John W. Koontz

Diazoxide down-regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats

We have previously reported that attenuation of hyperinsulinemia by diazoxide (DZ), an inhibitor of glucose‐mediated insulin secretion, increased insulin sensitivity and reduced body weight in obese Zucker rats. These findings prompted us to investigate the effects of DZ on key insulin‐sensitive enzymes regulating adipose tissue metabolism, fatty acid synthase (FAS), and lipoprotein lipase (LPL), as well as on circulating levels of leptin. We also determined the direct effects of diazoxide on FAS in 3T3‐L1 adipocytes. Seven‐week‐old female obese and lean Zucker rats were treated with DZ (150 mg/kg/d) or vehicle (C, control) for a period of 6 wk. Changes in plasma parameters by DZ include significant decreases in triglycerides, free fatty acids, glucose, and insulin, consistent with our previous reports. DZ obese rats exhibited lower plasma leptin levels (P<0.03) compared to their C animals. DZ significantly reduced adipose tissue FAS activity in both lean (P<0.0001) and obese (P<0.01) animals. LPL mRNA content was also decreased significantly in DZ‐treated obese animals (P≪ 0.009) as compared to their respective controls without a significant effect on lean animals. The possibility that DZ exerted a direct effect on adipocytes was further tested in cultured 3T3‐L1 adipocytes. Although diazoxide (5 μM) alone did not change FAS activity in cultured 3T3‐L1 adipocytes, it significantly attenuated insulins effect on FAS activity (P<0.001). We demonstrate that DZ regulates key insulin‐sensitive enzymes involved in regulation of adipose tissue metabolism. These findings suggest that modification of insulin‐sensitive pathways can be therapeutically beneficial in obesity management.—Standridge, M., Alemzadeh, R., Zemel, M., Koontz, J., Moustaid‐Moussa, N. Diazoxide down‐regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats FASEB J. 14, 455–460 (2000)

The role of the insulin receptor in mediating the insulin-stimulated growth response in Reuber H-35 cells

SummaryInsulin is able to stimulate a growth response in a variety of different cell types. However, the role of the insulin receptor in mediating this response is not clear. Indeed, it has been reported that the ability of insulin to stimulate a growth response is a result of its interaction with other growth factor receptors rather than the insulin receptor.We have previously reported that the H-35 hepatoma cell line responded to physiological concentrations of insulin as a growth factor and that the relative potency of proinsulin suggested that this response was mediated by the insulin receptor. In this report, two experimental approaches are used to demonstrate the involvement of the insulin receptor in mediating the growth response. Two different preparations of antibody to the insulin receptor are found to be capable of stimulating this response. In addition, the human insulin-like growth factors (IGF-I and II) show very low cross-reactivity with the insulin receptor and are significantly less potent than insulin in stimulating the growth response.

Insulin-mediated regulation of tyrosine aminotransferase in rat hepatoma cells: inhibition of transcription and inhibition of enzyme degradation.

Insulin induces the enzyme tyrosine aminotransferase (TAT) in Reuber H-35 rat hepatoma cells. A clone of these cells (KRC-7) was used to study the relationship between changes in enzyme activity and hybridizable mRNA, and rates of transcription for TAT in response to insulin. Our results indicate that enzyme activity is inducible by insulin in the presence of an inhibitor of RNA synthesis, suggesting that insulin functions post-transcriptionally to increase enzyme activity. Unexpectedly, insulin causes a decrease in the level of hybridizable TAT mRNA. Glucocorticoids cause an increase in TAT mRNA and insulin inhibits this increase when added either subsequent to or simultaneous with the addition of this agonist. Transcriptional runoffs demonstrate that insulin inhibits transcription of TAT to account for the aforementioned decrease in hybridizable mRNA. To examine the possibility that a post-translational mechanism is responsible for the increase in TAT activity caused by insulin, the rate of degradation of TAT protein was measured using polyclonal antibody. These experiments indicate that the rate of degradation of TAT is decreased about twofold in the presence of insulin, which suggests that part of the observed increase in TAT activity is due to selective post-translational stabilization of TAT. Therefore, insulin regulates TAT in KRC-7 cells by both transcriptional and post-translational mechanisms, the latter being responsible for the increase in activity.

Phorbol ester inhibition of hormonal induction of tyrosine aminotransferase

The liver specific enzyme, tyrosine aminotransferase, can be induced by glucocorticoids, cAMP analogs, or insulin. Each of these different inducing agents is believed to act through a separate pathway. The tumor promoting phorbol esters have been reported to stimulate phosphorylation of the insulin receptor and thereby decrease the ability of insulin to induce tyrosine aminotransferase. Our results demonstrate that TPA will not only inhibit the insulin stimulated increase in tyrosine aminotransferase, but will also inhibit induction of the enzyme by glucocorticoids or by cAMP.

Insulin and phorbol myristic acetate induce ornithine decarboxylase in Reuber H35 rat hepatoma cells by different mechanisms

Reuber H35 rat hepatoma cells respond to insulin or to tumor promoting phorbol esters with an increase in ornithine decarboxylase enzyme activity. This occurs in a time- and dose-dependent manner with both types of agonist. We report here that the increase in ornithine decarboxylase activity with optimal concentrations of both agonists is additive. Furthermore, the initial increase is dependent on continued RNA and protein synthesis. We also find that both of these agonists cause an increase in mRNA coding for ornithine decarboxylase in a time- and dose-dependent manner which suggests that the increase in enzyme activity can be accounted for by the increase in transcript levels. The difference in the time course of induction by the agonists, the additivity of induction by the two agonists, the differential sensitivity of induction to cycloheximide and RNA synthesis inhibitors, and the observation that phorbol myristic acetate causes a further increase in ornithine decarboxylase activity and transcript levels in cells already maximally induced by insulin suggest that these two agonists act through separate mechanisms.

Insulin-responsive tyrosine aminotransferase transcription requires multiple promoter regions

This study used transient transfection analysis to determine the DNA regions which mediate basal and insulin-sensitive transcription from the gene encoding tyrosine aminotransferase (TAT; EC 2.6.1.5). Basal expression requires at least parts of two regions: a region at -3600 and a region from -208 to + 62. Insulin sensitivity requires at least one region of the promoter not required for basal expression. Thus, insulin cannot act solely by direct modification of any of the components required for basal transcription. Previous results from this laboratory suggest that the insulin effects on basal and glucocorticoid-induced TAT transcription require different regions of the proximal promoter.

Nanomolar cyclic adenosine and guanosine monophosphates stimulate macrophage colony-stimulating factor responsiveness by murine transitional progenitors.

Both 3:5′ cyclic adenosine monophosphate (cAMP) and 3′:5′ cyclic guanosine monophosphate (cGMP) stimulated colony‐stimulating factor 1 (CSF‐1)–dependent colony formation by murine two‐signal–dependent progenitors without influencing colony formation by committed CSF‐1–responsive progenitors. The stimulatory effect was optimal at 10‐9 M and did not diminish with increasing concentrations of the cyclic nucleotides. The membrane‐permeating analogs dibutyryl cAMP and 8‐Br‐cGMP similarly augmented colony formation by the transitional progenitors at 10‐9 M; however, with increasing concentration, enhancement diminished with eventual inhibition of total colony formation at micromolar concentrations. Stimulation by the two cyclic nucleotides was mutually incompatible. The results indicate that physiological levels of extracellular cyclic nucleotides may significantly influence myelopoiesis. Furthermore, the results introduce the interesting possibility that stimulation, unlike inhibition, may be initiated through an extracytoplasmic mechanism that does not require direct activation of cytoplasmic cyclic nucleotide–dependent protein kinases.