Leptin Mediates a Glucose-Fatty Acid Cycle to Maintain Glucose Homeostasis in Starvation.

The transition from the fed to the fasted state necessitates a shift from carbohydrate to fat metabolism that is thought to be mostly orchestrated by reductions in plasma insulin concentrations. Here, we show in awake rats that insulinopenia per se does not cause this transition but that both hypoleptinemia and insulinopenia are necessary. Furthermore, we show that hypoleptinemia mediates a glucose-fatty acid cycle through activation of the hypothalamic-pituitary-adrenal axis, resulting in increased white adipose tissue (WAT) lipolysis rates and increased hepatic acetyl-coenzyme A (CoA) content, which are essential to maintain gluconeogenesis during starvation. We also show that in prolonged starvation, substrate limitation due to reduced rates of glucose-alanine cycling lowers rates of hepatic mitochondrial anaplerosis, oxidation, and gluconeogenesis. Taken together, these data identify a leptin-mediated glucose-fatty acid cycle that integrates responses of the muscle, WAT, and liver to promote a shift from carbohydrate to fat oxidation and maintain glucose homeostasis during starvation.

Leptin's hunger-suppressing effects are mediated by the hypothalamic-pituitary-adrenocortical axis in rodents.

Leptin informs the brain about sufficiency of fuel stores. When insufficient, leptin levels fall, triggering compensatory increases in appetite. Falling leptin is first sensed by hypothalamic neurons, which then initiate adaptive responses. With regard to hunger, it is thought that leptin-sensing neurons work entirely via circuits within the central nervous system (CNS). Very unexpectedly, however, we now show this is not the case. Instead, stimulation of hunger requires an intervening endocrine step, namely activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Increased corticosterone then activates AgRP neurons to fully increase hunger. Importantly, this is true for 2 forms of low leptin-induced hunger, fasting and poorly controlled type 1 diabetes. Hypoglycemia, which also stimulates hunger by activating CNS neurons, albeit independently of leptin, similarly recruits and requires this pathway by which HPA axis activity stimulates AgRP neurons. Thus, HPA axis regulation of AgRP neurons is a previously underappreciated step in homeostatic regulation of hunger.

Dissociation of Muscle Insulin Resistance from Alterations in Mitochondrial Substrate Preference.

Alterations in muscle mitochondrial substrate preference have been postulated to play a major role in the pathogenesis of muscle insulin resistance. In order to examine this hypothesis, we assessed the ratio of mitochondrial pyruvate oxidation (VPDH) to rates of mitochondrial citrate synthase flux (VCS) in muscle. Contrary to this hypothesis, we found that high-fat-diet (HFD)-fed insulin-resistant rats did not manifest altered muscle substrate preference (VPDH/VCS) in soleus or quadriceps muscles in the fasting state. Furthermore, hyperinsulinemic-euglycemic (HE) clamps increased VPDH/VCS in both muscles in normal and insulin-resistant rats. We then examined the muscle VPDH/VCS flux in insulin-sensitive and insulin-resistant humans and found similar relative rates of VPDH/VCS, following an overnight fast (∼20%), and similar increases in VPDH/VCS fluxes during a HE clamp. Altogether, these findings demonstrate that alterations in mitochondrial substrate preference are not an essential step in the pathogenesis of muscle insulin resistance.

A suite of activity-based probes for human cytochrome P450 enzymes.

Cytochrome P450 (P450) enzymes regulate a variety of endogenous signaling molecules and play central roles in the metabolism of xenobiotics and drugs. We recently showed that an aryl alkyne serves as an effective activity-based probe for profiling mouse liver microsomal P450s in vitro and in vivo. However, individual P450s display distinct substrate and inhibitor specificities, indicating that multiple probe structures may be required to achieve comprehensive coverage of this large and diverse enzyme family. Here, we have synthesized a suite of P450-directed, activity-based protein profiling (ABPP) probes that contain: (1) varied chemical architectures validated as mechanism-based inhibitors of the P450 enzyme family, and (2) terminal alkyne groups for click chemistry conjugation of reporter tags. This set of probes was screened against a wide cross-section of human P450s, leading to the discovery of an optimal set of probes that provide broad coverage of this enzyme family. We used these probes to profile the effects on P450 activity of aromatase inhibitors in current clinical use for the treatment of breast cancer. We describe the surprising discovery that one of these aromatase inhibitors, anastrozole, significantly increases probe-labeling of P450 1A2, indicative of a heterotypic cooperativity effect on a central P450 isozyme involved in metabolizing numerous drugs and xenobiotics. The results presented herein greatly expand the suite of ABPP probes for profiling P450s and illuminate new applications for these tools to understand P450-drug interactions.

Dehydration and insulinopenia are necessary and sufficient for euglycemic ketoacidosis in SGLT2 inhibitor-treated rats.

Sodium-glucose transport protein 2 (SGLT2) inhibitors are a class of anti-diabetic agents; however, concerns have been raised about their potential to induce euglycemic ketoacidosis and to increase both glucose production and glucagon secretion. The mechanisms behind these alterations are unknown. Here we show that the SGLT2 inhibitor (SGLT2i) dapagliflozin promotes ketoacidosis in both healthy and type 2 diabetic rats in the setting of insulinopenia through increased plasma catecholamine and corticosterone concentrations secondary to volume depletion. These derangements increase white adipose tissue (WAT) lipolysis and hepatic acetyl-CoA content, rates of hepatic glucose production, and hepatic ketogenesis. Treatment with a loop diuretic, furosemide, under insulinopenic conditions replicates the effect of dapagliflozin and causes ketoacidosis. Furthermore, the effects of SGLT2 inhibition to promote ketoacidosis are independent from hyperglucagonemia. Taken together these data in rats identify the combination of insulinopenia and dehydration as a potential target to prevent euglycemic ketoacidosis associated with SGLT2i.

Mechanisms by which a Very-Low-Calorie Diet Reverses Hyperglycemia in a Rat Model of Type 2 Diabetes.

Caloric restriction rapidly reverses type 2 diabetes (T2D), but the mechanism(s) of this reversal are poorly understood. Here we show that 3 days of a very-low-calorie diet (VLCD, one-quarter their typical intake) lowered plasma glucose and insulin concentrations in a rat model of T2D without altering body weight. The lower plasma glucose was associated with a 30% reduction in hepatic glucose production resulting from suppression of both gluconeogenesis from pyruvate carboxylase (VPC), explained by a reduction in hepatic acetyl-CoA content, and net hepatic glycogenolysis. In addition, VLCD resulted in reductions in hepatic triglyceride and diacylglycerol content and PKCɛ translocation, associated with improved hepatic insulin sensitivity. Taken together, these data show that there are pleotropic mechanisms by which VLCD reverses hyperglycemia in a rat model of T2D, including reduced DAG-PKCɛ-induced hepatic insulin resistance, reduced hepatic glycogenolysis, and reduced hepatic acetyl-CoA content, PC flux, and gluconeogenesis.