Lipid sensing and insulin resistance in the brain

Lipid sensing and insulin signaling in the brain independently triggers a negative feedback system to lower glucose production and food intake. Here, we discuss the underlying molecular and neuronal mechanisms of lipid sensing and insulin signaling in the hypothalamus and how these mechanisms are affected in response to high-fat feeding. We propose that high-fat feeding concurrently disrupts hypothalamic insulin-signaling and lipid-sensing mechanisms and that experiments aimed to restore both insulin action and lipid sensing in the brain could effectively lower glucose production and food intake to restore metabolic homeostasis in type 2 diabetes and obesity.     

A high-fat diet raises fasting plasma CCK but does not affect upper gut motility, PYY, and ghrelin, or energy intake during CCK-8 infusion in lean men

There is evidence from studies in animals that the effects of both fat and CCK on gastrointestinal function and energy intake are attenuated by consumption of a high-fat diet. In humans, the effects of exogenous CCK-8 on antropyloroduodenal motility, plasma CCK, peptide YY (PYY), and ghrelin concentrations, appetite, and energy intake are attenuated by a high-fat diet. Ten healthy lean males consumed isocaloric diets (~15,400 kJ per day), containing either 44% (high-fat, HF) or 9% (low-fat, LF) fat, for 21 days in single-blind, randomized, cross-over fashion. Immediately following each diet (i.e., on day 22), subjects received a 45-min intravenous infusion of CCK-8 (2 ng.kg(-1).min(-1)), and effects on antropyloroduodenal motility, plasma CCK, PYY, ghrelin concentrations, hunger, and fullness were determined. Thirty minutes after commencement of the infusion, subjects were offered a buffet-style meal, from which energy intake (in kilojoules) was quantified. Body weight was unaffected by the diets. Fasting CCK (P < 0.05), but not PYY and ghrelin, concentrations were greater following the HF, compared with the LF, diet. Infusion of CCK-8 stimulated pyloric pressures (P < 0.01) and suppressed antral and duodenal pressures (P < 0.05), with no difference between the diets. Energy intake also did not differ between the diets. Short-term consumption of a HF diet increases fasting plasma CCK concentrations but does not affect upper gut motility, PYY and ghrelin, or energy intake during CCK-8 infusion, in a dose of 2 ng.kg(-1).min(-1), in healthy males.     

A Lipoprotein Lipase–Promoting Agent,NO-1886, Improves Glucose and Lipid Metabolism in High Fat,High Sucrose–Fed New Zealand White Rabbits

The synthetic compound NO-1886 is a lipoprotein lipase activator that lowers plasma triglycerides and elevates high-density lipoprotein cholesterol (HDL-C). Recently, the authors found that NO-1886 also had an action of reducing plasma glucose in high-fat/high-sucrose diet–induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 on insulin resistance and β-cell function in rabbits. Our results showed that high-fat/high-sucrose feeding increased plasma triglyceride, free fatty acid (FFA), and glucose levels and decreased HDL-C level. This diet also induced insulin resistance and impairment of acute insulin response to glucose loading. Supplementing 1% NO-1886 into the high-fat/high-sucrose diet resulted in decreased plasma triglyceride, FFA, and glucose levels and increased HDL-C level. The authors also found a clear increased glucose clearance and a protected acute insulin response to intravenous glucose loading by NO-1886 supplementation. These data suggest that NO-1886 suppresses the elevation of blood glucose in rabbits induced by feeding a high-fat/high-sucrose diet, probably through controlling lipid metabolism and improving insulin resistance.     

Both afferent and efferent nerves are implicated in cholecytokinin motor actions in the small intestine of the rat.

Cholecystokinin (CCK) regulates intestinal motility after being released by several luminal nutrients. However the mechanism of action of CCK is still not well known. The aim of our study was to establish the mechanism of action of CCK in the rat intestine using an in vivo model and focusing on the nervous pathways involved in the response as well as type of receptors. Anesthetized rats were prepared with two strain-gauges, in duodenum and jejunum, to record circular muscle motor activity. A group of animals was also prepared with a catheter to infuse capsaicin inside the duodenum. Responses to CCK-octapeptide (CCK-8) as well as to CCK agonists were studied. CCK-8 was also infused after CCK antagonists, atropine, hexamethonium or L-nitroarginine. Results show that duodenal response to CCK-8 is excitatory although inhibitory responses can be induced by gastrin. In the jejunum, CCK-8 induces an inhibitory response that is mediated by both CCK-A and -B receptors. Excitatory responses to CCK-8 are due to stimulation of preganglionic receptors while inhibitory responses are NO mediated through stimulation of postganglionic CCK-B receptors. Capsaicin locally applied in duodenal mucosa significantly decreased CCK-8 response, whereas mucosal exposure to lidocaine completely blocked CCK-8 response. In conclusion our results show that CCK response varies along the intestine according to the predominance of excitatory or inhibitory efferent innervation. Moreover, CCK-8 actions are mediated through both extrinsic and intrinsic afferent fibres.     

Peptides that regulate food intake: norepinephrine is not required for reduction of feeding induced by cholecystokinin

CCK octapeptide (CCK-8) is released by the gut in response to a meal and acts via CCK(A) receptors on vagal afferents to induce satiety. However, the central neural pathways by which peripheral CCK-8 affects feeding are poorly understood. In the present study, we tested the hypothesis that norepinephrine (NE) is necessary for satiety induced by peripheral CCK-8 by using mice lacking dopamine beta-hydroxylase (Dbh(-/-)), the enzyme responsible for synthesizing NE and epinephrine from dopamine. We found that Dbh(-/-) mice are as responsive to the satiating effects of CCK-8 as their normal littermates.     

Hormonal Signaling in the Gut

The gut is anatomically positioned to play a critical role in the regulation of metabolic homeostasis, providing negative feedback via nutrient sensing and local hormonal signaling. Gut hormones, such as cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1), are released following a meal and act on local receptors to regulate glycemia via a neuronal gut-brain axis. Additionally, jejunal nutrient sensing and leptin action are demonstrated to suppress glucose production, and both are required for the rapid antidiabetic effect of duodenal jejunal bypass surgery. Strategies aimed at targeting local gut hormonal signaling pathways may prove to be efficacious therapeutic options to improve glucose control in diabetes.     

Ileal interposition attenuates the satiety responses evoked by cholecystokinin-8 and -33

One of the possible mechanisms by which the weight-reducing surgical procedure ileal interposition (II) works is by increasing circulating levels of lower gut peptides that reduce food intake, such as glucagon like peptide-1 and peptide YY. However, since this surgery involves both lower and upper gut segments, we tested the hypothesis that II alters the satiety responses evoked by the classic upper gut peptide cholecystokinin (CCK). To test this hypothesis, we determined meal size (MS), intermeal interval (IMI) and satiety ratio (SR) evoked by CCK-8 and -33 (0, 1, 3, 5 nmol/kg, i.p.) in two groups of rats, II and sham-operated. CCK-8 and -33 reduced MS more in the sham group than in the II group; CCK-33 prolonged IMI in the sham group and increased SR in both groups. Reduction of cumulative food intake by CCK-8 in II rats was blocked by devazepide, a CCK₁ receptor antagonist. In addition, as previously reported, we found that II resulted in a slight reduction in body weight compared to sham-operated rats. Based on these observations, we conclude that ileal interposition attenuates the satiety responses of CCK. Therefore, it is unlikely that this peptide plays a significant role in reduction of body weight by this surgery.     

Effect of exogenous and endogenous insulin on the secretory response of the pancreas to the octapeptide of cholecystokinin (CCK8) in normal rats

The author studied the effect of insulin on CCK8-stimulated secretion by the pancreas. CCK8 (0.6 nmol.kg-1) was administered to normal anaesthetized rats 30 min after the intravenous injection of insulin (10 U.kg-1), glucose (2 g.kg-1) or NaCl (controls). Pancreatic juice was collected from the intubated common bile duct. In rats given exogenous insulin, there were no statistically significant differences in total protein, amylase and trypsinogen output after CCK8 compared with the controls. In rats in which endogenous insulin secretion was stimulated with glucose, the amylase response to CCK8 was not significantly different from the control animals, but the trypsinogen response was significantly lower. The results show that insulin, in some still unknown manner, inhibits the trypsinogen secretory response to CCK8. In addition, they confirm data claiming that the synthesis and secretion of pancreatic amylase require a given critical ratio of insulin to glucose, or of insulin to the factor stimulating pancreatic secretion.     

Cholecystokinin impact on rainbow trout glucose homeostasis: possible involvement of central glucosensors

Although the role of cholecystokinin (CCK) on fish appetite regulation has been widely studied, its involvement in the regulation of glucose metabolism had been little explored to date. In the present study we have carried out different experimental approaches to study CCK effects in rainbow trout (a so-called 'glucose intolerant' fish species) glucose homeostasis. We have found that for the first time in a vertebrate species, systemic or central CCK administration causes hyperglycemia, which is at least in part related to the presence of an ancestral gut-brain axis in which CCK is involved. By using capsaicin we have found that part of the action of CCK on glucose homeostasis is mediated by vagal and splanchnic afferents. Changes in hepatic metabolism after systemic CCK administration suggest that the effects are not directly taking place on the liver, but probably in other tissues, while after the central CCK administration, the glycogenolytic response observed in liver could be mediated by the activation of the sympathetic system. In hypothalamus and hindbrain changes elicited by CCK-8 treatment are likely related to the glucosensor response to the increased glycemia and/or vagal/splanchnic afferences whereas in hindbrain a possible action through specific CCK-1 receptors cannot be excluded. All these processes result in changes in metabolic parameters related with glucose homeostasis control. Further studies are needed to fully understand the role of this peptide on glucose homeostasis control in fish.     

Role of PYK2 in the development of obesity and insulin resistance

Non-receptor proline-rich tyrosine kinase-2 (PYK2), which is activated by phosphorylation of one or more of its tyrosine residues, has been implicated in the regulation of GLUT4 glucose transporter translocation and glucose transport. Some data favor a positive role of PYK2 in stimulating glucose transport, whereas other studies suggest that PYK2 may participate in the induction of insulin resistance. To ascertain the importance of PYK2 in the setting of obesity and insulin resistance, we (1) evaluated the regulation of PYK2 in mice fed a high-fat diet and (2) characterized body and glucose homeostasis in wild type (WT) and PYK2(-/-) mice on different diets. We found that both PYK2 expression and phosphorylation were significantly increased in liver and adipose tissues harvested from high-fat diet fed mice. Wild type and PYK2(-/-) mice were fed a high-fat diet for 8 weeks to induce insulin resistance/obesity. Surprisingly, in response to this diet PYK2(-/-) mice gained significantly more weight than WT mice (18.7+/-1.2g vs. 9.5+/-0.6g). Fasting serum leptin and insulin and blood glucose levels were significantly increased in high-fat diet fed mice irrespective of the presence of PYK2 protein. There was a close correlation between serum leptin and body weight. Intraperitoneal glucose tolerance tests revealed that as expected, the high-fat diet resulted in increased blood glucose levels following glucose administration in wild type mice compared to those fed normal chow. An even greater increase in blood glucose levels was observed in PYK2(-/-) mice compared to wild type mice. These results demonstrate that a lack of PYK2 exacerbates weight gain and development of glucose intolerance/insulin resistance induced by a high-fat diet, suggesting that PYK2 may play a role in slowing the development of obesity, insulin resistance, and/or frank diabetes.     

Endogenous cholecystokinin reduces food intake and increases Fos-like immunoreactivity in the dorsal vagal complex but not in the myenteric plexus by CCK1 receptor in the adult rat

We hypothesized that endogenous CCK reduces food intake by activating the dorsal vagal complex (DVC) and the myenteric neurons of the gut. To test this hypothesis, adult rats were given camostat mesilate; a nonnutrient releaser of endogenous CCK, by orogastric gavage, and Fos-like immunoreactivity (Fos-LI) was quantified in the DVC and the myenteric plexus. The results for endogenous CCK were compared with those for exogenous CCK-8. Exogenous CCK-8 reduced food intake and stimulated Fos-LI in the DVC and in myenteric neurons of the duodenum and jejunum. In comparison, endogenous CCK reduced food intake and increased DVC Fos-LI but did not increase Fos-LI in the myenteric plexus. Similar to CCK-8, devazepide, a specific CCK(1) receptor antagonist, and not L365,260, a specific CCK(2) receptor antagonist, attenuated the reduction of food intake by camostat. In addition, Fos-LI in the DVC in response to both exogenous CCK-8 and camostat administration was significantly attenuated by vagotomy, as well as by blocking CCK(1) receptors. These results demonstrate for the first time that reduction of food intake in adult rats by endogenous CCK released by a nonnutrient mechanism requires CCK(1) receptors, the vagus nerve, and activation of the DVC, but not the myenteric plexus.     

Nonsulfated cholecystokinins in the small intestine of pigs and rats

Cholecystokinin (CCK) is a gut hormone that acts via two receptors. The CCK_A-receptor requires the tyrosyl residue in the C-terminal bioactive site of CCK to be O-sulfated, whereas, the CCK_B-receptor binds irrespective of sulfation. Consequently, unsulfated CCK peptides – if present – may constitute a hormone system that acts only through the CCK_B-receptor. Therefore, we have now examined whether, CCK peptides occur in nonsulfated form in the small intestine of pigs and rats. The concentrations of sulfated and nonsulfated CCK were measured by RIAs, one specific for sulfated CCKs and a new two-step assay specific for nonsulfated CCK. For further characterization, the intestinal extracts were subjected to size- and ion exchange-chromatography.The intestinal concentrations of sulfated and nonsulfated CCK were highest in the duodenum and the proximal part of jejunum both in the pig and the rat. The porcine duodenal mucosa contained 193 ± 84 pmol/g sulfated CCK and 31 ± 10 pmol/g nonsulfated CCK, and the upper rat intestine 70 ± 19 pmol/g and 8 ± 2 pmol/g, respectively. The degree of sulfation correlated with the endoproteolytic proCCK processing. Thus, 38% of porcine CCK-58 was unsulfated, whereas, only 12% of CCK-8 was unsulfated.The results show that a substantial part of intestinal CCK peptides in rats and pigs are not sulfated, and that the longer peptides (CCK-58 and CCK-33) are less sulfated than the shorter (CCK-22 and CCK-8). Hence, the results demonstrate that proCCK in the gut is processed both to sulfated and unsulfated α-amidated peptides of which the latter are assumed to act via the CCK_B-receptor.     

A1 adenosine receptor partial agonist lowers plasma FFA and improves insulin resistance induced by high-fat diet in rodents

There is substantial evidence in the literature that elevated plasma free fatty acids (FFA) play a role in the pathogenesis of type 2 diabetes. CVT-3619 is a selective partial A(1) adenosine receptor agonist that inhibits lipolysis and lowers circulating FFA. The present study was undertaken to determine the effect of CVT-3619 on insulin resistance induced by high-fat (HF) diet in rodents. HF diet feeding to rats for 2 wk caused a significant increase in insulin, FFA, and triglyceride (TG) concentrations compared with rats fed chow. CVT-3619 (1 mg/kg) caused a time-dependent decrease in fasting insulin, FFA, and TG concentrations. Acute administration of CVT-3619 significantly lowered the insulin response, whereas glucose response was not different with an oral glucose tolerance test. Treatment with CVT-3619 for 2 wk resulted in significant lowering of FFA, TG, and insulin concentrations in rats on HF diet. To determine the effect of CVT-3619 on insulin sensitivity, hyperinsulinemic euglycemic clamp studies were performed in C57BL/J6 mice fed HF diet for 12 wk. Glucose infusion rate was decreased significantly in HF mice compared with chow-fed mice. CVT-3619 treatment 15 min prior to the clamp study significantly (P < 0.01) increased glucose infusion rate to values similar to that for chow-fed mice. In conclusion, CVT-3619 treatment lowers FFA and TG concentrations and improves insulin sensitivity in rodent models of insulin resistance.     

Effects of forebrain microinjection of cholecystokinin on dopamine cell firing rate.

In anesthetized rats, midbrain dopamine (DA) neuronal firing rate was differentially sensitive to focal brain microinjection of cholecystokinin peptides (CCK-4 and CCK-8) and N-methyl-D-aspartate (NMDA) into nucleus accumbens, amygdala and prefrontal cortex. Whereas changes in DA neuronal firing rate were frequently observed in response to intra-amygdalar microinjection of CCK peptides, NMDA was most effective in eliciting changes in DA neuronal activity following intra-accumbal microinjection. Thus, stimulation of amygdalar CCK receptors and accumbal excitatory amino acid receptors may participate in the afferent regulation of midbrain DA neuronal function.     

Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.     

Probiotics modulate gut microbiota and improve insulin sensitivity in DIO mice

Obesity and type 2 diabetes are characterized by subclinical inflammatory process. Changes in composition or modulation of the gut microbiota may play an important role in the obesity-associated inflammatory process. In the current study, we evaluated the effects of probiotics (Lactobacillus rhamnosus, L. acidophilus and Bifidobacterium bifidumi) on gut microbiota, changes in permeability, and insulin sensitivity and signaling in high-fat diet and control animals. More importantly, we investigated the effects of these gut modulations on hypothalamic control of food intake, and insulin and leptin signaling. Swiss mice were submitted to a high-fat diet (HFD) with probiotics or pair-feeding for 5 weeks. Metagenome analyses were performed on DNA samples from mouse feces. Blood was drawn to determine levels of glucose, insulin, LPS, cytokines and GLP-1. Liver, muscle, ileum and hypothalamus tissue proteins were analyzed by Western blotting and real-time polymerase chain reaction. In addition, liver and adipose tissues were analyzed using histology and immunohistochemistry. The HFD induced huge alterations in gut microbiota accompanied by increased intestinal permeability, LPS translocation and systemic low-grade inflammation, resulting in decreased glucose tolerance and hyperphagic behavior. All these obesity-related features were reversed by changes in the gut microbiota profile induced by probiotics. Probiotics also induced an improvement in hypothalamic insulin and leptin resistance. Our data demonstrate that the intestinal microbiome is a key modulator of inflammatory and metabolic pathways in both peripheral and central tissues. These findings shed light on probiotics as an important tool to prevent and treat patients with obesity and insulin resistance.     

Long-term high-fat feeding leads to severe insulin resistance but not diabetes in Wistar rats

Although lipid excess can impair beta-cell function in vitro, short-term high-fat feeding in normal rats produces insulin resistance but not hyperglycemia. This study examines the effect of long-term (10-mo) high polyunsaturated fat feeding on glucose tolerance in Wistar rats. The high fat-fed compared with the chow-fed group was 30% heavier and 60% fatter, with approximately doubled fasting hyperinsulinemia (P < 0.001) but only marginal fasting hyperglycemia (7.5 +/- 0.1 vs. 7.2 +/- 0.1 mmol/l, P < 0.01). Insulin sensitivity was approximately 67% lower in the high-fat group (P < 0.01). The acute insulin response to intravenous arginine was approximately double in the insulin-resistant high-fat group (P < 0.001), but that to intravenous glucose was similar in the two groups. After the intravenous glucose bolus, plasma glucose decline was slower in the high fat-fed group, confirming mild glucose intolerance. Therefore, despite severe insulin resistance, there was only a mildly elevated fasting glucose level and a relative deficiency in glucose-stimulated insulin secretion; this suggests that a genetic or congenital susceptibility to beta-cell impairment is required for overt hyperglycemia to develop in the presence of severe insulin resistance.     

The molecular nature of vascularly released cholecystokinin from the isolated perfused porcine duodenum

Using sequence-specific radioimmunoassays, the quantities and molecular nature of cholecystokinin (CCK) have been determined in extracts of porcine duodenal mucosa and in the vascular perfusate from the isolated porcine duodenum. The basal concentration of CCK in the perfusate was 84 pM equiv. CCK-8 (mean; range: 32–173 pM, n = 5). After intraluminal stimulation with amino acids, acidified fat emulsions and hydrochloric acid, the concentrations increased 2–5-fold. Both in the basal and stimulated state the concentrations of the related hormone, gastrin, were below 5 pM equiv. gastrin-17. CCK in the perfusate was concentrated by affinitychromatography using antibodies directed against the bioactive C-terminus. Subsequent gel chromatography revealed a form with a size like or slightly larger than the C-terminal dodecapeptide (CCK-12), a predominant form resembling the C-terminal octapeptide (CCK-8), and a form resembling the C-terminal tetrapeptide (CCK-4). The duodenal mucosa contained in addition CCK-33, -39 and CCK-peptides with further N-terminal extensions. The results suggest that small CCK peptides are the principal circulating forms, while CCK-33 and larger forms are biosynthetic precursors.     

Effects of peripheral CCK receptor blockade on gastric emptying in rats

Type A CCK receptor (CCKAR) antagonists differing in blood-brain barrier permeability [devazepide penetrates; the dicyclohexylammonium salt of Nalpha-3-quinolinoyl-d-Glu-N,N-dipentylamide (A-70104) does not] were used to test the hypothesis that duodenal nutrient-induced inhibition of gastric emptying is mediated by CCKARs located peripheral to the blood-brain barrier. Rats received A-70104 (700 or 3,000 nmol. kg(-1). h(-1) iv) or devazepide (2.5 micromol/kg iv) and either a 15-min intravenous infusion of CCK-8 (3 nmol. kg(-1). h(-1)) or duodenal infusion of casein, peptone, Intralipid, or maltose. Gastric emptying of saline was measured during the last 5 min of each infusion. A-70104 and devazepide abolished the gastric emptying response to a maximal inhibitory dose of CCK-8. Each of the macronutrients inhibited gastric emptying. A-70104 and devazepide attenuated inhibitory responses to each macronutrient. Intravenous injection of a CCK antibody to immunoneutralize circulating CCK had no effect on peptone or Intralipid-induced responses. Thus endogenous CCK appears to act in part by a paracrine or neurocrine mechanism at CCKARs peripheral to the blood-brain barrier to inhibit gastric emptying.