Ciglitazone, a new hypoglycemic agent. I. Studies in ob/ob and db/db mice, diabetic Chinese hamsters, and normal and streptozotocin-diabetic rats.

Ciglitazone, 5-[4-(1-methylcyclohexylmethoxy) benzyl]-thiazolidine-2,4-dione, is a new hypoglycemic agent orally active in the obese-hyperglycemic animal models. In C57BL/6J-ob/ob mice, treatment with 100 mg/kg ciglitazone for 2 days elicited a drastic fall in blood glucose. It also decreased plasma insulin, triglycerides, and free fatty acids and food intake without affecting the body weight. Its hypoglycemic activity was independent of its effect on food intake. Regranulation of islet beta-cells and increased pancreatic insulin content were observed in ob/ob mice treated for 41-44 days with 100 mg/kg/day ciglitazone. Ciglitazone showed no effect on food intake, blood glucose, or pancreatic islet beta-cells in a group of lean C57BL/6J-+/? mice concomitantly treated at a dose of 150 mg/kg/day. In C57BL/KsJ-db/db mice, ciglitazone decreased blood glucose and food intake. The untreated db/db mice lost weight despite hyperphagia, whereas the ciglitazone-treated db/db mice gained weight. In the spontaneously diabetic Chinese hamsters, ciglitazone showed no significant effect on food intake, body weight, blood glucose, or insulin content in either plasma or pancreas, but it lowered plasma lipids. In normal rats, ciglitazone failed to affect fasting blood glucose but improved glucose tolerance without increasing insulin secretory response to glucose. In streptozotocin-diabetic rats, it showed no effect on blood glucose or glycemic response to exogenous insulin. The hypoglycemic activity of ciglitazone was specific for obese-hyperglycemic and insulin-resistant animals.

Immunocytochemical localization of androgen receptors in the scalp of the stumptail macaque monkey, a model of androgenetic alopecia.

The purpose of this study was to identify the distribution of androgen receptors in the bald and hairy scalp of adult male and female stumptail macaque monkeys by light microscopic biotin-avidin immunocytochemistry with a highly purified rat monoclonal antibody against the cloned human androgen receptor. Consistent, intense nuclear and minimal cytoplasmic immunostaining was observed in several distinct cell populations of the pilosebaceous unit including the dermal papilla, hair epithelium, outer root sheath, dermal sheath, and sebaceous gland. A similar distribution of androgen receptors was found in miniaturized and terminal anagen and telogen follicles of the bald and hairy scalp, respectively. Binding of androgen receptor antibody was also detected in dermal fibroblasts, basal and intermediate layers of the interfollicular epidermis, and duct and glandular cells of eccrine sweat glands. This investigation demonstrates the presence of androgen receptors in the pilosebaceous unit of the scalp of the stumptail macaque and also shows that their distribution is comparable to that previously reported for humans.

Localization of minoxidil sulfotransferase in rat liver and the outer root sheath of anagen pelage and vibrissa follicles.

The precise biochemical mechanism and site(s) of action by which minoxidil stimulates hair growth are not yet clear. Minoxidil sulfate is the active metabolite of minoxidil, with regard to smooth muscle vasodilation and hair growth. Formation of minoxidil sulfate is catalyzed by specific PAPS-dependent sulfotransferase(s) and minoxidil-sulfating activities have been previously reported to be present in liver and hair follicles. One of these minoxidil-sulfating enzymes has been purified from rat liver (rat minoxidil sulfotransferase, MST) and a rabbit anti-MST antibody has been prepared. Using this anti-MST antibody, we have immunohistochemically localized minoxidil sulfotransferase in the liver and anagen hair follicles from rat. In rat pelage and vibrissa follicles, this enzyme is localized within the cytoplasm of epithelial cells in the lower outer root sheath. Although the immunolocalization of MST might not necessarily correlate with the MST activity known to be present in anagen follicles, the results of this study strongly suggest that the lower outer root sheath of the hair follicle may serve as a site for the sulfation of topically applied minoxidil.

Hair growth effects of oral administration of finasteride, a steroid 5 alpha-reductase inhibitor, alone and in combination with topical minoxidil in the balding stumptail macaque.

A 5 alpha-reductase inhibitor, finasteride, was administered orally at 0.5 mg/day, alone or in combination with topical 2% minoxidil, for 20 weeks to determine the effects on scalp hair growth in balding adult male stumptail macaque monkeys. A 7-day dose-finding study showed that both 0.5- and 2.0-mg doses of the drug produced a similar diminution in serum dihydrotestosterone (DHT) in male stumptails. Hair growth was evaluated by shaving and weighing scalp hair at baseline and at 4-week intervals during treatment to obtain cumulative delta hair weight (sum of the 4-week changes in hair weight from baseline) for the 20-week study. The activity of the 5 alpha-reductase enzyme was assessed by RIA of serum testosterone (T) and DHT at 4-week intervals. The combination of finasteride and minoxidil generated significant augmentation of hair weight (additive effect) compared to either drug alone. Finasteride increased hair weight in four of five monkeys. When the data of the one nonresponsive monkey were excluded, finasteride elicited a significant elevation in hair weight compared to topical vehicle alone. Minoxidil also evoked a significant increase in hair weight compared to vehicle alone. Serum T was unchanged, whereas serum DHT was significantly depressed in monkeys that received either finasteride or the combination of finasteride and minoxidil. These data suggest that inhibition of the conversion of T to DHT by this 5 alpha-reductase inhibitor reverses the balding process and enhances hair regrowth by topical minoxidil in the male balding stumptail macaque.

Localization of S-100 protein in Müller cells of the retina--1. Light microscopical immunocytochemistry.

S-100 is an acidic brain protein previously found to be present in glial cells of the brain and the nervous system of gut and respiratory tract. Immunocytochemistry at the light microscopical level localized immunoreactivity for S-100 in the Müller cells in the retina of rat, guinea pig, and Chinese hamster. The Müller cells represent the main glial component of the retina, with a structural role in the support and insulation of neurons and sensory elements. The use of S-100 protein as an immunocytochemical marker of Müller cells may be useful in the study of pathologic conditions of the retina where glial cell proliferation could reflect the index of neuronal injury.

Morphometric evaluation of capillary basement membrane thickness in the quadriceps muscle of diabetic and nondiabetic Chinese hamsters.

Quadriceps muscle capillaries from 19-23 month old genetically diabetic (XA and AC) and nondiabetic (M) subline Chinese hamsters were morphometrically evaluated to determine if capillary basement membrane thickening (CBMT) is a quantifiable complication of diabetes. Significant CBMT was present in the diabetic XA Chinese hamsters (49.37 nm +/- 17.81, p less than 0.007) in comparison with the nondiabetic M hamsters (34.08 nm +/- 9.98). Although there was a trend towards expansion of the muscle capillary basement membranes in the diabetic AC Chinese hamsters, the value was not statistically significant. A nested analysis of variance showed that the greatest source of variation in basement membrane thickness occurred among capillaries within each animal. In addition, a positive correlation (r = 0.62; p less than 0.002) existed between blood glucose levels and CBMT in the XA subline. These data should serve as guidelines for evaluation of antimicrovascular disease compounds which will be tested to determine if they prevent or retard microangiopathy in the diabetic Chinese hamster.

Ciglitazone, a hypoglycemic agent: early effects on the pancreatic islets of ob/ob mice.

Chronic administration of ciglitazone (5-4[1-methyl-cyclohexylmethoxy)-benzyl]-thiazolidine-2,4 dione) decreased both plasma glucose and insulin concentrations in ob/ob mice. When given as an admixture to the feed, blood glucose levels were reduced as early as 12 hours after initiation of treatment. Concomitant with the decrease in circulating insulin, there was an increased hormone content in the beta-cells as judged by RIA and aldehyde-fuchsin staining. Acute oral dosing with ciglitazone produced a 41% reduction in circulating insulin at a time when glucose concentrations were as yet unaffected. Ciglitazone also inhibited glucose-stimulated insulin secretion in vitro. The results suggest that the hypoglycemic agent, ciglitazone, may reduce plasma glucose and insulin concentrations at least partially as the result of independent mechanisms.

Characterization of glucagon-like peptide-1-(7-36)amide in the hypothalamus.

The distribution of glucagon-like peptide-1-(7-36)amide like immunoreactivity (GLP-1-(7-36)NH2 IR) in rat brain was determined. Highest concentrations were found in the hypothalamus. A combination of gel chromatography and anion exchange chromatography showed that the majority of hypothalamic immunoreactivity exactly corresponded in position to synthetic GLP-1-(7-36)NH2. Chromatographic analyses of rat ileum demonstrated a similar pattern, whereas in rat pancreas mainly a large proglucagon fragment and GLP-1 were indicated. Determination of the subcellular distribution by differential centrifugation of hypothalamic tissue revealed that most of the GLP-1-(7-36)NH2 IR was present in the synaptosome fraction. GLP-1-(7-36)NH2 was released from hypothalamic tissue slices in a calcium-dependent fashion by potassium-induced depolarization. Thus GLP-1-(7-36)NH2 appears to fulfil two criteria for a neurotransmitter. No change was found in its hypothalamic content in streptozocin-induced diabetic rats compared to normal controls but a decrease was seen in hyperinsulinemic hyperglycemic KKAy mice compared to KK mice.

Morphometric analysis of the vagus nerve in non-diabetic and ketonuric diabetic Chinese hamsters.

Morphometric analysis of axons from the ventral division of the vagus nerve of ketonuric diabetic Chinese hamsters and age-sex matched non-diabetic controls was performed to determine the frequency distribution and numerical and volume density. Myelinated fibres of diabetics displayed a significant reduction in diameter (P less than 0.001) compared with controls, which was correlated inversely with progressive ketonuria (P less than 0.05). The reduced calibre of myelinated fibres was the result of thin myelin sheaths rather than a reduction in axon diameter. A marked decrease in numerical density (P less than 0.05) and volume density (P less than 0.005) was found in the myelinated fibres of diabetics compared with controls. Non-myelinated axons showed a significant shift to smaller diameter (P less than 0.001) in diabetics, which was correlated inversely with duration of ketonuria (P less than 0.05). Numerical density of non-myelinated axons was increased (P less than 0.01) in diabetic hamsters whereas volume density was comparable in diabetic and control animals. These data provide morphological evidence of impairment in the parasympathetic nervous system which may be a major factor underlying previously reported gastrointestinal and pancreatic islet dysfunction in the diabetic Chinese hamster.

Road traffic accidents with vehicular entrapment: incidence of major injuries and need for advanced life support.

Road traffic accidents (RTAs) with entrapment are perceived as a challenge to emergency systems because of the severity of the ensuing traumas and the inherent complexity of the rescue procedures. To clarify these two aspects this prospective cohort study enrolling 244 entrapped trauma patients was conducted by a Regional Medical Helicopter Service. Forty-six victims (18.9%) were found dead, 101 (51%) of the 198 patients who reached the hospital alive had an injury severity score (ISS) > or = 16. The use of seat belts was associated with lower trauma severity. Out of the 101 severely traumatized patients (ISS > or = 16), 46 (42.6%) were intubated at road side, 12 required decompression of a tension pneumothorax on the scene and in 15 cases a pneumothorax was drained during the early intrahospital phase. Thirty-six (34.7%) patients had the first systolic blood pressure (SBP) < or = 90 mmHg and were then aggressively infused: in 75% of these cases, the SBP on arrival at the emergency department increased. The first SBP was significantly correlated with mortality. There was no correlation of extrication time, total rescue time and mortality. Fourteen patients (13.9%) died during hospitalization. These data demonstrate that a high percentage of entrapped patients require advanced life support (ALS), including on scene intubation and chest decompression. Aggressive field resuscitation and immediate transport to a level 1 trauma centre is associated with a mortality lower than that predicted by TRISS in spite of the prolonged prehospital time.

Islet innervation of nondiabetic and diabetic Chinese hamsters. I. Acetylcholinesterase histochemistry and norepinephrine fluorescence.

Cholinergic and adrenergic innervation of pancreatic islets from age- and sex-matched nondiabetic (M subline) and diabetic (AC subline) Chinese hamsters was analyzed by acetylcholinesterase (AChE) histochemistry and norepinephrine fluorescence. The AChE activity was significantly diminished in islets of diabetic animals compared with that of nondiabetic controls. The reduction in cholinergic innervation displayed an inverse relationship with respect to nonfasting plasma glucose and ketone levels. On the basis of qualitative analysis, adrenergic activity also appeared to be depressed in islets of diabetic animals. These date suggest that autonomic control of islet function is altered in diabetic Chinese hamsters when plasma glucose and ketone levels arae elevated and may exacerbate metabolic complications in this animal model.

Capillary basement membrane thickening associated with the small intestine of the ketonuric diabetic Chinese hamster.

Morphometric evaluation of intestinal capillary basement membranes demonstrated a significant thickening in those of ketonuric diabetic Chinese hamsters compared to age-matched nondiabetic controls. A highly significant correlation was found between increased capillary basement membrane thickness and progression of ketonuria. Age was also positively related to elevation in capillary basement membrane thickness of control and diabetic hamsters. Capillary basement membrane thickness of diabetic animals was not significantly related to a combination of progredient ketonuria and advance in age.

The penetration enhancer SEPA augments stimulation of scalp hair growth by topical minoxidil in the balding stumptail macaque.

The purpose of this study was to determine if the penetration enhancer SEPA (2-n-nonyl-1,3-dioxolane) would augment the scalp hair growth effects of topical minoxidil in the balding stumptail macaque. A 1-in2 area on the balding scalp of 40 adult female monkeys (four drug-treated and four vehicle-treated groups of 5 monkeys each) was topically treated 5 days/week, q.d. or b.i.d., with approximately 250 microliters of minoxidil-SEPA (2.5% minoxidil, weight/volume in 10% SEPA, 25% propylene glycol and 65% isopropyl alcohol), Rogaine topical solution (TS, 2% minoxidil, weight/volume in 20% propylene glycol, 60% ethanol and 20% water) or respective vehicles (without drug) for 16 weeks via paintbrush application. Scalp hair was collected by shaving and vacuuming the dosed area at baseline and at 4-week intervals. The shaved hair was filtered, weighed and recorded as the change from baseline. The q.d. and b.i.d. minoxidil-SEPA groups displayed a significant increase in hair weight compared to their respective vehicles at week 4 whereas q.d. and b.i.d. Rogaine TS groups were not active until week 8 and 12, respectively. Both minoxidil-SEPA treatments produced significantly greater cumulative hair weight over the entire 16-week study compared to either of the Rogaine TS treatments. Comparable increases in cumulative hair weight were evident between q.d. and b.i.d. minoxidil-SEPA groups and between q.d. and b.i.d. Rogaine TS groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural localization and quantification of extracellular calcium binding sites in mouse vibrissa and human scalp follicles.

The purpose of these experiments was to determine if an extracellular calcium binding site gradient is evident in freshly dissected or cultured mouse vibrissa and human scalp follicles and to measure possible drug effects on this gradient. Mouse vibrissae were cultured with or without either minoxidil or pinacidil, and human scalp follicles were cultured with or without epidermal growth factor. Anagen vibrissa and scalp follicles were dissected and placed in culture for 4 h to 4 days, then fixed in a solution containing lanthanum chloride and prepared for either quantitative energy-dispersive X-ray microanalysis (X-ray) or qualitative transmission electron microscopy (TEM). Since lanthanum has a high charge density it displaces Ca2+ ions from anionic binding sites. TEM analysis revealed extensive accumulation of electron-dense lanthanum deposits in the intercellular compartment of differentiating cells in the hair shaft and inner root sheath in the apex of the follicular bulb. Sparse lanthanum precipitate was observed in the intercellular space of the proliferative cells at the base of the bulb. This gradient of lanthanum precipitate was evident in both freshly dissected and cultured vibrissa and scalp hair follicles, irrespective of treatment with drugs that grow hair or epidermal growth factor. X-ray microanalysis indicated that percent by weight of lanthanum was markedly higher in the apex compared to the base of the follicular bulb in vibrissa and scalp follicles. These qualitative and quantitative data demonstrate that an extracellular calcium binding site gradient exists in cultured vibrissa and scalp hair follicles, and that this gradient is not significantly affected by hair growth altering drugs including minoxidil or pinacidil, and epidermal growth factor.

Reduced hypothalamic somatostatin and neuropeptide Y concentrations in the spontaneously-diabetic Chinese hamster.

Hypothalamic concentrations of six regulatory peptides having central effects on appetite and/or glucoregulation were measured by radioimmunoassay in spontaneously-diabetic Chinese hamsters and in age- and sex-matched non-diabetic control animals. In the diabetic hamsters, hypothalamic concentrations of somatostatin and neuropeptide Y were significantly reduced by 25-30% below controls. None of the other four peptides examined (bombesin, galanin, neurotensin and vasoactive intestinal peptide) differed significantly between the two groups. Disturbances in neuropeptide Y (the most potent central appetite stimulant yet discovered) and in somatostatin could be related to hyperphagia, an early and possibly primary abnormality of the diabetic syndrome in the Chinese hamster.

Morphometric analysis of autonomic neuropathology in the abdominal sympathetic trunk of the ketonuric diabetic Chinese hamster.

Fibers from the infradiaphragmatic portion of the sympathetic trunk of ketonuric diabetic Chinese hamsters were quantitatively analyzed by light and electron microscopy to determine frequency distribution and numerical density. Myelinated and unmyelinated fibers displayed a significant reduction in diameter which was exacerbated by increased duration of ketonuria. Mean numerical density of myelinated fibers was reduced whereas that of unmyelinated fibers was increased. The alterations in sympathetic nerve populations are believed to be a manifestation of previously observed demyelination and axonal degeneration. On the basis of axon diameter, it appeared that both visceral afferent and efferent fibers were affected. These data strongly suggest that autonomic neuropathology in the sympathetic trunk of the diabetic Chinese hamster may be a critical factor underlying gastrointestinal dysfunction.

Depopulation of the ventromedial hypothalamic nucleus in the diabetic Chinese hamster.

The relationship between diabetes and the size, density and area of the ventromedial hypothalamic nucleus (VMH) was studied in the genetically diabetic Chinese hamster. Matched diabetic and non-diabetic control chinese hamsters were perfused, the hypothalamus collected, sectioned and stained for light microscopy. The mid-point of each VMH nucleus was located, photographed and enlarged for morphometric analysis. Each neuron that possessed a nucleolus and was located within the confines of a VMH was counted, and subsequently the area of each nucleus and the density of neurons per area of VMH were calculated. The results indicated that both the area and absolute number of neurons within the VMH of diabetic hamsters were significantly reduced compared to control values (P less than 0.01) The density of neurons per unit area of VMH was similar in both groups. These data suggest that the VMH experiences a neuronal depopulation in diabetic hamsters which may have a functional influence on the hypothalamic-pancreatic axis in this species.

Morphometric evaluation of the hypothalamic-ovarian axis of the ketonuric, diabetic Chinese hamster: relationship to the reproductive cycle.

The relationship between diabetes and the morphological alterations which occur in hypothalamic and ovarian tissue was examined in the long-term, ketonuric-diabetic Chinese hamster. Matched diabetic and non-diabetic control hamsters were inspected daily for changes in the reproductive cycle by vaginal lavage. On dioestrus, animals were perfused, the hypothalamus and ovaries collected, prepared for microscopy and morphometrically analyzed. The nuclei in the medial basal hypothalamus of diabetic hamsters exhibited a decreased area (p less than 0.01) and neuronal population (p less than 0.05-0.01) compared with controls. The ovaries of the diabetic animals had a reduced follicular population (p less than or equal to 0.05) and an increased atresia rate (p less than or equal to 0.05) compared with controls. In addition, all diabetic hamsters were acyclic. In diabetic animals, the corpora luteal cells contained a reduced lipid content (p less than or equal to 0.001) which was possibly functionally related to a significant decline in serum progesterone levels (p less than or equal to 0.01). Based on these results it is suggested that the hypothalamic-ovarian axis is both morphologically and functionally impaired in the diabetic hamster.

A study of the morphological changes in the small intestine of the spontaneously diabetic Chinese hamster.

Several morphological changes were observed microscopically in the small intestine of some diabetic Chinese hamsters. Although some alterations lacked statistical significance due to variation, most diabetics displayed a greater incidence and severity compared with nondiabetic controls. The following structural deviations were seen in the small intestines of some diabetics: increased surface area, elevated number of goblet cells per villus, decreased muscle thickness with connective tissue infiltration, reduced number of Auerbach's plexuses, lymphocyte aggregations accompanied by blunted villi, blood vascular lesions and deformed villi due to excessive loss of epithelial cells.

Pioglitazone preserves pancreatic islet structure and insulin secretory function in three murine models of type 2 diabetes.

Thiazolidinediones may slow the progression of type 2 diabetes by preserving pancreatic beta-cells. The effects of pioglitazone (PIO) on structure and function of beta-cells in KKA(y), C57BL/6J ob/ob, and C57BL/KsJ db/db mice (genetic models of type 2 diabetes) were examined. ob/ob (n = 7) and db/db (n = 9) mice were randomly assigned to 50-125 mg.kg body wt-1.day-1 of PIO in chow beginning at 6-10 wk of age. Control ob/ob (n = 7) and db/db mice (n = 9) were fed chow without PIO. KKA(y) mice (n = 15) were fed PIO daily at doses of 62-144 mg.kg body wt-1.day-1. Control KKA(y) mice (n = 10) received chow without PIO. Treatment continued until euthanasia at 14-26 wk of age. Blood was collected at baseline (before treatment) and just before euthanasia and was analyzed for glucose, glycosylated hemoglobin, and plasma insulin. Some of the splenic pancreas of each animal was resected and partially sectioned for light or electron microscopy. The remainder of the pancreas was assayed for insulin content. Compared with baseline and control groups, PIO treatment significantly reduced blood glucose and glycosylated hemoglobin levels. Plasma insulin levels decreased significantly in ob/ob mice treated with PIO. All groups treated with PIO exhibited significantly greater beta-cell granulation, evidence of reduced beta-cell stress, and 1.5- to 15-fold higher levels of pancreatic insulin. The data from these studies suggest that comparable effects would be expected to slow the progression of type 2 diabetes, either delaying or possibly preventing progression to an insulin-dependent state.