␤3-Adrenergic receptors (␤3-ARs) are expressed predominantly on white and brown adipocytes, and acute treatment of mice with CL 316,243, a potent and highly selective ␤3-AR agonist, produces a 2-fold increase in energy expenditure, a 50 -100-fold increase in insulin levels, and a 40 -50% reduction in food intake. Recently, we generated gene knockout mice lacking functional ␤3-ARs and demonstrated that each of these responses were mediated exclusively by ␤3-ARs. However, the tissue site responsible for producing these actions is unknown. In the present study, genetically engineered mice were created in which ␤3-ARs are expressed exclusively in white and brown adipocytes (WAT؉BAT-mice), or in brown adipocytes only (BAT-mice). This was accomplished by injecting tissue-specific ␤3-AR transgenic constructs into mouse zygotes homozygous for the ␤3-AR knockout allele. Control, knockout, WAT؉BAT, and BAT-mice were then treated acutely with CL, and the effects on various parameters were assessed. As previously observed, all effects of CL were completely absent in gene knockout mice lacking ␤3-ARs. The effects on O 2 consumption, insulin secretion, and food intake were completely rescued with transgenic re-expression of ␤3-ARs in white and brown adipocytes (WAT؉BAT-mice), demonstrating that each of these responses is mediated exclusively by ␤3-ARs in white and/or brown adipocytes, and that ␤3-ARs in other tissue sites were not required. Importantly, transgenic re-expression of ␤3-ARs in brown adipocytes only (BAT-mice) failed to rescue, in any way, CL-mediated effects on insulin levels and food intake and only minimally restored effects on oxygen consumption, indicating that any effect on insulin secretion and food intake, and a full stimulation of oxygen consumption required the presence of ␤3-ARs in white adipocytes. The mechanisms by which ␤3-AR agonist stimulation of white adipocytes produces these responses are unknown but may involve novel mediators not previously known to effect these processes.Obesity is a prevalent condition frequently associated with diabetes, hypertension, and cardiovascular disease. Because available treatments are minimally effective, substantial efforts have been directed toward the discovery of new, effective, anti-obesity drugs. The ␤3-adrenergic receptor (␤3-AR) 1 represents one of a number of potential anti-obesity drug targets for which selective agonists have been developed (1-3). The ␤3-AR is encoded by a distinct gene that is expressed predominantly in white and brown adipocytes (4 -7), important sites for energy storage and energy expenditure, respectively. Selective activation of ␤3-ARs leads to marked increases in triglyceride breakdown (lipolysis) and energy expenditure (1-3), and long term treatment of obese rodents with ␤3-selective agonists reduces fat stores and improves obesity-induced insulin resistance (1-3). Thus, ␤3-selective agonists are promising anti-obesity compounds.Acute treatment of rodents with ␤3-selective agonists causes a number of diverse metabolic effects in...

Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.

"Spot 14" (S14) was originally identified as a mRNA from rat liver that responded rapidly to thyroid hormone, and has now been shown to play a key role in the tissue-specific regulation of lipid metabolism. In addition to its responsiveness to thyroid hormone, S14 gene transcription is controlled by dietary substrates, such as glucose and polyunsaturated fatty acids, and by fuel-related hormones including insulin and glucagon. The S14 protein forms homodimers via a carboxyl-terminal "zipper" domain. The protein is located primarily in the cell nucleus, and its expression in liver is limited to the perivenous portion of the hepatic lobule, the site of fatty acid synthesis. S14 protein is critical for the induction of key enzymes involved in the switching of hepatic metabolism from the fasted to the fed state. S14 antisense oligonucleotides inhibit both the intracellular production of lipids and their export as very low-density lipoprotein (VLDL) particles. S14 acts at the level of transcription to regulate expression of genes encoding key metabolic enzymes, including those required for long-chain fatty acid synthesis. The human S14 gene is located at 11q13.5, a region that is amplified in a subset of aggressive breast cancers. S14 mRNA is expressed in most breast cancer-derived cell lines, and the protein is found in the nuclei of two thirds of human breast cancer specimens, but not in normal nonlactating mammary glands. S14 expression in breast tumors is highly concordant with overabundance of a key lipogenic enzyme. This indicates the association of S14 with enhanced tumor lipogenesis, an established marker of poor prognosis. In addition to the utility of S14 as a model system for elucidation of the mechanism of thyroid hormone action, studies of its regulation and function have provided insights into tissue-specific metabolic control by hormones and dietary substrates in both normal and neoplastic tissues.

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)

It has been reported that protein kinase C (PKC) interacts at multiple sites in ␤-cell stimulus-secretion coupling. Nevertheless, there is still controversy concerning the importance of this enzyme in glucose-induced insulin release. The present study was undertaken to clarify whether glucose, directly, or through changes in cytoplasmic free Ca