Dietary citrate acutely induces insulin resistance and markers of liver inflammation in mice.

Citrate is widely used as a food additive being part of virtually all processed foods. Although considered inert by most of the regulatory agencies in the world, plasma citrate has been proposed to play immunometabolic functions in multiple tissues through altering a plethora of cellular pathways. Here, we used a short-term alimentary intervention (24 hours) with standard chow supplemented with citrate in amount corresponding to that found in processed foods to evaluate its effects on glucose homeostasis and liver physiology in C57BL/6J mice. Animals supplemented with dietary citrate showed glucose intolerance and insulin resistance as revealed by glucose and insulin tolerance tests. Moreover, animals supplemented with citrate in their food displayed fed and fasted hyperinsulinemia and enhanced insulin secretion during an oral glucose tolerance test. Citrate treatment also amplified glucose-induced insulin secretion in vitro in INS1-E cells. Citrate supplemented animals had increased liver PKCα activity and altered phosphorylation at serine or threonine residues of components of insulin signaling including IRS-1, Akt, GSK-3 and FoxO1. Furthermore, citrate supplementation enhanced the hepatic expression of lipogenic genes suggesting increased de novo lipogenesis, a finding that was reproduced after citrate treatment of hepatic FAO cells. Finally, liver inflammation markers were higher in citrate supplemented animals. Overall, the results demonstrate that dietary citrate supplementation in mice causes hyperinsulinemia and insulin resistance both in vivo and in vitro, and therefore call for a note of caution on the use of citrate as a food additive given its potential role in metabolic dysregulation.

Food restriction promotes damage reduction in rat models of type 2 diabetes mellitus.

There are several animal models of type 2 diabetes mellitus induction but the comparison between models is scarce. Food restriction generates benefits, such as reducing oxidative stress, but there are few studies on its effects on diabetes. The objective of this study is to evaluate the differences in physiological and biochemical parameters between diabetes models and their responses to food restriction. For this, 30 male Wistar rats were distributed in 3 groups (n = 10/group): control (C); diabetes with streptozotocin and cafeteria-style diet (DE); and diabetes with streptozotocin and nicotinamide (DN), all treated for two months (pre-food restriction period). Then, the 3 groups were subdivided into 6, generating the groups CC (control), CCR (control+food restriction), DEC (diabetic+standard diet), DER (diabetic+food restriction), DNC (diabetic+standard diet) and DNR (diabetic+food restriction), treated for an additional two months (food restriction period). The food restriction (FR) used was 50% of the average daily dietary intake of group C. Throughout the treatment, physiological and biochemical parameters were evaluated. At the end of the treatment, serum biochemical parameters, oxidative stress and insulin were evaluated. Both diabetic models produced hyperglycemia, polyphagia, polydipsia, insulin resistance, high fructosamine, hepatic damage and reduced insulin, although only DE presented human diabetes-like alterations, such as dyslipidemia and neuropathy symptoms. Both DEC and DNC diabetic groups presented higher levels of protein carbonyl groups associated to lower antioxidant capacity in the plasma. FR promoted improvement of glycemia in DNR, lipid profile in DER, and insulin resistance and hepatic damage in both diabetes models. FR also reduced the protein carbonyl groups of both DER and DNR diabetic groups, but the antioxidant capacity was improved only in the plasma of DER group. It is concluded that FR is beneficial for diabetes but should be used in conjunction with other therapies.

Oral lactate intensifies insulin toxicity during severe insulin-induced hypoglycemia in mice

ABSTRACT We investigated whether oral lactate could prevent seizures and deaths in mice with severe hypoglycemia induced by a high dose of insulin. For this purpose, mice were fasted for 15 h and then given an intraperitoneal injection of regular insulin (5.0 U/kg or 10.0 U/kg). Immediately after insulin injection, the mice received an oral dose of saline (control), glucose (5.5 mmol/kg), or lactate (18.0 mmol/kg). Glucose and lactate levels were measured in the blood and brain before and after the seizures began. Glucose and lactate delayed (p < 0.05) the onset of seizures associated with severe insulin-induced hypoglycemia. Elevated (p < 0.05) brain levels of lactate were associated with an absence of seizures in mice that received glucose or lactate, suggesting that lactate could prevent convulsions associated with severe insulin-induced hypoglycemia. However, the same oral dose of lactate that delayed the onset of convulsions also increased the mortality rate. In contrast, diazepam (3.0 mg/kg) prevented seizures and markedly decreased the frequency of death during severe insulin-induced hypoglycemia. The results demonstrated that in contrast to oral glucose, oral lactate intensifies insulin toxicity.

Early-onset obesity and food restriction alter hepatocyte metabolism in adult Wistar rats.

CONTEXT: Caloric restriction (CR) is suggested for overweight control. OBJECTIVE: Systemic and liver glucose metabolism in the reduced-litter (RL) rat model under 30% CR was investigated. MATERIALS AND METHODS: Newborn litters were organised in control (G9); RL with free diet (G3L); and RL with CR (G3R). Assessments were made at the age of 90 d. RESULTS: Higher liver glycogen content and changes in systemic glucose handling were found in the RL groups. Hepatocyte glucose metabolism was similar in groups G9 and G3L, but basal glucose production and glycogenolysis were higher, while gluconeogenesis and basal glycolysis were lower in the G3R. Urea production was lower in the RL groups. DISCUSSION: The altered glucose handling of the RL adult rats was not reversed by moderate (30%) CR. Hepatocyte glucose and nitrogen metabolism were changed by both early overfeeding and current feeding conditions. CONCLUSIONS: RL and CR alter systemic and liver glucose metabolism.

Glutamine dipeptide and cortisol change the liver glucose metabolism and reduce the severity of insulin-induced hypoglycaemia in untreated T1DM Swiss mice.

CONTEXT: Glutamine is conditionally essential in type 1 diabetes mellitus, and might be useful to counteract hypoglycaemia. OBJECTIVE: To investigate the systemic and hepatic effects of counter-regulatory hormones and glutamine dipeptide (GDP) during hypoglycemic episodes. MATERIALS AND METHODS: Diabetic Swiss mice made hypoglycaemic by insulin injection (1 U/kg) were given counter-regulatory hormones and/or GDP. Sixty minutes later, liver histology, liver glucose metabolism and plasma were assessed. RESULTS: Combined, cortisol and GDP improved the hypoglycemic profile. During liver perfusion, gluconeogenesis was possibly the major pathway leading to glucose release. Perfusion with gluconeogenic precursors after glycogen depletion by adrenaline increased liver glucose and urea release. DISCUSSION: The less severe hypoglycaemia could result from cortisol stimulating periportal gluconeogenesis and GDP inhibiting pericentral glycogenolysis, both favouring liver glucose release. CONCLUSIONS: At least some benefits of GDP and cortisol during hypoglycaemia came from their hepatic actions, and their use in diabetic patients should be explored.

Supplementation with L-Glutamine and L-Alanyl-L-Glutamine Changes Biochemical Parameters and Jejunum Morphophysiology in Type 1 Diabetic Wistar Rats.

We evaluated the effects of the supplementation with L-glutamine and glutamine dipeptide (GDP) on biochemical and morphophysiological parameters in streptozotocin-diabetic rats. For this purpose, thirty animals were distributed into six groups treated orally (gavage) during thirty days: non diabetic rats (Control) + saline, diabetic + saline; Control + L-glutamine (248 mg/kg), Diabetic + L-glutamine (248 mg/kg), Control + GDP (400 mg/kg), Diabetic + GDP (400 mg/kg). Diabetes was induced by an intravenous injection of streptozotocin (60 mg/kg) and confirmed by fasting glucose ≥ 200 mg/dL. Physiological parameters, i.e., body mass, food intake, blood glucose, water intake, urine and faeces were evaluated during supplementation. After the period of supplementation, the animals were euthanized. The blood was collected for biochemical assays (fructosamine, transaminases, lipid profile, total protein, urea, ammonia). Moreover, the jejunum was excised and stored for morphophysiological assays (intestinal enzyme activity, intestinal wall morphology, crypt proliferative index, number of serotoninergic cells from the mucosa, and vipergic neurons from the submucosal tunica). The physiological parameters, protein metabolism and intestinal enzyme activity did not change with the supplementation with L-glutamine or GDP. In diabetic animals, transaminases and fructosamine improved with L-glutamine and GDP supplementations, while the lipid profile improved with L-glutamine. Furthermore, both forms of supplementation promoted changes in jejunal tunicas and wall morphometry of control and diabetic groups, but only L-glutamine promoted maintenance of serotoninergic cells and vipergic neurons populations. On the other hand, control animals showed changes that may indicate negative effects of L-glutamine. Thus, the supplementation with L-glutamine was more efficient for maintaining intestinal morphophysiology and the supplementation with GDP was more efficient to the organism as a whole. Thus, we can conclude that local differences in absorption and metabolism could explain the differences between the supplementation with L-glutamine or GDP.

Acute exercise and caffeine improve insulin-induced hypoglycemia in normal and malnourished rats / Exercício agudo e cafeína melhoram a hipoglicemia induzida por insulina em ratos normais e desnutridos

In food restriction, hypoglycemic episodes can be more severe and persistent. This study assessed the influence of acute exercise and caffeine on the insulin-induced hypoglycemia in freely-fed or malnourished (50% food restriction) young rats. At the age of 60 days, rats under overnight fasting received an insulin injection to cause an episode of hypoglycemia. In some animals, hypoglycemia was preceded by an acute session of exercise, in others, caffeine was orally given 15 minutes after insulin injection; or exercise and caffeine were combined. Blood samples were collected at regular intervals for five hours after insulin injection. A beneficial effect of both exercise or caffeine on the hypoglycemic episode in the malnourished rats was found. In the control rats, the association of exercise+caffeine was more beneficial than either intervention alone. It is discussed that exercise and caffeine, alone or combined, can be used as exogenous anti-hypoglycemic resources, but considering the nutritional status of the subject.

Na restrição alimentar, os episódios de hipoglicemia podem ser mais severos e persistentes. Este trabalho avaliou a influência do exercício agudo e da cafeína sobre a hipoglicemia induzida por insulina em ratos jovens alimentados livremente ou desnutridos (50% de restrição alimentar) desde o nascimento. Aos 60 dias de idade, os ratos em jejum noturno receberam injeção de insulina para causar um episódio de hipoglicemia. Em alguns animais, a hipoglicemia foi precedida por sessão aguda de exercício; em outros foi administrada a cafeína via oral, 15 min. após a injeção de insulina; ou foi associada a exercício e cafeína. Amostras de sangue foram coletadas em intervalos regulares por 5h desde a aplicação da insulina. Registrou-se efeito benéfico do exercício e da cafeína isolados sobre o episódio hipoglicêmico nos ratos desnutridos. Nos ratos controle, a associação de exercício+cafeína teve efeito benéfico mais acentuado do que as intervenções separadas. É discutido que exercício físico e cafeína, isolados ou combinados, podem ser explorados como recursos anti-hipoglicêmicos exógenos, levando-se em consideração porém o estado nutricional do indivíduo.

Sustained Liver Glucose Release in Response to Adrenaline Can Improve Hypoglycaemic Episodes in Rats under Food Restriction Subjected to Acute Exercise.

Background. As the liver is important for blood glucose regulation, this study aimed at relating liver glucose release stimulated by glucagon and adrenaline to in vivo episodes of hypoglycaemia. Methods. The blood glucose profile during an episode of insulin-induced hypoglycaemia in exercised and nonexercised male Wistar control (GC) and food-restricted (GR, 50%) rats and liver glucose release stimulated by glucagon and adrenaline were investigated. Results. In the GR, the hypoglycaemic episodes showed severe decreases in blood glucose, persistent hypoglycaemia, and less complete glycaemic recovery. An exercise session prior to the episode of hypoglycaemia raised the basal blood glucose, reduced the magnitude of the hypoglycaemia, and improved the recovery of blood glucose. In fed animals of both groups, liver glucose release was activated by glucagon and adrenaline. In fasted GR rats, liver glycogenolysis activated by glucagon was impaired, despite a significant basal glycogenolysis, while an adrenaline-stimulated liver glucose release was recorded. Conclusions. The lack of liver response to glucagon in the GR rats could be partially responsible for the more severe episodes of hypoglycaemia observed in vivo in nonexercised animals. The preserved liver response to adrenaline can partially account for the less severe hypoglycaemia in the food-restricted animals after acute exercise.