m. Concentrations of TNF-α or IL-1β were significantly higher in both interstitial fluid and serum for LPS challenged rats compared with GIK treated and control. This is described as † on the figure. P ≤ 0.001 for all groups, except for IL-1β in serum (LPS compared with GIK-treated), with a value of 0.042. The lowering in IL-1β concentration in GIK treated was only from 203 to 106 pg ml−1 explaining the high P-value. In GIK-treated rats the concentration of TNF-α or IL-1β was not different from control for interstitial fluid, but significantly different from control for serum, described as * on the figure (P < 0.001 and P = 0.007, respectively).

The rats receiving LPS had a significant increase in TNF-α and IL-1β in the interstitial fluid to 3800 ± 1100 and 16500 ± 5000 pg ml−1, respectively (P = 0.001 and P < 0.001, respectively, compared with control). Serum levels of TNF-α increased to 9400 ± 700 pg ml−1 and for IL-1β to 203 ± 34 pg ml−1 (P < 0.001 for both compared with control). In GIK-treated rats the levels in interstitial fluid were 3760 ± 120 pg ml−1 for TNF-α (P = 0.001 compared with experimental group and P > 0.05 compared with control) and for IL-1β 1050 ± 300 pg ml−1 (P < 0.001 compared with experimental group and P > 0.05 compared with control). In the GIK-treated rats the serum concentration of TNF-α was 4300 ± 950 pg ml−1 (P < 0.001 compared with experimental and control) and for IL-1β 106 ± 20 pg ml−1 (P = 0.042 compared with experimental and P = 0.007 compared with control). The control rats had serum levels of TNF-α and IL-1β of 160 ± 100 and 8.3 ± 8 pg ml−1, respectively, and levels in interstitial fluid of 150 ± 80 and 44 ± 44 pg ml−1, respectively.

Glucose measurements (Table 2)

Table 2.

Serum levels of glucose in experimental and control rats (n = 6)

Glucose (mmol l−1)
LPS + saline 6.3 ± 0.5
LPS + GIK 4.6 ± 0.3*
Control (saline) 6.2 ± 0.3
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Mean ± s.e.m.

*

P = 0.01 compared with control.

In the untreated control rats, non-fasted serum glucose averaged 6.2 ± 0.3 mmol l−1, and in the experimental rats receiving LPS the glucose level was 6.3 ± 0.5 mmol l−1. In the GIK-treated group the glucose level was lowered significantly to 4.6 ± 0.3 mmol l−1 (P = 0.01 compared with control).

Discussion

Our data demonstrate a novel function of insulin in dermis. After lowering of Pif with LPS or pro-inflammatory cytokines, insulin given subdermally in the skin reversed this lowering, and in fact raised Pif back to control values. This effect was repeated in all experimental groups. However, insulin given together with a PI3K inhibitor had no such effect. A continuous GIK infusion given to rats with endotoxaemia not only significantly attenuated oedema formation, measured as Ealb, but significantly reduced the amount of TNF-α and IL-1β in interstitial fluid in skin and in serum. These findings indicate both a local oedema preventing effect of insulin in dermis, and also an anti-inflammatory effect by inhibiting pro-inflammatory cytokine production.

Methodological consideration

We have earlier presented data demonstrating that Pif is lowered in different experimental models of inflammation in rat skin (Nedrebø et al. 1999). The method involves induction of circulatory arrest after inducing acute inflammation. This is done in order to avoid an increase in IFV, potentially causing an underestimation of Pif. It has been shown that reliable values of Pif can be obtained up to 90 min after circulatory arrest without significant change (Wiig et al. 1981).

Ealb and TTW were measured with a double tracer method using two radioactive isotopes. During a short experiment the concentration of the tracer in the interstitium is low enough for there only to be blood-tissue transport (Reed et al. 1993; Renkin & Tucker, 1998). Also, by having a short experimental period, as here, we avoid underestimation of extravasation due to removal of the tracer by lymph. Even though skin from both paw and back can be evaluated at the same time, only one time point per animal is obtained. There is a risk of measurement errors since Ealb is small compared to the distribution volumes of both tracers used. Due to possible higher degree of degradation by 131I-HSA, this tracer was chosen as the reference tracer for intravascular volume.

Different methods are being used for sampling of interstitial fluid in different organs. The interstitial fluid is important, as it is the fluid bathing the cells. Included among the methods used for sampling IF are microdialysis (Winter et al. 2002), wicks (Wiig et al. 1991), micropipettes (Sylven & Bois, 1960) and centrifugation. The problem with sampling of interstitial fluid has been the risk of contamination by intravascular or intracellular fluid. Wiig et al. (2003a) demonstrated that centrifugation of skin at a G-force of 424 or less was very reliable for sampling undisturbed interstitial fluid from skin and tumour. This method was successfully used for extracting interstitial fluid during experimental inflammation in rat skin, thereby being able to quantify the amount of cytokines found in interstitial fluid (Nedrebø et al. 2004).

Insulin and oedema-preventing effects

The control of Pif is partly regulated by connective tissue cells and the adhesion receptors that anchor them to structural connective tissue components (Berg, 1999). Integrins are transmembrane heterodimeric cell surface receptors mediating cell–cell or cell–matrix contact. By using polyclonal anti-β1-integrin antibodies locally in skin of rats, Pif was lowered (Reed et al. 1992). It was reasoned that the connective tissue cells via the β1-integrin receptors could exert tension on these fibre networks and thereby influence Pif. A reverse change in Pif was seen when platelet-derived growth factor BB was given subcutanously after lowering of Pif had been induced. PDGF-BB brought Pif back to control values. This was further investigated in mice homozygous for the mutated PDGF-β receptor (Heuchel et al. 1999). In these mice PDGF-BB could not restore a lowering of Pif, showing an in vivo role for the PDGF-β receptor via the PI3K pathway in control of tissue fluid homeostasis (Ahlen et al. 1998).

Insulin exerts important metabolic, but also cellular mitogenic, effects (Pelegrinelli et al. 2001). The tyrosine kinase activity of the cell surface insulin receptor (IR) is required to mediate its many biological actions (Guilherme & Czech, 1998). Binding of insulin stimulates the intrinsic tyrosine kinase of the IR, which results in phosphorylation of the β subunits on tyrosine residues and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) (White & Kahn, 1994). After stimulation, IRS-1 associates with several proteins, including PI3K. An increase in PI3K activity constitutes a key signalling pathway in growth factor-induced mitogenesis, chemotaxis, membrane ruffling and adhesion to fibronectin, and PDGF-BB stimulated fibroblast-mediated collagen gel contraction (Ahlen et al. 1998; Kinashi et al. 1995; Wennstrom et al. 1994). The latter suggests that one function of activated PI3K in PDGF-BB-stimulated cellular interactions with collagenous extracellular matrices is to elicit and/or maintain the Ca2+ response. In our present study, insulin given s.c. increased Pif after it had been lowered by LPS, TNF-α or IL-1β. By introducing a PI3K inhibitor together with insulin, this effect was blocked, suggesting a role of insulin signalling via the PI3K pathway (Table 1). LPS and TNF-α/IL-1β significantly increased Ealb and TTW in skin (Fig. 2), while this was not observed in rats treated with a continuous GIK infusion. These results together with reversal of Pif indicate that insulin could have an oedema-preventing effect.

Insulin and anti-inflammatory effects

Insulin resistance occurs in septic shock (Lang & Dobrescu, 1989; Glauser et al. 1991), and a typical observation in sepsis is an early hyperglycaemic phase and a later hypoglycaemic phase. In addition to elevated levels of serum glucose an increase in free fatty acids and glycerol is seen. This is indicative of an altered glucose and lipid metabolism, both of which are under the influence of insulin. The disturbances in glucose and lipid metabolism in sepsis can be related to changes in secretion of insulin (Yelich & Filkins, 1982), perturbation by endotoxin of insulin action at the cellular level (Holley & Spitzer, 1980) or development of insulin receptor autoantibodies (Spitzer et al. 1984). Taken together this indicates that insulin cannot bring out its optimum action in sepsis. To restore the physiological action of insulin, it can be given as a continuous infusion together with glucose and potassium. Glucose and potassium are given simultaneously to avoid hypoglycaemia and hypokalaemia. Over the last years there has been more focus on the anti-inflammatory effects of insulin, and its role has been investigated in sepsis, AMI, ischaemia–reperfusion and cardiac dysfunction. The results obtained in these studies indicate that insulin improves cardiac dysfunction in sepsis and septic shock. Studies were performed by giving insulin in a GIK regimen. A study performed on patients admitted to an intensive care unit showed that giving a GIK regimen focusing on a low and strict level of serum glucose had a significant effect on mortality and morbidity compared to less intensively treated patients (van den Berghe et al. 2001). One question arising is whether the effect is due to glucose or insulin. There is evidence indicating that the effect is not dependent on glucose (Rao et al. 1998) since insulin given at reperfusion of AMI in rats showed myocardial protection, and that the insulin protection acts via Akt prosurvival signalling (Jonassen et al. 2001). By using wortmannin the insulin protection via Akt was inhibited. Also there are studies indicating that a condition of hyperglycaemia is pro-inflammatory, as hyperglycaemia increases the production of reactive oxygen in cultured aortic endothelial cells (Giardino et al. 1996). Also high concentrations of glucose cause increased leucocyte rolling and adherence, together with an increased expression of P-selectin on endothelial surfaces in rats (Booth et al. 2001). Insulin on the other hand exerts anti-inflammatory effects by suppressing the expression of the transcription factor nuclear factor-κB (NF-κB) (Dandona et al. 2001) and free radical generation. LPS binding to its Toll-like receptor 4 (TLR4) stimulates induction of NF-κB. This again leads to the production of TNF-α (Wright et al. 2002) and subsequently IL-1β. The observation of blocking NF-κB and thereby reducing the amount of TNF-α is also observed in rats with hypovolaemic haemorrhagic shock (Altavilla et al. 2001). By suppressing NF-κB expression, insulin would not only attenuate production of pro-inflammatory cytokines, it will also reduce and inhibit effects of the same pro-inflammatory cytokines as this transcription factor is involved in the signalling pathway of inflammatory responses induced by TNF-α and IL-1β (Karin & Ben-Neriah, 2000). When analysing pro-inflammatory cytokines in interstitial fluid of skin and in serum of GIK-treated rats in our experiment, we found that they had significantly lower concentrations of TNF-α and IL-1β compared to the animals receiving LPS alone. It has been shown by others that insulin inhibits TNF-α found in serum (Satomi et al. 1985), but not in interstitial fluid. Previous studies performed in our laboratory have shown that pro-inflammatory cytokines increase oedema formation in skin, and also that the concentrations of the same cytokines increase in serum and interstitial fluid in skin in different inflammatory models. Based on these results insulin could act anti-inflammatorily by attenuating production of pro-inflammatory cytokines.

Concluding remarks

This study demonstrates a possible novel function of insulin in dermis. Insulin raises a lowered Pif to normal values when injected locally in the skin. Insulin given in a GIK regimen to rats challenged with LPS attenuates both an increased protein extravasation in skin, and production of TNF-α and IL-1β in interstitial fluid in skin and in serum. These results support other studies that favour the use of insulin as an anti-inflammatory agent, but also imply that insulin will attenuate the fluid flux from the circulation to the interstitium seen under endotoxaemia.

Acknowledgments

Financial support from The Norwegian Research Council, The Norwegian Heart Association and Locus on Circulatory Research University of Bergen are gratefully acknowledged.

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