Robert G. Spanheimer

Decreased collagen production in diabetic rats

Many of the chronic complications of diabetes mellitus involve defects in the connective tissue such as poor wound healing, diminished bone formation, and decreased linear growth. Because collagen is the major protein component of these connective tissues, we examined collagen production in diabetic rats as a probe of this generalized defect in connective tissue metabolism. Doses of streptozocin ranging from 35 to 300 mg/kg were used to induce diabetes of graded metabolic severity in rats. Parietal bone or articular cartilage was removed and incubated at 37°C with 5 μCi L-[5-3H]proline for 2 h, and collagen and noncollagen protein production were quantitated after separation with purified bacterial collagenase. Within 2 wk after induction of diabetes, collagen production was significantly reduced in bone and cartilage from diabetic rats to 52% (P < .01) and 51% (P < .01) of control (buffer-injected) levels, respectively. In contrast, noncollagen protein production in bone and cartilage from diabetic animals was no different from in tissue from control rats. The correlation between collagen relative to total protein production (relative rate) and the degree of hyperglycemia was highly significant for both bone (r = −.77, P < .001) and cartilage (r = −.87, P < .001). Other factors found to correlate with altered collagen production were the duration of diabetes and the amount of weight loss. Thus, diabetes is associated with a marked decrease in collagen production, which was seen early after induction of diabetes and was specific when compared with noncollagen protein production. Cumulative effects of these marked changes in collagen production may contribute to the chronic connective tissue complications in diabetes.

Differential Binding of Insulin to Human Arterial and Venous Endothelial Cells in Primary Culture

The properties of 125I-insulin binding were assessed in endothelial cells prepared from the veins and the arteries of human umbilical cords. The endothelial nature of both the arterial and venous cultures was documented by the presence of characteristic endothelial features, including Weibel-Palade bodies, factor VIII antigen, and morphology. Both arterial and venous cells possessed typical receptors for insulin on the basis of specificity of binding, curvilinear Scatchard plots, affinity profiles, pH dependency, and dissociation kinetics. Arterial cells bound at least 2.5 times more insulin than did venous cells, whether studied at 4 h, 24 h, or 72 h after in vitro plating. We conclude that (1) specific receptors for insulin are present on human arterial as well as human venous endothelial cells and (2) the concentration of insulin receptors varies among endothelial cells derived from different vascular sources.

Correlation between decreased collagen production in diabetic animals and in cells exposed to diabetic serum : response to insulin

Collagen production has been shown to be decreased in costal cartilage from nondiabetic animals after incubation with diabetic rat serum. Since collagen was decreased to a similar degree in tissues from diabetic animals, we questioned whether altered collagen production in vivo could be related to altered production induced in vitro. Collagen and noncollagen protein production in articular cartilage from diabetic animals (production in vivo) was compared to protein production in dermal fibroblasts from non-diabetic rats exposed to serum from the same diabetic rats (production in vitro). Diabetes was induced by intravenous administration of 90 mg/kg of streptozotocin into male Sprague-Dawley rats. Cartilage was removed and incubated with [3H]-proline for 2 hours at 37 degrees C (in vivo), while fibroblasts were exposed to experimental serum from individual animals for 24 hours with addition of 5 microCi of [5-3H]-proline for the final 6 hours (in vitro). Collagen and noncollagen protein production were quantitated using purified bacterial collagenase. Collagen production in cartilage decreased to 46% (p less than .01) and noncollagen to 68% (p less than .05) of levels in control animals. Fibroblasts exposed to 2.5% diabetic serum decreased collagen and noncollagen protein production to levels of 30% (p less than .01) and 54% (p less than .05) of production in cells incubated in 2.5% normal rat serum. Correlation between defective collagen production in cartilage from individual rats and the effects of their own serum on collagen production in fibroblasts was significant (r = 0.84, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of growth factor mRNA levels in the eyes of diabetic rats

The underlying etiology of diabetic microvascular disease remains unknown. To examine the potential contribution of basic fibroblast growth factor (bFGF), which is an angiogenic factor, and insulin-like growth factor-I (IGF-I) to the development of diabetic microvascular disease, bFGF and IGF-I mRNA levels were measured in tissues of control, diabetic, and insulin-treated diabetic rats. Diabetes was induced in rats by intravenous injection of streptozotocin (STZ) 65 mg/kg, and the rats were maintained for 21 days. bFGF mRNA levels increased threefold in the eyes of diabetic versus control rats, whereas a consistent change in bFGF mRNA levels was not observed in other tissues. In contrast, IGF-I mRNA levels decreased in the eyes and other tissues, including kidney, lung, and skeletal muscle, of diabetic as compared with control rats. Insulin treatment prevented the diabetes-induced increase in bFGF and decrease in IGF-I mRNA levels. Acidic FGF (aFGF) mRNA levels were unchanged in eyes from diabetic versus control rats. In partially purified retinas, diabetes increased bFGF mRNA levels twofold as compared with levels in control retinas, whereas IGF-I mRNA levels decreased to 58% of control levels in retinas from diabetic rats. Insulin treatment again prevented the diabetes-induced increase in IGF-I mRNA levels in the retina but had no effect on the diabetes-induced increase in bFGF mRNA levels. bFGF peptide levels were minimally increased in diabetic versus control retinas.(ABSTRACT TRUNCATED AT 250 WORDS)

Correction of Altered Collagen Metabolism in Diabetic Animals with Insulin Therapy3

Skeletal abnormalities commonly reported in both human and experimental diabetes include impaired linear growth and osteopenia. In the present study we examined the effect of diabetes and insulin therapy on collagen, the major protein constituent of extracellular matrix. Insulin-deficient diabetes was induced in growing rats by injection of 90 mg/kg of streptozotocin. Articular cartilage and long bone (femur) were removed, and tissues incubated with [3H]-proline in vitro for 2 hours at 37 degrees C. Uptake of [3H]-proline into both collagen and noncollagen proteins was determined using purified bacterial collagenase. In cartilage, collagen production decreased to 46% of buffer-injected control animals within one week of induction of diabetes (p less than 0.01), and remained at this level for three weeks. A similar degree of suppression was found in long bone from untreated animals, in which collagen production decreased to 58% of control (p less than 0.01). Insulin administration at the onset of diabetes prevented the expected decrease in collagen production such that after one week of therapy, collagen production in bone and cartilage was 98% and 93% of control (p +/- 0.01 vs. untreated), respectively. When insulin therapy was delayed for one week after the induction of diabetes, collagen production in articular cartilage increased from 46% to 92% and 97% of control at the end of one and two weeks of therapy (p less than 0.01 vs. untreated), respectively. Noncollagen protein production in untreated rats decreased to 49% of control in long bone and to 72% of control in articular cartilage after one week (p less than 0.01), with correction to control levels in both tissues within one week of insulin therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-Specific Regulation of Basic Fibroblast Growth Factor mRNA Levels by Diabetes

Because basic fibroblast growth factor (bFGF) is recognized as an angiogenic factor and diabetes is characterized by multiple vascular complications, including diabetic microangiopathy, we examined the regulation of tissue bFGF mRNA levels by diabetes. Diabetes was induced in male Sprague-Dawley rats by injection of 125 mg/kg body wt i.v. streptozocin (STZ), with intensive insulin therapy initiated in half of the diabetic rats. Rats were killed 96 h postinjection of STZ. Tissue bFGF and insulinlike growth factor I (IGF-I) mRNA levels were measured simultaneously with a solution hybridization–RNase protection assay. bFGF mRNA levels increased from 1.7- to 2.7-fold in eye, heart, lung, and brain from diabetic compared with buffer-injected control rats. In skeletal muscle, bFGF mRNA levels decreased to 23% of control levels, whereas bFGF mRNA levels were unchanged in kidneys from diabetic versus control rats. Changes in tissue bFGF mRNA levels were partially reversed by insulin treatment in all tissues. In contrast, IGF-I mRNA levels were significantly decreased from 15 to 50% of control levels in all tissues studied except those in brain, which decreased to only 85% of control levels. These data demonstrate that bFGF mRNA levels are altered by diabetes in a tissue-specific fashion and are consistent with the hypothesis that increased production of bFGF may contribute to the development of diabetic microangiopathy in some tissues.

Reducing cardiovascular risk in diabetes. Which factors to modify first

PREVIEW Diabetic patients are at high risk for morbidity and mortality from vascular complications. Therefore, it is important to identify and modify cardiovascular risk factors early in these patients. In this article, Dr Spanheimer discusses the major risk factors as well as other atherogenic factors and presents a practical approach for achieving recommended treatment goals.

Nutritional and Hormonal Regulation of Articular Collagen Production in Diabetic Animals

Although changes in collagen production probably play a major role in the connective tissue defects of diabetes, we do not know to what extent these changes are attributable to hormonal/metabolic versus nutritional alterations. To study collagen production as influenced separately by nutrition versus hormonal/metabolic factors, rats were given 50 mg/kg i.v. streptozocin (STZ) (mild weight-gaining diabetes) or 100 mg/kg STZ (severe weight-losing diabetes) and compared with nondiabetic food-restricted rats to match weight changes in diabetic animals. Articular cartilage was incubated with [3H]proline, and uptake of [3H]proline into both collagen and noncollagen proteins was determined with purified bacterial collagenase. Collagen decreased to 49% in mildly diabetic rats and 16% in severely diabetic rats, compared with control rats fed ad libitum and decreased to 85 and 73%, respectively, in food-restricted rats (both P < .01 vs. diabetes). Diabetes induced a greater defect in collagen production than food restriction and a greater decrease in collagen than noncollagen protein production within each group, suggesting a specific effect on collagen. With comparable levels of metabolic severity (glucose, β-hydroxybutyrate), diabetic animals that lost weight produced significantly less collagen than animals that gained weight, suggesting separate mechanisms. Quantitation of the impact of undernutrition on collagen production in diabetes demonstrated that ∼31 to 32% of the defect was due to undernutrition, leaving ∼68–69% of the defect due to the diabetic state. Multivariate analysis of metabolic (glucose, β-hydroxybutyrate), hormonal(insulin, insulinlike growth factor I [IGF-I]), and nutritional (weight change) factors revealed that altered collagen production was correlated only with the degree of weight change (P <.01) in food-restricted animals; reduced collagen production was correlated only with circulating IGF-I (P <.01) in diabetic animals.

Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin

The [35S]glycosaminoglycans ([35S]GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with 35SO4 for 72 h, the [35S]glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of [35S]GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of [35S]GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans.

Hyperthyroidism Caused by Inappropriate Thyrotropin Hypersecretion: Studies in Patients With Selective Pituitary Resistance to Thyroid Hormone

We have identified the condition of thyrotropin (thyroid-stimulating hormone [TSH])-induced hyperthyroidism secondary to selective pituitary insensitivity to thyroid hormone in three patients. Each patient was clinically hyperthyroid, with elevated serum levels of thyroxine (T4) and triiodothyronine (T3) and detectable levels of serum TSH before therapy. After therapy each patient had notably elevated TSH levels at a time that peripheral levels of thyroid hormones were in the hyperthyroid range. Before and after therapy, serum levels of TSH were suppressed by therapy with liothyronine sodium and were stimulated by protirelin (thyrotropin-releasing hormone) both before and after liothyronine and dexamethasone treatment. Dexamethasone therapy decreased the levels of TSH, protirelin-stimulated TSH, and circulating T4 and T3. Serum levels of glycoprotein alpha-subunit were 0.6 to 2.4 ng/ml, values considerably lower than found in patients with TSH-secreting pituitary tumors. We suggest that the frequency of TSH-induced hyperthyroidism secondary to pituitary insensitivity to thyroid hormone may be higher than presently indicated in the medical literature.