Michael Schneir

Enhanced Collagenase Activity in Diabetic Rat Gingiva: In Vitro and In Vivo Evidence

Gingiva from alloxan and streptozotocin-diabetic rats exhibited markedly enhanced collagenolytic activity in tissue culture. This effect was eliminated by puromycin or by repeated freeze-thawing of the tissue prior to incubation. Soluble extracts of the diabetic gingiva in situ were found to contain breakdown products of collagen similar in size to the reaction products generated by tissue collagenase. These fragments were not detected in the control tissue. This study indicates that experimental diabetes stimulates the synthesis of gingival collagenase in culture and that a similar effect occurs in vivo.

Skin Collagen Metabolism in the Streptozotocin-induced Diabetic Rat: Enhanced Catabolism of Collagen Formed Both Before and During the Diabetic State

Collagen catabolism has been measured in skins of streptozotocin-induced diabetic rats. For measuring catabolism of collagen synthesized de novo during the diabetic state, we measured the amounts of [3H]hydroxyproline-containing degradation products in skins of diabetic rats, killed 4 h after [3H]proline injection (protocol 1); degradation products were isolated in TCA-soluble fractions of skin homogenates. For measuring catabolism of collagen preexisting before the induction of the diabetic state, we measured the 21-day loss of [3H]hydroxyproline (and hydroxyproline) in entire skins of rats that were streptozotocin-treated after [3H]proline injection (protocol 2). A 2.5-fold increase in the relative amounts of [3H]hydroxyproline-containing degradation products was measured in the TCA-soluble fractions of skins from diabetic rats (protocol 1). These degradation products had a low molecular weight (as evident from their diffusibility), and they were derived from recently synthesized collagen, possibly procollagen (as evident from their high [3H]hydroxyproline specific activity). Furthermore, they were not derived from the degradation of [3H]hydroxyproline-labeled collagen present before induction of the diabetic state (protocol 2). Evidence for this conclusion is as follows: the amounts of [3H]hydroxyproline-containing degradation products in skins of diabetic rats were not greater than that in skins of control rats, despite a 50% resorption of collagen in skins of diabetic rats. Overall, the catabolism of collagen formed de novo during the diabetic state was distinguished from the catabolism of collagen formed before, and both catabolic processes were enhanced in rat skins of streptozotocin-induced diabetic rats.

Response of rat connective tissues to streptozotocin-diabetes. Tissue-specific effects on collagen metabolism.

Abstract We assessed the effect of streptozotocin-diabetes on in vivo collagen metabolism in skin, aorta and intestine by injecting [ 3 H]proline into rats, 20 days after administering the diabetogen, streptozotocin. One day after [ 3 H]proline injection, diabetic and control animals were killed, their tissues analyzed for both 3 H-labeled and unlabeled hydroxyproline and results expressed per entire tissue. Thereby, the effect of diabetes on net collagen synthesis and tissue collagen mass, respectively, was evaluated. Diabetes resulted in a lower content of [ 3 H]collagen in skin and aorta, suggesting decreased net collagen synthesis. This decrease in net synthesis was accompanied by a decrease of collagen mass in skin, whereas aortic collagen mass was unaffected. Consequently, an acceleration of collagen degradation in skin is postulated to have accompanied the expected depression of collagen synthesis; alterations of the physiochemical properties of skin from diabetic rats support this interpretation. For intestine, both net collagen synthesis and mass increased in diabetic rats, reflecting increased collagen synthesis—possibly associated with polyphagy. In conclusion, with regard to collagen metabolism, representative connective tissues respond differently to experimental diabetes, and we suggest that this insight will be useful in future studies aimed at understanding the pathophysiology of connective tissues affected by diabetes.

A chemically modified tetracycline inhibits streptozotocin-induced diabetic depression of skin collagen synthesis and steady-state type I procollagen mRNA

Wasting of connective tissues including skin, bone, and cartilage have been closely associated with elevated matrix metalloproteinase (MMP) activity and depressed collagen content in the streptozotocin (STZ)-induced diabetic rat, while tetracyclines have been reported to normalize total body weight, skin hydroxyproline and collagen content in this model, in part through inhibition of MMPs. In the present study, we report the effect of CMT-1, a chemically modified tetracycline that lacks antimicrobial properties but retains divalent cation binding and MMP inhibitory activity, on diabetic skin collagen synthesis and steady-state levels of procollagen alpha 1(I) mRNA. Male, 4-month old Sprague-Dawley rats received a single injection of 75 mg/kg STZ or citrate vehicle alone and diabetic status was confirmed by positive glucosuria. Some diabetic animals received 10 mg/day of CMT-1 by oral gavage and, 28 days after STZ treatment, body weight, blood glucose values and the in vivo rates of skin collagen production were measured using the pool-expansion technique. Steady-state levels of procollagen alpha 1(I) mRNA were analyzed 21 days after STZ treatment by hybridization of total RNA with a 32P labelled cDNA to rat type I procollagen alpha 1(I) mRNA in a dot-blot assay. STZ treatment was found to significantly depress body weight, skin collagen hydroxyproline content, the in vivo rate of collagen production, and hybridizable levels of type I procollagen alpha 1(I) mRNA. CMT-1 administered daily to STZ-treated rats inhibited the diabetic depression of these parameters but had little or no effect on non-diabetic controls or on STZ-induced hyperglycemia. Thus, in addition to the inhibition of MMP mediated extracellular collagen degradation, these results suggest CMT-1 also acts to inhibit diabetic connective tissue breakdown in STZ-induced diabetes by increasing both steady-state levels of type I procollagen mRNA and collagen synthesis through mechanism(s) that are independent of the antibacterial properties of tetracyclines.

Minocycline-treatment of diabetic rats normalizes skin collagen production and mass : possible causative mechanisms

Daily minocycline-treatment of streptozotocin-induced diabetic rats not only prevented a diabetes-caused atrophy of skin collagen mass (10-mos old rats), but also normalized skin collagen mass to match that of growing (ca. 1%/d) non-diabetic controls (4- and 5-mos old rats). The causative mechanism by which minocycline-treatment normalizes skin collagen mass must, in part, be related to a general anabolic effect on growth (body weight) because the effect on skin collagen mass correlates strongly to that on body weight. Consequently, a minocycline-stimulated increase of a systemic factor (such as insulin-like growth factor) is not unlikely. The anabolic effect of minocycline-treatment of diabetic rats is also expressed as a normalized cellular ribosome mass (an index of total protein synthetic capacity) and a normalized absolute rate of collagen production. (Calculation of an absolute rate was justified by an apparent maximum saturation of the prolyl-tRNA pool(s) of skin, maximum saturation obtained by the pool-flooding approach). The normalized skin ribosome amount does not, however, explain a selective effect of minocycline-treatment on collagen production as opposed to that for non-collagen protein, this selective effect measured as relative collagen production. To explain such selectivity, the inhibition of diabetes-induced excess skin collagenase activity seems unlikely. (This inference is based on results from a preliminary study indicating that recently [less than 2 h] synthesized collagen is not degraded by the excess collagenase in skin of diabetic rats). Thus, the principal collagen fraction acted on by pathologically excess collagenase might be collagen at a later stage (greater than 2 h after synthesis) in its life cycle. (Another possibility for the selective effect of minocycline on collagen production, as yet untested, is reduced intracellular procollagen degradation.) Overall, this is the first study aimed at discerning the mechanism(s) by which minocycline-treatment enhances the rate of collagen production in tissues of a diabetic rat. For future studies, the extent to which the positive effect on growth, ribosome mass, and rate of collagen production contributes to the change of collagen mass must, along with the known minocycline-inhibition of collagenase activity, be quantified. Such quantification is a prerequisite for evaluating the chemotherapeutic efficacy of minocycline-treatment on collagen-degradative diseases.

Extensive Degradation of Recently Synthesized Collagen in Gingiva of Normal and Streptozotocin-induced Diabetic Rats

The degradation of recently synthesized collagen (probably procollagen) in rat incisor gingiva was three times greater than that in skin. Concomitantly, the formation of undegraded (intact) collagen molecules in gingiva was slower than that in skin. This high basal rate of degradation in gingiva was just slightly increased in streptozotocin-induced diabetic rats, whereas the low basal rate in skin was dramatically increased by the diabetic state. The degradation of recently synthesized collagen was measured by the relative amounts(%) of [3H]hydroxyproline-containing material in the TCA-soluble fraction of a tissue, compared with the total amount (TCA-soluble + TCA-insoluble) of [3H]hydroxyproline-containing material. Separation of the TCA fractions allowed the formation of collagen degradation products (TCA-soluble) to be viewed separately from the formation of undegraded collagen molecules (TCA-insoluble). The [3H]hydroxyproline-containing material in the TCA-soluble fraction was greatest in amount and in specific activity, 30 min after [ 3H] proline injection, supporting the origin of this material as being procollagen. At this time period, the relative amounts of TCA-soluble [ 3H] hydroxyproline-containing material were 40.3% (gingiva) and 12.7% (skin). For diabetic rats, the values were 55% and 48.8%, respectively. For the [3H] hydroxyproline-containing material in the TCA-insoluble fraction, at 30 min, the specific activity of [3H] hydroxyproline was 4.3 for gingiva and 7.4 for skin. At all other time periods, the values were also greater for skin than for gingiva, making it unlikely that the formation of intact collagen molecules occurred faster in gingiva than in skin. Overall, the prevailing concept of rapid collagen turnover in oral tissues seems applicable to procollagen, but not to collagen of rat incisor gingiva.

BASIC BIOLOGICAL SCIENCES Confirmation that Neither Phenotype Nor Hydroxylation of Collagen Is Altered In Overgrown Gingiva From Diphenylhydantoin-Treated Patients

Collagens, solubilized by pepsin-digestion of diphenylhydantoin-induced overgrown gingiva, appeared similar to collagens solubilized from inflamed gingiva with regard to: ratio of type I to type III collagen, ratio of α1 to α2 of type I collagen, and degree of hydroxylation of type I collagen.

Diabetes stimulated procollagen degradation in rat tendon in vitro

To identify the mechanisms responsible for the paucity of recently synthesized collagen in connective tissues during diabetes, in vitro procollagen metabolism was studied in non-diabetic (control) and diabetic rats. Achilles tendons from the two groups were incubated for 1-8 h (35 degrees C) in medium containing [14C]proline and the radiolabeled collagen in the tissue, and that released into the media, were examined by SDS-polyacrylamide gel electrophoresis and fluorography. The bulk of the radiolabeled collagen in tendon from the diabetics was recovered as degradation products; these, but also procollagen and collagen components, were prominent in the control tissues. Moreover, the collagenous components synthesized by the diabetic rat tendons were more readily digested in vitro by trypsin than those produced by control tissues. We conclude that diabetes reduces collagen accretion in connective tissues in part due to increased intracellular degradation of procollagen.

Streptozotocin-induced Diabetes and the Rat Periodontium: Decreased Relative Collagen Production

This is the first study concerning the extent to which relative collagen production (RCP) in rat periodontal tissues is affected by diabetes. Determination of RCP, rather than individual production rates for collagen or for non-collagen protein, was deemed necessary because saturation of all proline pools in tissues of diabetics (and non-diabetic controls) was not achieved. Such non-saturation occurred despite the injection of a pool-expanding dose of proline (400-1150 mg/rat), non-saturation indicated by the lesser specific radioactivity (S.R.) of free-[3H]proline in tissues than that of the injected solution. RCP was decreased in five periodontal tissues (incisor and molar gingiva, incisor and molar periodontal ligament, antemolar palatal mucosa) and in skin. Diabetes-decreased RCP seems to result from decreased collagen synthesis and increased intracellular degradation, although some evidence is presented for increased extracellular degradation of recently secreted collagen.

Skin Collagen Metabolism in the Streptozotocin-Induced Diabetic Rat: Free Hydroxyproline, the Principal In Vivo Degradation Product of Newly Synthesized Collagen - Probably Procollagen

We characterized the degradation products of recently synthesized collagen present in skins of control and diabetic rats. Specifically, the TCA-soluble fractions of homogenized skins from control and diabetic rats (killed 1 and 4 hours after [3H]-proline injection) were fractionated by molecular sieve chromatography, and eluted fractions were analyzed for hydroxyproline and [3H]-hydroxyproline. Free [3H]-hydroxyproline was the principal (greater than 95%), low molecular weight (greater than 2000 daltons), [3H]-hydroxyproline-containing material eluted from the molecular sieve column, this amount representing approximately 80% (controls) and approximately 87% (diabetics) of [3H]-hydroxyproline-containing material in TCA-soluble fractions of skin homogenates. These observations are similar to those from the intracellular degradation of cellular and secretory proteins in that the principal--almost exclusive--degradation product was the free amino acid. The free hydroxyproline had a greater specific radioactivity than that in any other [3H]-hydroxyproline-containing fraction (soluble and insoluble, see below); furthermore, the total radioactivity of free [3H]-hydroxyproline was greater at 1 hour than 3 hours later. These two properties (identity with free amino acid; time-dependent decrease in amounts) are consistent with [3H]-hydroxyproline arising from the intracellular degradation of procollagen. The [3H]-hydroxyproline-containing material eluting before free hydroxyproline (designated peptidyl [3H]-hydroxyproline) was similar to free [3H]-hydroxyproline in terms of specific radioactivity and the time-dependent decreases of specific and total radioactivities, these similarities indicating that the peptidyl [3H]-hydroxyproline are intermediates in the degradative pathway of procollagen to free amino acids. Results for control and diabetic rats were qualitatively similar, with regard to the inter-fraction ratios of specific radioactivities and their time-dependent changes. However, the degradative process, as assessed by the release of free and peptidyl [3H]-hydroxyproline, was dramatically enhanced by the diabetic state, extending our previous results based on analyses of uncharacterized degradation products.