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In vivo collagen metabolism in spontaneously diabetic (dbdb ) mice
EXPERIMENTAL
AND
MOLECULAR
In Viva Collagen
PATHOLOGY
45, 221-226 (1986)
Metabolism (db/db)
in Spontaneously Mice
Diabetic
JON C. BOWERSOX’ Vascular
Research University
Laboratory,
of Southern Received
Estelle Doheny Eye Foundation California School of Medicine, March
26, 1986, and in revised
and Department of Ophthalmology. Los Angeles, California 90033
form April
10, 1986
To further define the pathogenesis of diabetic connective tissue lesions, collagen synthesis and degradation were measured in viva in spontaneously diabetic dbidb mice. A double isotopic labeling technique, in which “T-labeled and 3H-labeled proline were injected into the same mouse 7 days apart, was applied. Collagen synthesis and degradation were assessed in skins, intestines, hearts, and kidneys. There were no changes in collagen metabolism in the intestines of the diabetic mice. in all other tissues, collagen degradation was accelerated. Collagen synthesis was decreased in skins, but increased in the hearts and kidneys of the diabetic mice. These tissue-specific changes in collagen metabolism resulted in a net loss of collagen in all tissues examined except intestines. The results of this study provide insight into the mechanisms leading to connective tissue defects occurring in diabetes ItIe~~itUS. 0 1986 Academic Press. Inc.
INTRODUCTION Changes in collagen metabolism occur in experimental diabetes mellitus. In vivo collagen synthesis is decreased in skins of aortas, but increased in intestines of rats rendered diabetic by streptozotocin injection (Schneir ef al., 1979). There is also evidence that there is accelerated degradation of newly synthesized skin collagen in streptozotocin-diabetic rats (Schneir et al., 1982, 1984). Furthermore, both streptozotocin- and alloxan-induced diabetes result in increased rates of collagen synthesis in glomerular basement membranes of rats (Cohen and Khalifa, 1977; Hasselachler and Wahl, 1980). These studies have all focused on the acute effects of chemically induced insulin deprivation as a model of insulin-dependent diabetes mellitus. In such systems, the genetic components and long-term effects of diabetes on connective tissues cannot be evaluated. Alternatively, animals that spontaneously develop diabetes may prove more useful for delineating chronic connective tissue changes occurring in this disease. The “dbidb” mouse is an animal model of non-insulin-dependent diabetes mellitus (NIDDM), in which tissue insensitivity to circulating insulin develops. The db/db mice are obese, have normal or elevated levels of plasma insulin, and develop chronic tissue changes analogous to those observed in NIDDM (Hummel ef al., 1966; Coleman, 1983). Biochemical defects in the cells of db/db mice (Kahn et al., 1973; Posner et al., 1978) and the pattern of inheritance of the disease traits (Coleman, 1978, 1982) support the importance of the genetic components in this animal model. This report describes experiments using spontaneously diabetic db/db mice to assess collagen synthesis and degradation in viva, after prolonged periods of hyperglycemia. ’ Present address: Department of Surgery, Box 126, Madigan Army Medical Center, Tacoma, Wash. 9843 1.
221 0014-4800/86 $3.00 Copyright All rights
0 1986 by Academic Press. Inc. of reproduction in any form reserved.
222
JON
MATERIALS
C. BOWERSOX
AND METHODS
Diabetic C57BL db/db mice and normoglycemic littermate controls (n = 6 in each group) were obtained from Jackson Laboratories, Bar Harbor, Maine. Diabetes was confirmed by observing glycosuria and increased body weights in the diabetic mice, but not in the litermate controls (diabetics, 49.3 & 1.7 g; controls, 22.5 -+ 1.9 g). Collagen synthesis and degradation were measured concurrently in the same animal by a double-label isotopic technique (Dice et al., 1978). Five-month-old mice were injected intraperitoneally with [14C]proline (Schwarz-Mann, Inc., 260 mCi/mmole). Control mice received a dose of 0.45 &i/g body wt; diabetic animals received 0.32 &i/g body wt. This dose adjustment was made based on the estimated pool of distribution of the water-soluble, radiolabeled amino acid and accounted for the increased fat content in the diabetic mice (Rettori et al., 1964). The total doses of isotope administered were 10.1 l&i in the control mice and 15.8 pCi in the diabetic mice. Seven days after the mice received the [*4C]-proline injection, they were injected intraperitoneally with [3H]proline (29.3 Ci/mmole) (controls, 4.6 lKi/g; diabetics, 3.2 $X/g). Four hours later the mice were killed by cervical dislocation. Entire shaved skins, as well as intestines, hearts, and kidneys were excised and processed for hydroxyproline analysis. Total hydroxyproline was measured in acid hydrolysates using an automated procedure as previously described (Grant, 1964). Radiolabeled hydroxyproline was separated by ion-exchange chromatography (Schneir et al., 1979) and was assayed using a Beckman LS-9000 liquid scintillation counter. Using this experimental design, two time points on a curve representing collagen turnover were obtained in the same animal: [3H]hydroxyproline values, obtained after 4 hr of radiolabeling, provided the initial points describing collagen synthesis; [14C]hydroxyproline levels were measured 7 days after isotope injection and reflected the amount of collagen degradation occurring over 7 days. The [3H]/[14C]hydroxyproline ratios provided an indicator of relative rates of degradation. In an adult animal, with no net tissue accretion, tissues in which there is accelerated collagen degradation will have elevated [3H]/[14C]hydroxyproline ratios. RESULTS Body weights of the control and diabetic mice did not change over the 2 months preceding the radiolabeling period. Diabetic mice consistently weighed over twice as much as control animals. As expected, increased organ masses were also observed in the diabetic mice: dry weights were 53-81% greater than controls (Table I). Although a similar increase in tissue collagen mass was also anticipated, such was not the case. In skins, hearts, and kidneys of the diabetic mice tissue collagen concentrations were reduced by 32-40%; only in intestines were collagen concentrations unchanged (Table I). The net result of the changes in collagen concentration was a relatively decreased organ collagen mass in all diabetic tissues except intestines (Fig. 1). The observed reduction in collagen mass could have resulted from decreased collagen synthesis, increased collagen degradation, or both. The double-isotope radiolabeling technique used in this experiment allowed the measurement of each component independently.
COLLAGEN
METABOLISM
IN DIABETIC
TABLE I Effects of Diabetes on Tissue Mass, Collagen Concentration,
Tissue Skin Control Diabetic Intestine Control Diabetic Heart Control Diabetic Kidney Control Diabetic
Tissue collagen concentration0
Tissue dry wt (mg)
OLdmd
223
MICE
and Collagen Metabolism in db/db Mice Specific radioactivitiesb [3H]HP
[“‘CIHP
3H/‘4C
472.0 k 33.4’ 854.3 k 77.0
184.3 f 3.7 110.0 * 7.0
2.6 2 0.2 2.3 2 0.2
3.3 2 0.2 2.0 e 0.2
0.8 1.2
80.4 2 4.1 123.1 ? 7.0
41.8 i 4.6 44.7 k 2.1
49.6 k 0.4 49.2 ? 3.6
24.2 rt 1.6 24.5 k 2.9
2.0 2.0
29.3 ct 2.1 47.9 + 0.9
18.7 t 1.4 12.7 t 1.6
37.2 4 0.5 51.1 4 I.0
50.4 2 0.4 59.5 -t 0.8
0.7 0.9
62.1 f 1.3 103.6 2 6.7
9.0 _’ 0.6 6.0 2 0.4
35.5 2 2.4 42.2 2 1.3
55.7 -e 2.1 54.1 k 1.8
0.6 0.8
0 Tissue collagen concentration = tissue hydroxyproline x 7.46. b Hydroxyproline specific radioactivities are expressed as dpm hydroxyproline/pg c Data are expressed as means 2 SD (n = 6 for each group).
hydroxyproline.
Intestinal collagen metabolism was as expected if there were no differences between control and diabetic mice. [3H]Hydroxyproline specific radioactivities, indicative of collagen synthesis, were equal in the intestines of the normal and diabetic mice, as were [14C]hydroxyproline specific radioactivities, which represented collagen degradation (Fig. 2). Furthermore, the [3H]l[*4C]hydroxyproline ratios were equal in the two groups, as expected for animals in a steady-state condition, without ongoing collagen gain or loss. In contrast, skin collagen synthesis in the diabetic mice was decreased, as shown by a 12% reduction in the [3H]hydroxyproline specific radioactivities (Fig. loo-
SO-
so-
40-
20P 2 o %
o-Skin -20
htestine
Kidney
-
.-40-
FIG. 1. Effect of diabetes on total organ mass (open bars) and collagen mass (crosshatched bars) in db/db mice. Data are expressed as percentage change from littermate controls. Collagen mass was calculated as tissue mass x collagen concentration.
224
JON C. BOWERSOX
20
10
P e
0
z
-10
-20
-30
-40
J
FIG. 2. Effect of diabetes on [3H]hydroxyproline specific radioactivities, indicative of collagen synthesis (open bars) and [14C]hydroxyproline specific radioactivities, representing collagen degradation (crosshatched bars). Data are expressed as percentage change from littermate controls.
2). Collagen degradation was accelerated in the skins of the diabetic mice, as demonstrated by the 36% reduction in the [i4C]hydroxyproline pool, which was out of proportion to that expected from decreased collagen synthesis (13H]/ [i4C]hydroxyproline ratio of 0.8 in skins of control mice and 1.2 in diabetic mice). In hearts and kidneys of the diabetic mice there was an increase in collagen synthesis relative to controls. However, collagen degradation was also accelerated in the diabetic tissues, resulting in net reductions in tissue collagen concentrations (Table I). This conclusion was supported by the observed increase in tissue [3H]/[14C]hydroxyproline ratios in diabetic hearts and kidneys. DISCUSSION One prominent hypothesis explaining the mechanisms of diabetic connective tissue changes is that accelerated aging of collagen occurs in diabetes, resulting in less salt-extractable collagen (Behera and Patnaik, 1979). These changes appear similar to those occurring in normal tissues during the aging process (Schnider and Kohn, 1982) and have been attributed to increased collagen crosslinking. Alternatively, connective tissue changes may result from perturbations in collagen turnover, as demonstrated in the present study. Collagen synthesis and degradation were assessed in chronically hyperglycemic mice over a 7-day period. Whereas intestinal collagen metabolism appeared to be unaffected in the db/db mice, collagen degradation was accelerated in other tissues that were examined. Collagen synthesis was also affected, but in a tissue-specific manner. In skins from the diabetic mice, collagen synthesis was decreased compared to littermate controls; hearts and kidneys demonstrated increased collagen synthesis. In streptozotocin-diabetic rats, tissue specific changes in collagen have also been observed (Schneir et al., 1979). In contrast to the db/db mice, however,
COLLAGEN
METABOLISM
IN DIABETIC
MICE
225
intestinal collagen synthesis was markedly increased after insulin deprivation and correlated well with organ hypertrophy. One consistent change observed in both the diabetic rats and mice was accelerated skin collagen degradation (Schneir et al., 1982). Enhanced protein degradation has also been reported in hindquarter muscle preparations from db/db mice (Shargill er al., 1984). As suggested by Dice et al. (1978) and others, increased protein degradation may be a uniform response by cells to insulin deprivation. Another possibility is that diabetes results in the biosynthesis of defective collagen molecules that are rapidly degraded before secretion (Bienkowski et al., 1978; Schneir et al., 1984). The reason for the tissue-specific nature of the changes observed in collagen metabolism is unclear, but the results emphasize the need to address each organ independently when evaluating diabetic connective tissue changes. One important question that remains to be addressed is whether vascular lesions observed in diabetics are also organ-specific or whether the effects on the vascular system are similar throughout the body. ACKNOWLEDGMENTS These studies were supported in part by NIH Grants DE-076006 and EY-3129. The helpful discussions with Dr. Michael Schneir and Dr. Nino Sorgente are gratefully appreciated.
REFERENCES H. N., and PATNAIK, B. K. (1979). In vivo and in vitro effects of alloxan on collagen characteristics of bone, skin and tendon of Swiss mice. Gerontology 25, 255-260. BIENKOWSKI, R. S., COWAN, M. J., MCDONALD, J. A., and CRYSTAL, R. G. (1978). Degradation of newly synthesized collagen. J. Biol. Chem. 253, 4356-4363. COHEN, M. P, and KHALIFA, A. (1977). Renal glomerular collagen synthesis in streptozotocin diabetes: Reversal of increased basement membrane synthesis with insulin therapy. Biochim. Biophys. Acta 500, 395-404. COLEMAN, D. L. (1978). Obesity and diabetes: Two mutant genes causing diabetes-obesity syndromes in mice. Diubetologia 14, 141- 148. COLEMAN, D. L. (1982). Diabetes-obesity syndromes in mice. Diabetes 31(Suppl 1). 1-6. COLEMAN, D. L. (1983). Lessons from studies with genetic forms of diabetes in the mouse. Metabolism 32(Suppl 1). 162-165. DICE, J. F., WALKER, C. D., BYRNE, B., and CARDIEL. A. (1978). General characteristics of protein degradation in diabetes and starvation. Proc. Nut/. Acad. Sci. USA 75, 2093-2097. GRANT, R. A. (1964). An automated procedure for the determination of hydroxyprohne. J. Clin. Pathol. 17, 685-686. HASSELACHLER, C. H.. and WAHL, P. (1980). Influence of diabetes control in synthesis of protein and basement membrane collagen in isolated glomeruli of diabetic rats. Res. Exp. Med. 176, 247-253. HUMMEL. K. P, DICKIE, M. M., and COLEMAN, D. L. (1966). Diabetes, a new mutation in the mouse. Science 153, 1127-l 128. KAHN, C. R., NEVILLE, D. M., JR., and ROTH, J. (1973). Insulin receptor interaction in the obese hyperglycemic mouse. A new model of insulin resistance. J. Biol. Chem. 248, 244-250. POSNER, B. I., RAQUIDAN, D., JOSEFSBERG, Z.. and BERGERON, J. J. (1978). Different regulation of insulin receptors in intracellular (Golgi) and plasma membranes from livers of obese and lean mice. Proc. Natl. Acad. Sri. USA 75, 3302-3305. REI-~ORI, 0.. MEJIA, R. H., and FERNANDEZ, L. A. (1964). Factors of variation in blood volume determinations in the rat. Acta Physiol. Lat. Amer. 14, 221-230. SCHNEIR, M., BOWERSOX, J., RAMAMURTHY, N., YAVELOW. J.. MURRAY, J., EDLIN-FOLZ, E., and GOLUB, L. (1979). Response of rat connective tissues to streptozotocin diabetes. Tissue specific effects on collagen metabolism. Biochim. Biophys. Acta 583, 95-102. SCHNEIR, M., RAMAMURTHY, N., and GOLUB. L. (1982). Skin collagen metabolism in the streptozoBEHERA,
226
JON C. BOWERSOX
tocin-induced diabetic rat: Enhanced catabolism of collagen formed before and during the diabetic state. Diabetes 31, 426-431. SCHNEIR, M. L., RAMAMURTHY, N. S., and GOLUB, L. M. (1984). Extensive degradation of recently synthesized collagen in gingiva of normal and streptozotocin-induced diabetic rats. J. Dent. Res. 63, 23-27.
SCHNIDER, S. L., and KOHN, R. R. (1982). Effects of age and diabetes mellitis on the solubility of collagen from human skin, tracheal cartilage and dura mater. Exp. Geroniol. 17, 185-194. SHARGILL, N. S., OSHIMA, K., BRAY, G. A., and CHAN, T. M. (1984). Muscle protein turnover in the perfused hindquarters of lean and genetically obese-diabetic (dbidb) mice. Diabetes 33, 1160- 1164.
-
AND
MOLECULAR
In Viva Collagen
PATHOLOGY
45, 221-226 (1986)
Metabolism (db/db)
in Spontaneously Mice
Diabetic
JON C. BOWERSOX’ Vascular
Research University
Laboratory,
of Southern Received
Estelle Doheny Eye Foundation California School of Medicine, March
26, 1986, and in revised
and Department of Ophthalmology. Los Angeles, California 90033
form April
10, 1986
To further define the pathogenesis of diabetic connective tissue lesions, collagen synthesis and degradation were measured in viva in spontaneously diabetic dbidb mice. A double isotopic labeling technique, in which “T-labeled and 3H-labeled proline were injected into the same mouse 7 days apart, was applied. Collagen synthesis and degradation were assessed in skins, intestines, hearts, and kidneys. There were no changes in collagen metabolism in the intestines of the diabetic mice. in all other tissues, collagen degradation was accelerated. Collagen synthesis was decreased in skins, but increased in the hearts and kidneys of the diabetic mice. These tissue-specific changes in collagen metabolism resulted in a net loss of collagen in all tissues examined except intestines. The results of this study provide insight into the mechanisms leading to connective tissue defects occurring in diabetes ItIe~~itUS. 0 1986 Academic Press. Inc.
INTRODUCTION Changes in collagen metabolism occur in experimental diabetes mellitus. In vivo collagen synthesis is decreased in skins of aortas, but increased in intestines of rats rendered diabetic by streptozotocin injection (Schneir ef al., 1979). There is also evidence that there is accelerated degradation of newly synthesized skin collagen in streptozotocin-diabetic rats (Schneir et al., 1982, 1984). Furthermore, both streptozotocin- and alloxan-induced diabetes result in increased rates of collagen synthesis in glomerular basement membranes of rats (Cohen and Khalifa, 1977; Hasselachler and Wahl, 1980). These studies have all focused on the acute effects of chemically induced insulin deprivation as a model of insulin-dependent diabetes mellitus. In such systems, the genetic components and long-term effects of diabetes on connective tissues cannot be evaluated. Alternatively, animals that spontaneously develop diabetes may prove more useful for delineating chronic connective tissue changes occurring in this disease. The “dbidb” mouse is an animal model of non-insulin-dependent diabetes mellitus (NIDDM), in which tissue insensitivity to circulating insulin develops. The db/db mice are obese, have normal or elevated levels of plasma insulin, and develop chronic tissue changes analogous to those observed in NIDDM (Hummel ef al., 1966; Coleman, 1983). Biochemical defects in the cells of db/db mice (Kahn et al., 1973; Posner et al., 1978) and the pattern of inheritance of the disease traits (Coleman, 1978, 1982) support the importance of the genetic components in this animal model. This report describes experiments using spontaneously diabetic db/db mice to assess collagen synthesis and degradation in viva, after prolonged periods of hyperglycemia. ’ Present address: Department of Surgery, Box 126, Madigan Army Medical Center, Tacoma, Wash. 9843 1.
221 0014-4800/86 $3.00 Copyright All rights
0 1986 by Academic Press. Inc. of reproduction in any form reserved.
222
JON
MATERIALS
C. BOWERSOX
AND METHODS
Diabetic C57BL db/db mice and normoglycemic littermate controls (n = 6 in each group) were obtained from Jackson Laboratories, Bar Harbor, Maine. Diabetes was confirmed by observing glycosuria and increased body weights in the diabetic mice, but not in the litermate controls (diabetics, 49.3 & 1.7 g; controls, 22.5 -+ 1.9 g). Collagen synthesis and degradation were measured concurrently in the same animal by a double-label isotopic technique (Dice et al., 1978). Five-month-old mice were injected intraperitoneally with [14C]proline (Schwarz-Mann, Inc., 260 mCi/mmole). Control mice received a dose of 0.45 &i/g body wt; diabetic animals received 0.32 &i/g body wt. This dose adjustment was made based on the estimated pool of distribution of the water-soluble, radiolabeled amino acid and accounted for the increased fat content in the diabetic mice (Rettori et al., 1964). The total doses of isotope administered were 10.1 l&i in the control mice and 15.8 pCi in the diabetic mice. Seven days after the mice received the [*4C]-proline injection, they were injected intraperitoneally with [3H]proline (29.3 Ci/mmole) (controls, 4.6 lKi/g; diabetics, 3.2 $X/g). Four hours later the mice were killed by cervical dislocation. Entire shaved skins, as well as intestines, hearts, and kidneys were excised and processed for hydroxyproline analysis. Total hydroxyproline was measured in acid hydrolysates using an automated procedure as previously described (Grant, 1964). Radiolabeled hydroxyproline was separated by ion-exchange chromatography (Schneir et al., 1979) and was assayed using a Beckman LS-9000 liquid scintillation counter. Using this experimental design, two time points on a curve representing collagen turnover were obtained in the same animal: [3H]hydroxyproline values, obtained after 4 hr of radiolabeling, provided the initial points describing collagen synthesis; [14C]hydroxyproline levels were measured 7 days after isotope injection and reflected the amount of collagen degradation occurring over 7 days. The [3H]/[14C]hydroxyproline ratios provided an indicator of relative rates of degradation. In an adult animal, with no net tissue accretion, tissues in which there is accelerated collagen degradation will have elevated [3H]/[14C]hydroxyproline ratios. RESULTS Body weights of the control and diabetic mice did not change over the 2 months preceding the radiolabeling period. Diabetic mice consistently weighed over twice as much as control animals. As expected, increased organ masses were also observed in the diabetic mice: dry weights were 53-81% greater than controls (Table I). Although a similar increase in tissue collagen mass was also anticipated, such was not the case. In skins, hearts, and kidneys of the diabetic mice tissue collagen concentrations were reduced by 32-40%; only in intestines were collagen concentrations unchanged (Table I). The net result of the changes in collagen concentration was a relatively decreased organ collagen mass in all diabetic tissues except intestines (Fig. 1). The observed reduction in collagen mass could have resulted from decreased collagen synthesis, increased collagen degradation, or both. The double-isotope radiolabeling technique used in this experiment allowed the measurement of each component independently.
COLLAGEN
METABOLISM
IN DIABETIC
TABLE I Effects of Diabetes on Tissue Mass, Collagen Concentration,
Tissue Skin Control Diabetic Intestine Control Diabetic Heart Control Diabetic Kidney Control Diabetic
Tissue collagen concentration0
Tissue dry wt (mg)
OLdmd
223
MICE
and Collagen Metabolism in db/db Mice Specific radioactivitiesb [3H]HP
[“‘CIHP
3H/‘4C
472.0 k 33.4’ 854.3 k 77.0
184.3 f 3.7 110.0 * 7.0
2.6 2 0.2 2.3 2 0.2
3.3 2 0.2 2.0 e 0.2
0.8 1.2
80.4 2 4.1 123.1 ? 7.0
41.8 i 4.6 44.7 k 2.1
49.6 k 0.4 49.2 ? 3.6
24.2 rt 1.6 24.5 k 2.9
2.0 2.0
29.3 ct 2.1 47.9 + 0.9
18.7 t 1.4 12.7 t 1.6
37.2 4 0.5 51.1 4 I.0
50.4 2 0.4 59.5 -t 0.8
0.7 0.9
62.1 f 1.3 103.6 2 6.7
9.0 _’ 0.6 6.0 2 0.4
35.5 2 2.4 42.2 2 1.3
55.7 -e 2.1 54.1 k 1.8
0.6 0.8
0 Tissue collagen concentration = tissue hydroxyproline x 7.46. b Hydroxyproline specific radioactivities are expressed as dpm hydroxyproline/pg c Data are expressed as means 2 SD (n = 6 for each group).
hydroxyproline.
Intestinal collagen metabolism was as expected if there were no differences between control and diabetic mice. [3H]Hydroxyproline specific radioactivities, indicative of collagen synthesis, were equal in the intestines of the normal and diabetic mice, as were [14C]hydroxyproline specific radioactivities, which represented collagen degradation (Fig. 2). Furthermore, the [3H]l[*4C]hydroxyproline ratios were equal in the two groups, as expected for animals in a steady-state condition, without ongoing collagen gain or loss. In contrast, skin collagen synthesis in the diabetic mice was decreased, as shown by a 12% reduction in the [3H]hydroxyproline specific radioactivities (Fig. loo-
SO-
so-
40-
20P 2 o %
o-Skin -20
htestine
Kidney
-
.-40-
FIG. 1. Effect of diabetes on total organ mass (open bars) and collagen mass (crosshatched bars) in db/db mice. Data are expressed as percentage change from littermate controls. Collagen mass was calculated as tissue mass x collagen concentration.
224
JON C. BOWERSOX
20
10
P e
0
z
-10
-20
-30
-40
J
FIG. 2. Effect of diabetes on [3H]hydroxyproline specific radioactivities, indicative of collagen synthesis (open bars) and [14C]hydroxyproline specific radioactivities, representing collagen degradation (crosshatched bars). Data are expressed as percentage change from littermate controls.
2). Collagen degradation was accelerated in the skins of the diabetic mice, as demonstrated by the 36% reduction in the [i4C]hydroxyproline pool, which was out of proportion to that expected from decreased collagen synthesis (13H]/ [i4C]hydroxyproline ratio of 0.8 in skins of control mice and 1.2 in diabetic mice). In hearts and kidneys of the diabetic mice there was an increase in collagen synthesis relative to controls. However, collagen degradation was also accelerated in the diabetic tissues, resulting in net reductions in tissue collagen concentrations (Table I). This conclusion was supported by the observed increase in tissue [3H]/[14C]hydroxyproline ratios in diabetic hearts and kidneys. DISCUSSION One prominent hypothesis explaining the mechanisms of diabetic connective tissue changes is that accelerated aging of collagen occurs in diabetes, resulting in less salt-extractable collagen (Behera and Patnaik, 1979). These changes appear similar to those occurring in normal tissues during the aging process (Schnider and Kohn, 1982) and have been attributed to increased collagen crosslinking. Alternatively, connective tissue changes may result from perturbations in collagen turnover, as demonstrated in the present study. Collagen synthesis and degradation were assessed in chronically hyperglycemic mice over a 7-day period. Whereas intestinal collagen metabolism appeared to be unaffected in the db/db mice, collagen degradation was accelerated in other tissues that were examined. Collagen synthesis was also affected, but in a tissue-specific manner. In skins from the diabetic mice, collagen synthesis was decreased compared to littermate controls; hearts and kidneys demonstrated increased collagen synthesis. In streptozotocin-diabetic rats, tissue specific changes in collagen have also been observed (Schneir et al., 1979). In contrast to the db/db mice, however,
COLLAGEN
METABOLISM
IN DIABETIC
MICE
225
intestinal collagen synthesis was markedly increased after insulin deprivation and correlated well with organ hypertrophy. One consistent change observed in both the diabetic rats and mice was accelerated skin collagen degradation (Schneir et al., 1982). Enhanced protein degradation has also been reported in hindquarter muscle preparations from db/db mice (Shargill er al., 1984). As suggested by Dice et al. (1978) and others, increased protein degradation may be a uniform response by cells to insulin deprivation. Another possibility is that diabetes results in the biosynthesis of defective collagen molecules that are rapidly degraded before secretion (Bienkowski et al., 1978; Schneir et al., 1984). The reason for the tissue-specific nature of the changes observed in collagen metabolism is unclear, but the results emphasize the need to address each organ independently when evaluating diabetic connective tissue changes. One important question that remains to be addressed is whether vascular lesions observed in diabetics are also organ-specific or whether the effects on the vascular system are similar throughout the body. ACKNOWLEDGMENTS These studies were supported in part by NIH Grants DE-076006 and EY-3129. The helpful discussions with Dr. Michael Schneir and Dr. Nino Sorgente are gratefully appreciated.
REFERENCES H. N., and PATNAIK, B. K. (1979). In vivo and in vitro effects of alloxan on collagen characteristics of bone, skin and tendon of Swiss mice. Gerontology 25, 255-260. BIENKOWSKI, R. S., COWAN, M. J., MCDONALD, J. A., and CRYSTAL, R. G. (1978). Degradation of newly synthesized collagen. J. Biol. Chem. 253, 4356-4363. COHEN, M. P, and KHALIFA, A. (1977). Renal glomerular collagen synthesis in streptozotocin diabetes: Reversal of increased basement membrane synthesis with insulin therapy. Biochim. Biophys. Acta 500, 395-404. COLEMAN, D. L. (1978). Obesity and diabetes: Two mutant genes causing diabetes-obesity syndromes in mice. Diubetologia 14, 141- 148. COLEMAN, D. L. (1982). Diabetes-obesity syndromes in mice. Diabetes 31(Suppl 1). 1-6. COLEMAN, D. L. (1983). Lessons from studies with genetic forms of diabetes in the mouse. Metabolism 32(Suppl 1). 162-165. DICE, J. F., WALKER, C. D., BYRNE, B., and CARDIEL. A. (1978). General characteristics of protein degradation in diabetes and starvation. Proc. Nut/. Acad. Sci. USA 75, 2093-2097. GRANT, R. A. (1964). An automated procedure for the determination of hydroxyprohne. J. Clin. Pathol. 17, 685-686. HASSELACHLER, C. H.. and WAHL, P. (1980). Influence of diabetes control in synthesis of protein and basement membrane collagen in isolated glomeruli of diabetic rats. Res. Exp. Med. 176, 247-253. HUMMEL. K. P, DICKIE, M. M., and COLEMAN, D. L. (1966). Diabetes, a new mutation in the mouse. Science 153, 1127-l 128. KAHN, C. R., NEVILLE, D. M., JR., and ROTH, J. (1973). Insulin receptor interaction in the obese hyperglycemic mouse. A new model of insulin resistance. J. Biol. Chem. 248, 244-250. POSNER, B. I., RAQUIDAN, D., JOSEFSBERG, Z.. and BERGERON, J. J. (1978). Different regulation of insulin receptors in intracellular (Golgi) and plasma membranes from livers of obese and lean mice. Proc. Natl. Acad. Sri. USA 75, 3302-3305. REI-~ORI, 0.. MEJIA, R. H., and FERNANDEZ, L. A. (1964). Factors of variation in blood volume determinations in the rat. Acta Physiol. Lat. Amer. 14, 221-230. SCHNEIR, M., BOWERSOX, J., RAMAMURTHY, N., YAVELOW. J.. MURRAY, J., EDLIN-FOLZ, E., and GOLUB, L. (1979). Response of rat connective tissues to streptozotocin diabetes. Tissue specific effects on collagen metabolism. Biochim. Biophys. Acta 583, 95-102. SCHNEIR, M., RAMAMURTHY, N., and GOLUB. L. (1982). Skin collagen metabolism in the streptozoBEHERA,
226
JON C. BOWERSOX
tocin-induced diabetic rat: Enhanced catabolism of collagen formed before and during the diabetic state. Diabetes 31, 426-431. SCHNEIR, M. L., RAMAMURTHY, N. S., and GOLUB, L. M. (1984). Extensive degradation of recently synthesized collagen in gingiva of normal and streptozotocin-induced diabetic rats. J. Dent. Res. 63, 23-27.
SCHNIDER, S. L., and KOHN, R. R. (1982). Effects of age and diabetes mellitis on the solubility of collagen from human skin, tracheal cartilage and dura mater. Exp. Geroniol. 17, 185-194. SHARGILL, N. S., OSHIMA, K., BRAY, G. A., and CHAN, T. M. (1984). Muscle protein turnover in the perfused hindquarters of lean and genetically obese-diabetic (dbidb) mice. Diabetes 33, 1160- 1164.
-