Non Insulin Dependent Diabetes in Sand Rat (Psammomys obesus) and Production of Collagen in Cultured Aortic Smooth Muscle Cells. Influence of Insulin

In this report, we have shown that the standard laboratory diet administered to Psammomys obesus (sand rat) from Beni Abbes in Algeria, induced a non-insulin dependant diabetes, characterised by increase of body weight (p<0.001) as well as hyperinsulinemia, hyperglycemia and hypercholesterolemia. In cultured aortic smooth muscle cells (SMC) of sand rats, type I and type III collagen biosynthesis and insulin effects, at low dose, on these parameters were investigated. In all experimental conditions of cultured SMC study, The α chains of type I collagen were analysed by immunoblotting in media and cells. Metabolic radiolabelling and Immunochemical procedures revealed that, in diabetic state, synthetic SMC (SMCs) actively produce type I and III collagen which are synthesised in the cells and secreted in the medium; type I collagen was predominant as compared with type III collagen. Diabetes enhanced the collagen synthesis. Low dose of Insulin added to the medium, during 48h of incubation, induced a marked reduction in the synthesis of collagen types, especially type I collagen.

In this report, we have shown that the standard laboratory diet administered to Psammomys obesus (sand rat) from Beni Abbes in Algeria, induced a non-insulin dependant diabetes, characterised by increase of body weight (p<0.001) as well as hyperinsulinemia, hyperglycemia and hypercholesterolemia. In cultured aortic smooth muscle cells (SMC) of sand rats, type and type III collagen biosynthesis and insulin effects, at low dose, on these parameters were investigated. In all experimental conditions of cultured SMC study, The x chains of type collagen were analysed by immunoblotting in media and cells.
Metabolic radiolabelling and Immunochemical procedures revealed that, in diabetic state, synthetic SMC (SMCs) actively produce type and III collagen which are synthesised in the cells and secreted in the medium; type I collagen was predominant as compared with type III collagen. Diabetes enhanced the collagen synthesis. Low dose of Insulin added to the medium, during 48h of incubation, induced a marked reduction in the INTRODUCTION The atherosclerotic process affects the intima and media; it concerns all the cells of the aortic wall especially smooth muscle cells (SMC). [1] The migration of SMC from media to intima and the change of the SMC phenotype from contractile state to synthetic state are considered as the major events in the atherosclerotic process. [2,3] They are caused by endothelial cell dysfunctioning [1,4] which is signalled to SMC by cell to cell interaction. [5,61 Thus, SMC undergo the change of their phenotype and become synthetic and proliferative (SMCs). [7] Cultured SMCs show analogies with the observed SMCs in atherosclerotic lesions; they spontaneously lose their contraction capacities and increase their secretion capacities. [ [9,10] representing greater cardiovascular risks.
During diabetes, atherosclerosis process is accelerated. The increased SMC proliferation is an abnormality, which aggravates the process [111 as well as the dysfunctioning of the extracellular matrix (ECM) especially the quantitative changes of the collagen. In fact, the SMC is engaged in repetitive divisions and in an ECM synthesis in non-conformity to the genetic coded program at a contractile. [12,13] Our study was carried out on aortic SMCs cultured from diabetic Psammomys obesus. This model has the characteristic of developing a nutritional diabetic syndrome comparable to clinical diabetes with degenerative and vascular complications. [14] This study involves morpho- When trapped, animals were allowed to adapt to laboratory conditions for 15 days and fed on natural vegetables only. In the animal room, animals were maintained in conditions previously described by Marqui6 et al. [161 Animals of matched ages (2-3 months) and adapted to laboratory conditions were divided into two groups: 7 were fed a natural plant diet (50g/day) during 6 months and the daily caloric intake was 20kcal. 7 animals were fed a laboratory diet (rich in carbohydrates) with salt water "ad libitum" (NaC1 0.9%) during the same period. Their food consumption (10g/day) represented a higher caloric intake (32.5kcal/ day/animal). At the time of killing, sand rats were 9 months old. Animals fed on high caloric diet showed a relative weight gain and insulin levels increased over the weeks that followed. After the third month, they developed a non insulin dependent diabetes as described by Marqui6 et al. [161 At the sixth month of experiments, animals of the two groups: the normal group placed on hypocaloric diet (control group) and the diabetic group feeding high caloric diet, were killed.

Analytical Techniques
The animals were bled from the retro-orbital venous plexus; this technique eliminates using anaesthesic agents which affect measurements of biochemical parameters. Blood collected in tubes containing heparin was centrifuged at 3000 rpm for 10 min and plasma was stored at 30oc. Blood glucose and cholesterol were measured by the enzymatic colorimetric method using a test kit of Boehringer. Blood Insulin was determined by radioimmunoassay using CIS test kit (ORIS INDUS). Aortic SMC Culture Cellular culture technique was used according to Ross, [17] modified by Bourdillon et al. [18] Figure 1. For cultured SMC, explants were obtained from normal and diabetic thoracic aorta. They were prepared after removing SMCs in normal state SMCs in diabetic state FIGURE 1 Microscopic appearance of cultured aortic SMC at synthetic phenotype, they were fixed in aqueous bath and stained in May Grunwald Giemsa. X 200. In proliferative phase, aortic SMC of normal and diabetic Psammomys obesus were in synthetic state and presented a polygonal aspect.

Morphometric Measurements
In proliferative state, the medium was eliminated from the patches and SMCs were washed with PBS at 10% then fixed in aqueous bath and coloured with May Grunwald Giemsa.
Morphometric analysis was carried out of on 100 measurements of cellular and nuclear great axes as well as nucleolus numeration.

Collagen Biosynthesis
According to the method of Peterkofsky et al., [191 cells were labeled with L-5-3H proline (10gCi/ml, specific activity 20Ci/mmol, Isotopchim) during 24h in medium culture without foetal calf serum containing 10gg/ml of ascorbic acid. Total collagen, in medium and cells, was estimated after three successive dialyses; the first two against water, the third against acetic acid 0,5M and pepsin (200tg/ml). Radioactivity was measured by liquid scintillation and results were expressed in cpm/106cells. The collagen biosynthesis relative to total protein synthesis was estimated according to Wiestner et al. [20] Collagen Types After pepsination, medium (ECC) and cells (ICC) were lyophilised and resuspended in buffer solution (tris HC1, 0.05M, SDS 14%, bromophenol blue 0,05%, glycerol 0,5% and EDTA 2mM). The different fractions of radiolabeled collagen chains were separated by vertical electrophoresis on polyacrylamide gel (SDS-PAGE) according to the procedure of Laemmli. [21] After the gel discolouration, 01 and 02 collagenous chains were evaluated by densitometric analysis. At the end of this step, the gel was dried for 3h at 80C and the 0 chains of collagen separated on SDS-PAGE were quantified by excision of each o band. The radioactivity was eluted in hydrogen peroxide at 12% during 24h. The radioactivity was measured by liquid scintillation and expressed in cpm/106 cells. The radioactivity recovered in type I and type III collagen was quantified by numeric integration.

Immunoblotting
This study was carried out on type I collagen. For immunological identification, c1 and chains of type I collagen, contained in medium (ECC) and cells matrix (ICC), were transferred to nitrocellulose sheets (0,45tm of diameter) in tris buffer (0,189%), glycin (0,912%), methanol 20% at pH 8,6 by the method of Towbin et al. [22] Transferred proteins were incubated with first antiserum (at 4/100 dilution). After an overnight incubation at 4C, a second specific antiserum (at 1/250 dilution, Sanofy) linked to the peroxidase was added to bind the antigenantibody complex during a 2-h period at room temperature; the bands were revealed by tetrachloride 3-3 DAB 0.5%.
We prepared in our laboratory the rabbit antiserum against bovine type I collagen (Sigma) and the goat,,antiserum against bovine type III collagen (Sigma) used in the immunological procedures.
Antisera were titrated by qualitative antigen diffusion (type I or III collagen) and corresponding antiserum by distribution in wells bored in 1.5% agar gel. The antigen-antibody reaction was accelerated under low voltage (25A, 150 V) during 3h.

Influence of Insulin
Human insulin (Mixtar) was added at low doses (4mU/ml) to the medium of diabetic Psammomys obesus SMCs, during 48-h, to analyse his effects on morphological variation cells and on type I and type III collagen production.

Statistical Analysis
Glycemia, lipidemia, cellular proteins and collagen indicated in Tables were analysed statistically with Student's test and are expressed as mean+SD; insulin was analysed with Mann Whitney test.

Biochemical Study
The biochemical parameters can be seen in Table I. Psammomys obesus on the high caloric diet became diabetic, exhibited hyperglycemia and hypercholesterolemia with increase of esterified and free cholesterol compared with the control group (natural diet). These metabolic changes were associated with excess body weight (p<0.001).

Morphometric Study
Morphometric results indicated in the Table II showed that cellular (LC) and nuclear (LN) great axes were increased in the diabetic state (p<0,0001 for LC p<0.001 for LN). The number of nucleoli augmented from 3.2___0.9 in control group to 4.9 _+ 1.3 in diabetic group. The insulin influence during 48 h caused a slight decrease of

Biosynthesis of Total Collagen
The results of these parameters were indicated in   Immunological procedures FIGURE 5 Immunodiffusion test of anti rabbit type and anti goat type III collagen serum with corresponding antigens (type and type III collagen from Sigma). In Figure 5a two specific arc corresponding to anti goat type III collagen serum (anti R1 and anti 0t2 chains serum), in Figure 5b one specific arc corresponding to anti goat type III collagen serum (anti o1 chains serum). and the cell-matrix extract from aortic SMCs were submitted to SDS PAGE electrophoresis and immunoblotted with anti-rabbit type collagen serum (at 4/100 dilution). Bound antibody was detected with a second specific antiserum: anti-anti-rabbit serum (at 1/250 dilution) linked to the peroxidase and visualised by tetrachloride 3-3 DAB 0.5%. Designated markers were sized between phosphorylase (94 Kda) and cytochrome C (12.5 Kda).

MEDIUM
increased in the ratio of 9,3 versus 8,1 for type III collagen. In cells, the respective increase ratios of type I and type III collagen were 13.1 and 11.6. The insulin influence, during 48-h at low dose (4mU/ml) has induced a strong decrease of the radioproline incorporated into both compart-ments (p <0.0001). However, this decrease was more pronounced for type I collagen than type III collagen (p<0.001 for medium, p<0.01 for cells).
Total type I and type III collagen decreased but remained higher than the normal values (p<0,0002 for type I collagen p<0,0001 for type III collagen).

Immunological Procedures
Examination of the medium and cells by the immunoblott procedure demonstrated in Figure 6 that cultures of SMCs synthesised type I collagen which was secreted in the medium. In fact, the cell matrix and the medium contained both c1 and c2 chains. In diabetic state, the immunological identification of type I collagen in medium and cellmatrix showed a pro-nounced increase of c1 and c2 chains in the two compartments as observed in Figure 6. Otherwise, the antiserum used is known to react favourably with both chains. Before the biosynthesis study procedure, a densitometric analysis was made SDS-PAGE gel. The profiles of c chains of collagen types, obtained from medium and cells are shown in

DISCUSSION
According to the works of Marqui6 et al. [16,231 and Lahfa et al., [24] Psammomys obesus under hypercaloric diet during one month developed obesity with hyperinsulinemia and after three months developed diabetic syndrome characterised by hyperglycemia and elevateddisturb lipid concentration.
The cultured SMCs of diabetic Psammomys obesus presented striking changes. The morphometric method revealed an increase of cellular and nuclear great axes compared with normal cells. The number of nucleoli was also increased by 1.6. These changes could be in relation to the functional state of the cells which develop many organelles in the synthetic state such as ribosome, ergastoplasm, Golgi, etc. [25] Simons and Vanderbroeck [26] have noted these morphometric changes in aged cells [26] and the early ageing characterised the diabetic angiopathy. [27] In diabetic state, collagen biosynthesis is marked by a statistical increase of the labelled proline pool in ECC (x5.6) and ICC (x9.3), corresponding to the anarchic synthesis and disorganisation of the ECM during the atherosclerotic process development. [12] The newly synthesised collagen that constitutes the major ECM component, [28] marks the aggravation of the cellular alteration due to diabetes and causes fibrosis state. [29,30,31] In fact, the fibrosis of the aortic wall would be due to the presence of collagen, [32] which is dominant in the proliferative intima. [33] Our qualitative and quantitative study on the phenotypic expression of collagen has shown that cultured SMCs of the normal and diabetic Psammomys obesus synthesise more type I than type III collagen, in accord with the results of Layman et al. [34] in SMCs of adult humans, pigs and rabbits. Carey et al. [351 and Lawrence et al. [36] have shown that SMC produced essentially collagen I and III. Ang et al. [37] have observed an activation of mRNA coding 1 (I) and czl (III) chains of procollagen in rabbit synthetic SMCs. Compared with control, the diabetic state induced a large increase in type I and type III collagen biosynthesis and secretion. In addition, comparatively to collagen III, collagen I appeared more important in the medium (x10.8 vs x9.3) than in the cells (x13.6 vs x11.6). Robert [29] indicated that the newly synthesised collagen in the atherosclerotic lesions is due to type I collagen. Karim et al. [33] have noted equally an increase of mRNA coding for 01 (I) zl (III) chains after 30 days of experimental angioplasty. Katsuda et al. [32] have shown the coexistence of the two phenotypes in the intimal lesions, but type I collagen would stimulate the SMC migration, t38] During the development of the atherosclerotic process, many authors showed that collagen molecules are also subject to qualitative alterations.
These alterations would correspond to the progressive insolubility [39] and to the nonenzymatic glycosylation [40] which accelerates the macroangiopathic progression. [1,6] These processes would aggravate with diabetes. [411 However, the qualitative alterations of collagen have no influence on the immunological identification of 01 and 02 chains of type I collagen during diabetes.
In our study, the low dose insulin (4 mU/ml) at short term (48h) presented a therapeutic effect. Insulin promptly reduced all studied morphological and biochemical parameters of the SMCs of diabetic Psammomys obesus. In fact, the ECM synthesis of type I collagen especially the elevated ratio type I/type III collagen which was observed in diabetic Psammomys obesus SMCs, pronouncedly decreased. Thus, our results suggest that insulin at low dose induced inhibitory effects as compared with high doses studied by Kjelst6m and Mainquist [421 on fibroblasts of diabetic patients according with Stout [43] and Sowers. [44] They observed that high doses used are comparable to hyper-insulinemia and accelerated the atherosclerotic process in obese and diabetic patients. In addition, we have observed that SMCs activities of diabetic Psammomys obesus decreased under insulin effect but remain more important than the controls