Michelle A. Puchowicz

Brain insulin controls adipose tissue lipolysis and lipogenesis

White adipose tissue (WAT) dysfunction plays a key role in the pathogenesis of type 2 diabetes (DM2). Unrestrained WAT lipolysis results in increased fatty acid release, leading to insulin resistance and lipotoxicity, while impaired de novo lipogenesis in WAT decreases the synthesis of insulin-sensitizing fatty acid species like palmitoleate. Here, we show that insulin infused into the mediobasal hypothalamus (MBH) of Sprague-Dawley rats increases WAT lipogenic protein expression, inactivates hormone-sensitive lipase (Hsl), and suppresses lipolysis. Conversely, mice that lack the neuronal insulin receptor exhibit unrestrained lipolysis and decreased de novo lipogenesis in WAT. Thus, brain and, in particular, hypothalamic insulin action play a pivotal role in WAT functionality.

Neuroprotection in diet-induced ketotic rat brain after focal ischemia.

Neuroprotective properties of ketosis may be related to the upregulation of hypoxia inducible factor (HIF)-1α, a primary constituent associated with hypoxic angiogenesis and a regulator of neuroprotective responses. The rationale that the utilization of ketones by the brain results in elevation of intracellular succinate, a known inhibitor of prolyl hydroxylase (the enzyme responsible for the degradation of HIF-1α) was deemed as a potential mechanism of ketosis on the upregulation of HIF-1α. The neuroprotective effect of diet-induced ketosis (3 weeks of feeding a ketogenic diet), as pretreatment, on infarct volume, after reversible middle cerebral artery occlusion (MCAO), and the upregulation of HIF-1α were investigated. The effect of β-hydroxybutyrate (BHB), as a pretreatment, via intraventricular infusion (4 days of infusion before stroke) was also investigated following MCAO. Levels of HIF-1α and Bcl-2 (anti-apoptotic protein) proteins and succinate content were measured. A 55% or 70% reduction in infarct volume was observed with BHB infusion or diet-induced ketosis, respectively. The levels of HIF-1α and Bcl-2 proteins increased threefold with diet-induced ketosis; BHB infusions also resulted in increases in these proteins. As hypothesized, succinate content increased by 55% with diet-induced ketosis and fourfold with BHB infusion. In conclusion, the biochemical link between ketosis and the stabilization of HIF-1α is through the elevation of succinate, and both HIF-1α stabilization and Bcl-2 upregulation play a role in ketone-induced neuroprotection in the brain.

Mutations in the MPV17 gene are responsible for rapidly progressive liver failure in infancy.

MPV17 is a mitochondrial inner membrane protein of unknown function recently recognized as responsible for a mitochondrial DNA depletion syndrome. The aim of this study is to delineate the specific clinical, pathological, biochemical, and molecular features associated with mitochondrial DNA depletion due to MPV17 gene mutations. We report 4 cases from 3 ethnically diverse families with MPV17 mutations. Importantly, 2 of these cases presented with isolated liver failure during infancy without notable neurologic dysfunction. Conclusion: We therefore propose that mutations in the MPV17 gene be considered in the course of evaluating the molecular etiology for isolated, rapidly progressive infantile hepatic failure. (HEPATOLOGY 2007.)

A self-defeating anabolic program leads to β-cell apoptosis in endoplasmic reticulum stress-induced diabetes via regulation of amino acid flux

Background: Protein synthesis control is important for β-cell fate during ER stress. Results: Increased protein synthesis during chronic ER stress in β-cells involves the transcriptional induction of an amino acid transporter network. Conclusion: Increased amino acid uptake in β-cells during ER stress promotes apoptosis. Significance: Induced expression of a network of amino acid transporters in islets can contribute to chronic ER stress-induced diabetes. Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats

Central nervous system oxygen toxicity (CNS-OT) seizures occur with little or no warning, and no effective mitigation strategy has been identified. Ketogenic diets (KD) elevate blood ketones and have successfully treated drug-resistant epilepsy. We hypothesized that a ketone ester given orally as R,S-1,3-butanediol acetoacetate diester (BD-AcAc(2)) would delay CNS-OT seizures in rats breathing hyperbaric oxygen (HBO(2)). Adult male rats (n = 60) were implanted with radiotelemetry units to measure electroencephalogram (EEG). One week postsurgery, rats were administered a single oral dose of BD-AcAc(2), 1,3-butanediol (BD), or water 30 min before being placed into a hyperbaric chamber and pressurized to 5 atmospheres absolute (ATA) O2. Latency to seizure (LS) was measured from the time maximum pressure was reached until the onset of increased EEG activity and tonic-clonic contractions. Blood was drawn at room pressure from an arterial catheter in an additional 18 animals that were administered the same compounds, and levels of glucose, pH, Po(2), Pco(2), β-hydroxybutyrate (BHB), acetoacetate (AcAc), and acetone were analyzed. BD-AcAc(2) caused a rapid (30 min) and sustained (>4 h) elevation of BHB (>3 mM) and AcAc (>3 mM), which exceeded values reported with a KD or starvation. BD-AcAc(2) increased LS by 574 ± 116% compared with control (water) and was due to the effect of AcAc and acetone but not BHB. BD produced ketosis in rats by elevating BHB (>5 mM), but AcAc and acetone remained low or undetectable. BD did not increase LS. In conclusion, acute oral administration of BD-AcAc(2) produced sustained ketosis and significantly delayed CNS-OT seizures by elevating AcAc and acetone.

Binge drinking induces whole-body insulin resistance by impairing hypothalamic insulin action.

Rats given alcohol to simulate binge drinking exhibit the first signs of diabetes, a result of altered insulin action in the brain. Worse than a Hangover For some college students, getting hammered on weekends is just part of life. But such binge drinking may be setting these young people up for diabetes. Lindtner et al. now show that rats given large doses of ethanol to simulate several wild nights of partying develop abnormal brain responses to insulin, even when the alcohol has been cleared from their system. When this brain system goes awry, the animals become generally insulin-resistant, the first step in acquiring diabetes. The authors gave Sprague-Dawley rats a daily dose of alcohol equivalent to 7 ounces for a person. As long as 54 hours after the last drink, the rats showed signs of impaired glucose tolerance—insulin at a particular concentration was not as effective in provoking glucose uptake into body tissues. This was largely a result of decreased insulin action in the liver, which in turn prevented normal glucose uptake into the liver. Insulin also failed to induce normal lipolysis in white adipose tissue, increasing gluconeogenic substrates to the liver and further impairing liver insulin action. Unexpectedly, insulin acted abnormally in the brain after binge drinking. Infusion of insulin directly into the hypothalamus usually suppresses liver and adipose tissue lipolysis, likely via the autonomic nervous system. But after ethanol administration, the authors found that direct treatment with insulin did not have this effect: Insulin infused into the hypothalamus did not suppress hepatic glucose production or lipolysis. Consistent with these data was the fact that, in ethanol-treated animals, biochemical markers of insulin signaling (phosphorylation of the insulin receptor and AKT phosphorylation) decreased in the brain but not in the liver. How does the ethanol harm the hypothalamus? The authors found that rounds of binge drinking apparently trigger inflammation, as assessed by interleukin-6 and tumor necrosis factor–α levels. The tyrosine phosphatase gene PTP1B was also elevated, playing a critical role in mediating alcohol’s effect because its inhibition can restore normal glucose tolerance after binge drinking. Whether these toxic effects of ethanol occur in partying people is not yet known, but the powerful effect of ethanol on the insulin-glucose regulatory system is certainly sobering. Individuals with a history of binge drinking have an increased risk of developing the metabolic syndrome and type 2 diabetes. Whether binge drinking impairs glucose homeostasis and insulin action is unknown. To test this, we treated Sprague-Dawley rats daily with alcohol (3 g/kg) for three consecutive days to simulate human binge drinking and found that these rats developed and exhibited insulin resistance even after blood alcohol concentrations had become undetectable. The animals were resistant to insulin for up to 54 hours after the last dose of ethanol, chiefly a result of impaired hepatic and adipose tissue insulin action. Because insulin regulates hepatic glucose production and white adipose tissue lipolysis, in part through signaling in the central nervous system, we tested whether binge drinking impaired brain control of nutrient partitioning. Rats that had consumed alcohol exhibited impaired hypothalamic insulin action, defined as the ability of insulin infused into the mediobasal hypothalamus to suppress hepatic glucose production and white adipose tissue lipolysis. Insulin signaling in the hypothalamus, as assessed by insulin receptor and AKT phosphorylation, decreased after binge drinking. Quantitative polymerase chain reaction showed increased hypothalamic inflammation and expression of protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signaling. Intracerebroventricular infusion of CPT-157633, a small-molecule inhibitor of PTP1B, prevented binge drinking–induced glucose intolerance. These results show that, in rats, binge drinking induces systemic insulin resistance by impairing hypothalamic insulin action and that this effect can be prevented by inhibition of brain PTP1B.

Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice

The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARγ binding site in the promoter of Pck1 (PPARE−/−). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE−/− mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE−/− mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention.

KETONES SUPPRESS BRAIN GLUCOSE CONSUMPTION

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as beta-hydroxybutyrate (beta HB) and acetoacetate (AcAc), as occurs with fasting, prolonged starvation or chronic feeding of a high fat/low carbohydrate diet (ketogenic diet). In this study, the local cerebral metabolic rate of glucose consumption (CMRglu; microM/min/100g) was calculated in the cortex and cerebellum of control and ketotic rats using Patlak analysis. Rats were imaged on a rodent PET scanner and MRI was performed on a 7-Tesla Bruker scanner for registration with the PET images. Plasma glucose and beta HB concentrations were measured and 90-minute dynamic PET scans were started simultaneously with bolus injection of 2-Deoxy-2[18F]Fluoro-D-Glucose (FDG). The blood radioactivity concentration was automatically sampled from the tail vein for 3 min following injection and manual periodic blood samples were taken. The calculated local CMRGlu decreased with increasing plasma BHB concentration in the cerebellum (CMRGlu = -4.07*[BHB] + 61.4, r2 = 0.3) and in the frontal cortex (CMRGlu = -3.93*[BHB] + 42.7, r2 = 0.5). These data indicate that, under conditions of ketosis, glucose consumption is decreased in the cortex and cerebellum by about 10% per each mM of plasma ketone bodies.

Targeting pyruvate carboxylase reduces gluconeogenesis and adiposity and improves insulin resistance

We measured the mRNA and protein expression of the key gluconeogenic enzymes in human liver biopsy specimens and found that only hepatic pyruvate carboxylase protein levels related strongly with glycemia. We assessed the role of pyruvate carboxylase in regulating glucose and lipid metabolism in rats through a loss-of-function approach using a specific antisense oligonucleotide (ASO) to decrease expression predominantly in liver and adipose tissue. Pyruvate carboxylase ASO reduced plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also reduced adiposity, plasma lipid concentrations, and hepatic steatosis in high fat–fed rats and improved hepatic insulin sensitivity. Pyruvate carboxylase ASO had similar effects in Zucker Diabetic Fatty rats. Pyruvate carboxylase ASO did not alter de novo fatty acid synthesis, lipolysis, or hepatocyte fatty acid oxidation. In contrast, the lipid phenotype was attributed to a decrease in hepatic and adipose glycerol synthesis, which is important for fatty acid esterification when dietary fat is in excess. Tissue-specific inhibition of pyruvate carboxylase is a potential therapeutic approach for nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes.

Ketosis Proportionately Spares Glucose Utilization in Brain

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as β-hydroxybutyrate and acetoacetate, as occurs with fasting, starvation, or chronic feeding of a ketogenic diet. The relationship between changes in cerebral metabolic rates of glucose (CMRglc) and degree or duration of ketosis remains uncertain. To investigate if CMRglc decreases with chronic ketosis, 2-[18F]fluoro-2-deoxy-D-glucose in combination with positron emission tomography, was applied in anesthetized young adult rats fed 3 weeks of either standard or ketogenic diets. Cerebral metabolic rates of glucose (μmol/min per 100 g) was determined in the cerebral cortex and cerebellum using Gjedde-Patlak analysis. The average CMRglc significantly decreased in the cerebral cortex (23.0 ±4.9 versus 32.9 ±4.7) and cerebellum (29.3 ± 8.6 versus 41.2 ±6.4) with increased plasma ketone bodies in the ketotic rats compared with standard diet group. The reduction of CMRg|c in both brain regions correlates linearly by ∼9% for each 1 mmol/L increase of total plasma ketone bodies (0.3 to 6.3 mmol/L). Together with our meta-analysis, these data revealed that the degree and duration of ketosis has a major role in determining the corresponding change in CMRglc with ketosis.