Activation of Short and Long Chain Fatty Acid Sensing Machinery in the Ileum Lowers Glucose Production in Vivo.

Evidence continues to emerge detailing the myriad of ways the gut microbiota influences host energy homeostasis. Among the potential mechanisms, short chain fatty acids (SCFAs), the byproducts of microbial fermentation of dietary fibers, exhibit correlative beneficial metabolic effects in humans and rodents, including improvements in glucose homeostasis. The underlying mechanisms, however, remain elusive. We here report that one of the main bacterially produced SCFAs, propionate, activates ileal mucosal free fatty acid receptor 2 to trigger a negative feedback pathway to lower hepatic glucose production in healthy rats in vivo We further demonstrate that an ileal glucagon-like peptide-1 receptor-dependent neuronal network is necessary for ileal propionate and long chain fatty acid sensing to regulate glucose homeostasis. These findings highlight the potential to manipulate fatty acid sensing machinery in the ileum to regulate glucose homeostasis.

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high-protein feeding to regulate glucose homeostasis in vivo.

High-protein feeding acutely lowers postprandial glucose concentration compared to low-protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr-PKA-ERK-KATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high- vs. low-protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high-protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.

MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases.

Mouse protein-25 (MO25) isoforms bind to the STRAD pseudokinase and stabilise it in a conformation that can activate the LKB1 tumour suppressor kinase. We demonstrate that by binding to several STE20 family kinases, MO25 has roles beyond controlling LKB1. These new MO25 targets are SPAK/OSR1 kinases, regulators of ion homeostasis and blood pressure, and MST3/MST4/YSK1, involved in controlling development and morphogenesis. Our analyses suggest that MO25α and MO25ß associate with these STE20 kinases in a similar manner to STRAD. MO25 isoforms induce approximately 100-fold activation of SPAK/OSR1 dramatically enhancing their ability to phosphorylate the ion cotransporters NKCC1, NKCC2 and NCC, leading to the identification of several new phosphorylation sites. siRNA-mediated reduction of expression of MO25 isoforms in mammalian cells inhibited phosphorylation of endogenous NKCC1 at residues phosphorylated by SPAK/OSR1, which is rescued by re-expression of MO25α. MO25α/ß binding to MST3/MST4/YSK1 also stimulated kinase activity three- to four-fold. MO25 has evolved as a key regulator of a group of STE20 kinases and may represent an ancestral mechanism of regulating conformation of pseudokinases and activating catalytically competent protein kinases.

Glucagon and lipid signaling in the hypothalamus.

Hyperglycemia, caused in part by elevated hepatic glucose production (GP), is a hallmark feature of diabetes and obesity. The hypothalamus responds to hormones and nutrients to regulate hepatic GP and glucose homeostasis. This invited perspective focuses on the molecular signaling and biochemical pathways involved in the gluco-regulatory action of hypothalamic glucagon signaling and lipid sensing in health and disease. Recent evidence generated via genetic, molecular and chemical experimental approaches indicates that glucagon and lipid signaling independently trigger complementary hypothalamic mechanisms to lower GP. Thus, targeting hypothalamic glucagon or lipid signaling may have therapeutic potential in diabetes and obesity.

Insulin action in the hypothalamus and dorsal vagal complex.

Insulin resistance is a hallmark feature of type 2 diabetes and obesity. In addition to the classical view that insulin resistance in the liver, muscle and fat disrupts glucose homeostasis, studies in the past decade have illustrated that insulin resistance in the hypothalamus dysregulates hepatic glucose production and food intake, leading to type 2 diabetes and obesity. This invited review argues that in addition to the hypothalamus, insulin signalling in the dorsal vagal complex regulates hepatic glucose production and food intake. A thorough understanding of the physiological and pathophysiological mechanisms of insulin action in the hypothalamus and dorsal vagal complex is necessary in order to identify therapeutic targets for obesity and type 2 diabetes.

Insulin and glucagon signaling in the central nervous system.

The prevalence of the obesity and diabetes epidemic has triggered tremendous research investigating the role of the central nervous system (CNS) in the regulation of food intake, body weight gain and glucose homeostasis. This invited review focuses on the role of two pancreatic hormones--insulin and glucagon--that trigger signaling pathways in the brain to regulate energy and glucose homeostasis. Unlike in the periphery, insulin and glucagon signaling in the CNS does not seem to have opposing metabolic effects, as both hormones exert a suppressive effect on food intake and weight gain. They signal through different pathways and alter different neuronal populations suggesting a complementary action of the two hormones in regulating feeding behavior. Similar to its systemic effect, insulin signaling in the brain lowers glucose production. However, the ability of glucagon signaling in the brain to regulate glucose production remains unknown. Future studies that aim to dissect insulin and glucagon signaling in the CNS that regulate energy and glucose homeostasis could unveil novel signaling molecules to lower body weight and glucose levels in obesity and diabetes.

Manipulating mitochondrial dynamics in the NTS prevents diet-induced deficits in brown fat morphology and glucose uptake.

AIMS: Brown adipose tissue (BAT) can produce heat by metabolizing glucose and fatty acids. Activation of BAT is controlled by the central nervous system (CNS) through sympathetic innervation. Dysregulation of signalling molecules in selective CNS areas such as the nucleus of tractus solitarius (NTS) are linked with altered BAT activity, obesity and diabetes. High-fat diet (HFD)-feeding increases mitochondrial fragmentation in the NTS, triggering insulin resistance, hyperphagia and weight gain. Here we sought to determine whether changes in mitochondrial dynamics in the NTS can affect BAT glucose uptake. MAIN METHODS: Rats received DVC stereotactic surgery for local brain administration of viruses that express mutated Drp1 genes. BAT glucose uptake was measured with PET/CT scans. Biochemical assays and immunohistochemistry determined altered levels of key signalling molecules and neural innervation of BAT. KEY FINDINGS: We show that short-term HFD-feeding decreases BAT glucose uptake. However, inhibiting mitochondrial fragmentation in NTS-astrocytes of HFD-fed rats partially restores BAT glucose uptake accompanied by lower blood glucose and insulin levels. Tyrosine Hydroxylase (TH) revealed that rats with inhibited mitochondrial fragmentation in NTS astrocytes had higher levels of catecholaminergic innervation in BAT compared to HFD-fed rats, and did not exhibit HFD-dependent infiltration of enlarged white fat droplets in the BAT. In regular chow-fed rats, increasing mitochondrial fragmentation in the NTS-astrocytes reduced BAT glucose uptake, TH immune-positive boutons and ß3-adrenergic receptor levels. SIGNIFICANCE: Our data suggest that targeting mitochondrial dynamics in the NTS-astrocytes could be a beneficial strategy to increase glucose utilization and protect from developing obesity and diabetes.

Inhibition of mitochondrial fission and iNOS in the dorsal vagal complex protects from overeating and weight gain.

OBJECTIVES: The dorsal vagal complex (DVC) senses insulin and controls glucose homeostasis, feeding behaviour and body weight. Three-days of high-fat diet (HFD) in rats are sufficient to induce insulin resistance in the DVC and impair its ability to regulate feeding behaviour. HFD-feeding is associated with increased dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in the DVC. We investigated the effects that altered Drp1 activity in the DVC has on feeding behaviour. Additionally, we aimed to uncover the molecular events and the neuronal cell populations associated with DVC insulin sensing and resistance. METHODS: Eight-week-old male Sprague Dawley rats received DVC stereotactic surgery for brain infusion to facilitate the localised administration of insulin or viruses to express mutated forms of Drp1 or to knockdown inducible nitric oxide synthase (iNOS) in the NTS of the DVC. High-Fat diet feeding was used to cause insulin resistance and obesity. RESULTS: We showed that Drp1 activation in the DVC increases weight gain in rats and Drp1 inhibition in HFD-fed rats reduced food intake, weight gain and adipose tissue. Rats expressing active Drp1 in the DVC had higher levels of iNOS and knockdown of DVC iNOS in HFD-fed rats led to a reduction of food intake, weight gain and adipose tissue. Finally, inhibiting mitochondrial fission in DVC astrocytes was sufficient to protect rats from HFD-dependent insulin resistance, hyperphagia, weight gain and fat deposition. CONCLUSION: We uncovered new molecular and cellular targets for brain regulation of whole-body metabolism, which could inform new strategies to combat obesity and diabetes.

Mitochondrial Dynamics in the Brain Are Associated With Feeding, Glucose Homeostasis, and Whole-Body Metabolism.

The brain is responsible for maintaining whole-body energy homeostasis by changing energy input and availability. The hypothalamus and dorsal vagal complex (DVC) are the primary sites of metabolic control, able to sense both hormones and nutrients and adapt metabolism accordingly. The mitochondria respond to the level of nutrient availability by fusion or fission to maintain energy homeostasis; however, these processes can be disrupted by metabolic diseases including obesity and type II diabetes (T2D). Mitochondrial dynamics are crucial in the development and maintenance of obesity and T2D, playing a role in the control of glucose homeostasis and whole-body metabolism across neurons and glia in the hypothalamus and DVC.

Dynamin-Related Protein 1-Dependent Mitochondrial Fission Changes in the Dorsal Vagal Complex Regulate Insulin Action.

Mitochondria undergo dynamic changes to maintain function in eukaryotic cells. Insulin action in parallel regulates glucose homeostasis, but whether specific changes in mitochondrial dynamics alter insulin action and glucose homeostasis remains elusive. Here, we report that high-fat feeding in rodents incurred adaptive dynamic changes in mitochondria through an increase in mitochondrial fission in parallel to an activation of dynamin-related protein 1 (Drp1) in the dorsal vagal complex (DVC) of the brain. Direct inhibition of Drp1 negated high-fat-feeding-induced mitochondrial fission, endoplasmic reticulum (ER) stress, and insulin resistance in the DVC and subsequently restored hepatic glucose production regulation. Conversely, molecular activation of DVC Drp1 in healthy rodents was sufficient to induce DVC mitochondrial fission, ER stress, and insulin resistance. Together, these data illustrate that Drp1-dependent mitochondrial fission changes in the DVC regulate insulin action and suggest that targeting the Drp1-mitochondrial-dependent pathway in the brain may have therapeutic potential in insulin resistance.

Inhibition of glycine transporter-1 in the dorsal vagal complex improves metabolic homeostasis in diabetes and obesity.

Impaired glucose homeostasis and energy balance are integral to the pathophysiology of diabetes and obesity. Here we show that administration of a glycine transporter 1 (GlyT1) inhibitor, or molecular GlyT1 knockdown, in the dorsal vagal complex (DVC) suppresses glucose production, increases glucose tolerance and reduces food intake and body weight gain in healthy, obese and diabetic rats. These findings provide proof of concept that GlyT1 inhibition in the brain improves glucose and energy homeostasis. Considering the clinical safety and efficacy of GlyT1 inhibitors in raising glycine levels in clinical trials for schizophrenia, we propose that GlyT1 inhibitors have the potential to be repurposed as a treatment of both obesity and diabetes.

A fatty acid-dependent hypothalamic-DVC neurocircuitry that regulates hepatic secretion of triglyceride-rich lipoproteins.

The brain emerges as a regulator of hepatic triglyceride-rich very-low-density lipoproteins (VLDL-TG). The neurocircuitry involved as well as the ability of fatty acids to trigger a neuronal network to regulate VLDL-TG remain unknown. Here we demonstrate that infusion of oleic acid into the mediobasal hypothalamus (MBH) activates a MBH PKC-δ→KATP-channel signalling axis to suppress VLDL-TG secretion in rats. Both NMDA receptor-mediated transmissions in the dorsal vagal complex (DVC) and hepatic innervation are required for lowering VLDL-TG, illustrating a MBH-DVC-hepatic vagal neurocircuitry that mediates MBH fatty acid sensing. High-fat diet (HFD)-feeding elevates plasma TG and VLDL-TG secretion and abolishes MBH oleic acid sensing to lower VLDL-TG. Importantly, HFD-induced dysregulation is restored with direct activation of either MBH PKC-δ or KATP-channels via the hepatic vagus. Thus, targeting a fatty acid sensing-dependent hypothalamic-DVC neurocircuitry may have therapeutic potential to lower hepatic VLDL-TG and restore lipid homeostasis in obesity and diabetes.

Metformin activates a duodenal Ampk-dependent pathway to lower hepatic glucose production in rats.

Metformin is a first-line therapeutic option for the treatment of type 2 diabetes, even though its underlying mechanisms of action are relatively unclear. Metformin lowers blood glucose levels by inhibiting hepatic glucose production (HGP), an effect originally postulated to be due to a hepatic AMP-activated protein kinase (AMPK)-dependent mechanism. However, studies have questioned the contribution of hepatic AMPK to the effects of metformin on lowering hyperglycemia, and a gut-brain-liver axis that mediates intestinal nutrient- and hormone-induced lowering of HGP has been identified. Thus, it is possible that metformin affects HGP through this inter-organ crosstalk. Here we show that intraduodenal infusion of metformin for 50 min activated duodenal mucosal Ampk and lowered HGP in a rat 3 d high fat diet (HFD)-induced model of insulin resistance. Inhibition of duodenal Ampk negated the HGP-lowering effect of intraduodenal metformin, and both duodenal glucagon-like peptide-1 receptor (Glp-1r)-protein kinase A (Pka) signaling and a neuronal-mediated gut-brain-liver pathway were required for metformin to lower HGP. Preabsorptive metformin also lowered HGP in rat models of 28 d HFD-induced obesity and insulin resistance and nicotinamide (NA)-streptozotocin (STZ)-HFD-induced type 2 diabetes. In an unclamped setting, inhibition of duodenal Ampk reduced the glucose-lowering effects of a bolus metformin treatment in rat models of diabetes. These findings show that, in rat models of both obesity and diabetes, metformin activates a previously unappreciated duodenal Ampk-dependent pathway to lower HGP and plasma glucose levels.

Resveratrol activates duodenal Sirt1 to reverse insulin resistance in rats through a neuronal network.

Resveratrol improves insulin sensitivity and lowers hepatic glucose production (HGP) in rat models of obesity and diabetes, but the underlying mechanisms for these antidiabetic effects remain elusive. One process that is considered a key feature of resveratrol action is the activation of the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase sirtuin 1 (SIRT1) in various tissues. However, the low bioavailability of resveratrol raises questions about whether the antidiabetic effects of oral resveratrol can act directly on these tissues. We show here that acute intraduodenal infusion of resveratrol reversed a 3 d high fat diet (HFD)-induced reduction in duodenal-mucosal Sirt1 protein levels while also enhancing insulin sensitivity and lowering HGP. Further, we found that duodenum-specific knockdown of Sirt1 expression for 14 d was sufficient to induce hepatic insulin resistance in rats fed normal chow. We also found that the glucoregulatory role of duodenally acting resveratrol required activation of Sirt1 and AMP-activated protein kinase (Ampk) in this tissue to initiate a gut-brain-liver neuronal axis that improved hypothalamic insulin sensitivity and in turn, reduced HGP. In addition to the effects of duodenally acting resveratrol in an acute 3 d HFD-fed model of insulin resistance, we also found that short-term infusion of resveratrol into the duodenum lowered HGP in two other rat models of insulin resistance--a 28 d HFD-induced model of obesity and a nicotinamide (NA)-streptozotocin (STZ)-HFD-induced model of mild type 2 diabetes. Together, these studies highlight the therapeutic relevance of targeting duodenal SIRT1 to reverse insulin resistance and improve glucose homeostasis in obesity and diabetes.

Hypothalamic glucagon signals through the KATP channels to regulate glucose production.

Insulin, leptin and GLP-1 signal in the mediobasal hypothalamus (MBH) to lower hepatic glucose production (GP). MBH glucagon action also inhibits GP but the downstream signaling mediators remain largely unknown. In parallel, a lipid-sensing pathway involving MBH AMPK→malonyl-CoA→CPT-1→LCFA-CoA→PKC-δ leading to the activation of KATP channels lowers GP. Given that glucagon signals through the MBH PKA to lower GP, and PKA inhibits AMPK in hypothalamic cell lines, a possibility arises that MBH glucagon-PKA inhibits AMPK, elevates LCFA-CoA levels to activate PKC-δ, and activates KATP channels to lower GP. We here report that neither molecular or chemical activation of MBH AMPK nor inhibition of PKC-δ negated the effect of MBH glucagon. In contrast, molecular and chemical inhibition of MBH KATP channels negated MBH glucagon's effect to lower GP. Thus, MBH glucagon signals through a lipid-sensing independent but KATP channel-dependent pathway to regulate GP.

Insulin signals through the dorsal vagal complex to regulate energy balance.

Insulin signaling in the hypothalamus regulates food intake and hepatic glucose production in rodents. Although it is known that insulin also activates insulin receptor in the dorsal vagal complex (DVC) to lower glucose production through an extracellular signal-related kinase 1/2 (Erk1/2)-dependent and phosphatidylinositol 3-kinase (PI3K)-independent pathway, it is unknown whether DVC insulin action regulates food intake. We report here that a single acute infusion of insulin into the DVC decreased food intake in healthy male rats. Chemical and molecular inhibition of Erk1/2 signaling in the DVC negated the acute anorectic effect of insulin in healthy rats, while DVC insulin acute infusion failed to lower food intake in high fat-fed rats. Finally, molecular disruption of Erk1/2 signaling in the DVC of healthy rats per se increased food intake and induced obesity over a period of 2 weeks, whereas a daily repeated acute DVC insulin infusion for 12 days conversely decreased food intake and body weight in healthy rats. In summary, insulin activates Erk1/2 signaling in the DVC to regulate energy balance.

Jejunal leptin-PI3K signaling lowers glucose production.

The fat-derived hormone leptin binds to its hypothalamic receptors to regulate glucose homeostasis. Leptin is also synthesized in the stomach and subsequently binds to its receptors expressed in the intestine, although the functional relevance of such activation remains largely unknown. We report here that intrajejunal leptin administration activates jejunal leptin receptors and signals through a phosphatidylinositol 3-kinase (PI3K)-dependent and signal transducer and activator of transcription 3 (STAT3)-independent signaling pathway to lower glucose production in healthy rodents. Jejunal leptin action is sufficient to lower glucose production in uncontrolled diabetic and high-fat-fed rodents and contributes to the early antidiabetic effect of duodenal-jejunal bypass surgery. These data unveil a glucoregulatory site of leptin action and suggest that enhancing leptin-PI3K signaling in the jejunum lowers plasma glucose concentrations in diabetes.

Hypothalamic glucagon signaling inhibits hepatic glucose production.

Glucagon activates hepatic protein kinase A (PKA) to increase glucose production, but the gluco-stimulatory effect is transient even in the presence of continuous intravenous glucagon infusion. Continuous intravenous infusion of insulin, however, inhibits glucose production through its sustained actions in both the liver and the mediobasal hypothalamus (MBH). In a pancreatic clamp setting, MBH infusion with glucagon activated MBH PKA and inhibited hepatic glucose production (HGP) in rats, as did central glucagon infusion in mice. Inhibition of glucagon receptor-PKA signaling in the MBH and hepatic vagotomy each negated the effect of MBH glucagon in rats, whereas the central effect of glucagon was diminished in glucagon receptor knockout mice. A sustained rise in plasma glucagon concentrations transiently increased HGP, and this transiency was abolished in rats with negated MBH glucagon action. In a nonclamp setting, MBH glucagon infusion improved glucose tolerance, and inhibition of glucagon receptor-PKA signaling in the MBH enhanced the ability of intravenous glucagon injection to increase plasma glucose concentrations. We also detected a similar enhancement of glucose concentrations that was associated with a disruption in MBH glucagon signaling in rats fed a high-fat diet. We show that hypothalamic glucagon signaling inhibits HGP and suggest that hypothalamic glucagon resistance contributes to hyperglycemia in diabetes and obesity.

Insulin activates Erk1/2 signaling in the dorsal vagal complex to inhibit glucose production.

Insulin activates PI3-kinase (PI3K)/AKT to regulate glucose homeostasis in the peripheral tissues and the mediobasal hypothalamus (MBH) of rodents. We report that insulin infusion into the MBH or dorsal vagal complex (DVC) activated insulin receptors. The same dose of insulin that activated MBH PI3K/AKT did not in the DVC. DVC insulin instead activated Erk1/2 and lowered glucose production in rats and mice. Molecular and chemical inhibition of DVC Erk1/2 negated, while activation of DVC Erk1/2 recapitulated, the effects of DVC insulin. Circulating insulin failed to inhibit glucose production when DVC Erk1/2 was inhibited in normal rodents, while DVC insulin action was disrupted in high-fat-fed rodents. Activation of DVC ATP-sensitive potassium channels was necessary for insulin-Erk1/2 and sufficient to inhibit glucose production in normal and high-fat-fed rodents. DVC is a site of insulin action where insulin triggers Erk1/2 signaling to inhibit glucose production and of insulin resistance in high-fat feeding.