beta-Adrenergic desensitization by chronic insulin exposure in 3T3-L1 cultured adipocytes.

Cultured 3T3-L1 cells provide a model system for studies of the long-term regulation of lipolysis. Insulin acutely inhibits isoproterenol-stimulated lipolysis primarily by decreasing the apparent affinity apparent Km for isoproterenol. In contrast, chronic insulin exposure inhibits lipolysis by a reduction in the maximal effect of isoproterenol Vmax. The decrease in Vmax can be observed with insulin concentrations that are as low as 10(-9) mol/L at the time of addition. The effect is stable to washing, and the cells' responsiveness to isoproterenol returns partially with continued culture. Chronic insulin exposure also markedly reduced dibutyryl-cAMP-stimulated lipolysis indicating an insulin-induced change distal to cAMP concentration in the cascade of reactions controlling lipolysis in these cells. Time course and insulin dose-response experiments indicate an additional proximal alteration. These results indicate that: (1) 3T3-L1 cells are a useful model for studying the long-term regulation of lipolysis. (2) Chronic insulin exposure inhibits lipolysis by a mechanism that differs from the acute effect of insulin. (3) The chronic effects of insulin may be mediated through changes at multiple levels in the lipolytic cascade.

Promotion of lipolysis in rat adipocytes by pertussis toxin: reversal of endogenous inhibition.

Pertussis toxin (PT), a protein produced by Bordetella pertussis, was studied for its effect on lipolysis in isolated rat epididymal adipocytes. Exposure of adipocytes to pertussis toxin resulted in a significant increase in cyclic AMP levels and lipolysis after a lag of 1-2 hr. Both the maximal rate of lipolysis and the time lag (beyond 1 hr) were PT concentration-dependent. Heat treatment (95 degrees C, 30 min) or incubation with specific antibody directed against PT eliminated the ability of toxin to increase lipolysis. Cell-free culture medium from B. pertussis, but not from nontoxigenic Bordetella species, had the same effect on lipolysis as purified toxin. Comparison of the PT effect with the known lipolytic effect of cholera toxin (CT) revealed that the two toxins elicited responses that were indistinguishable in time course and magnitude. In contrast, the adenylate cyclase (EC 4.6.1.1) activities in membranes prepared from PT- or CT-treated adipocytes were different. Adenylate cyclase activity in membranes from control (untreated) adipocytes was inhibited 35-64% by the adenosine analogue N6-(L-2-phenylisopropyl)-adenosine. As expected from previous studies, membranes from CT-treated adipocytes demonstrated an increased basal activity but showed the same proportional inhibition by N6-(L-2-phenylisopropyl)-adenosine as controls. On the other hand, membranes from adipocytes exposed to PT (400 ng/ml for 4 hr) showed no increase in basal adenylate cyclase activity but had reduced sensitivity to N6-(L-2-phenylisopropyl)-adenosine inhibition, with the maximal effect ranging from 11 to 30% at 10(-6) M N6-(L-2-phenylisopropyl)-adenosine. These data support the hypothesis that PT promotes cyclic AMP-dependent lipolysis in a manner quantitatively equivalent to CT, but by a different mechanism involving increased cyclic AMP levels resulting from loss of responsiveness to endogenous inhibitors such as adenosine.

Effects of thyroid hormones and thyrotropin-releasing hormone on thyrotropin biosynthesis by mouse pituitary tumor cells in vitro.

Mouse thyrotropic tumor cells grown in primary culture were shown to synthesize TSH and proteins, as determined by the incorporation of radioactive proline into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins. The net TSH content of the cells and medium determined by RIA is also increased during 24 h of incubation, and newly formed hormone is detected in the medium within 1 h after the addition of proline tracer. To study the effect of T4 and T3 on TSH synthesis, cultures were pulse-labeled with [3H]proline after they had been exposed to either T3 or T4. After 48 but not 24 h, exposure to either T3 or T4 was followed by inhibition. When studied after 48 h of incubation, T4, (10(-13) M) or T3 (10(-11) M) at the lowest concentration tested, was inhibitory to TSH synthesis. At concentrations of T4 and T3 greater than 10(-9) M, the inhibitory effects on TSH synthesis were partially reversed, suggesting a biphasic response. Incubation in TRH (10(-7) M) for 24 h led to a significant increase in TSH synthesis, total protein, acid-precipitable protein, and total DNA. The effect of TRH on TSH biosynthesis was a function of the logarithm of its concentration over the range of 10(-11)-10(-7) M. The inhibitory action of 10(-6) M T3 on TSH synthesis was reversed by exposure to 10(-10) or 10(-7) M TRH.

Cyclic nucleotides and lipolysis.

The recent literature regarding the mechanisms of regulation of lipolysis with emphasis on the role of cyclic nucleotides is reviewed. The following conclusions appear warranted at present. (1) Cyclic AMP (cAMP) is a major regulator of lipolysis. However, mechanisms other than the production and catabolism of cAMP also exist. (2) Insulin can lower adipocyte cyclic AMP levels, but this effect cannot explain all aspects of the antilipolytic effect of insulin. (3) Insulin stimulates cyclic AMP phosphodiesterase and inhibits adenylate cyclase in adipocytes. In addition, there are probably other targets of insulin action. The possibilities include cAMP dependent protein kinase, phosphoprotein phosphatase, and triacylglycerol lipase. (4) Cyclic GMP is probably not directly involved in the regulation of lipolysis. (5) Cytosolic Ca2+ probably plays an important role in the regulation of lipolysis. The nature of such a role for Ca2+ and the potential role of calmodulin in the regulation of lipolysis remain to be explored.

An evaluation of diabetic and pseudodiabetic glomerulosclerosis.

Diabetic glomerulosclerosis must be either a primary manifestation or a secondary consequence of the metabolic abnormalities of diabetes. Several earlier reports have attempted to support the former hypothesis by describing cases of pathognomonic renal lesions in nondiabetic subjects; however, the clinical and pathologic data in these reports are inconclusive. We have reviewed our experience at the University of Virginia Hospital with 447 percutaneous renal biopsies performed over a period of four years from July 1973 through July 1977. Of these cases, only two appeared to represent diabetic glomerulosclerosis occurring in nondiabetic subjects. Upon further investigation, one case provided to be light chain disease demonstrated by immunofluorescence staining. The other case, on repeat renal biopsy, proved to be membranoproliferative glomerulonephritis. We conclude that a diagnosis of diabetic glomerulosclerosis must be viewed with suspicion in nondiabetic subjects. Suspected cases should be labeled pseudodiabetic glomerulosclerosis and investigated further.

Do thyroid-stimulating immunoglobulins cause non-toxic and toxic multinodular goitre?

The prevalence of serum thyroid-stimulating immunoglobulins (T.S.I.) in a variety of thyroid diseases was determined in 96 patients and 35 normal controls. Significantly elevated levels of T.S.I. were found not only in patients with Graves' disease and Hashimoto's thyroiditis but also in those with non-toxic and toxic multinodular goitre, whereas patients with a single autonomously functioning thyroid nodule, with subacute thyroiditis, and with "hyperthyroiditis" had levels which did not differ from those in the controls. We postulate that non-toxic multinodular goitre, like Graves' disease, may result from increased circulating T.S.I., which in some cases may be present in sufficient concentration to cause thyrotoxicosis.