Regulation of receptor-type protein tyrosine phosphatases by their C-terminal tail domains.

Protein tyrosine phosphatases (PTPs) perform specific functions in vivo, despite being vastly outnumbered by their substrates. Because of this and due to the central roles PTPs play in regulating cellular function, PTP activity is regulated by a large variety of molecular mechanisms. We review evidence that indicates that the divergent C-terminal tail sequences (C-terminal domains, CTDs) of receptor-type PTPs (RPTPs) help regulate RPTP function by controlling intermolecular associations in a way that is itself subject to physiological regulation. We propose that the CTD of each RPTP defines an 'interaction code' that helps determine molecules it will interact with under various physiological conditions, thus helping to regulate and diversify PTP function.

Incretin, insulinotropic and glucose-lowering effects of whey protein pre-load in type 2 diabetes: a randomised clinical trial.

AIMS/HYPOTHESIS: Since protein ingestion is known to stimulate the secretion of glucagon-like peptide-1 (GLP-1), we hypothesised that enhancing GLP-1 secretion to harness its insulinotropic/beta cell-stimulating activity with whey protein pre-load may have beneficial glucose-lowering effects in type 2 diabetes. METHODS: In a randomised, open-label crossover clinical trial, we studied 15 individuals with well-controlled type 2 diabetes who were not taking any medications except for sulfonylurea or metformin. These participants consumed, on two separate days, 50 g whey in 250 ml water or placebo (250 ml water) followed by a standardised high-glycaemic-index breakfast in a hospital setting. Participants were randomised using a coin flip. The primary endpoints of the study were plasma concentrations of glucose, intact GLP-1 and insulin during the 30 min following meal ingestion. RESULTS: In each group, 15 patients were analysed. The results showed that over the whole 180 min post-meal period, glucose levels were reduced by 28% after whey pre-load with a uniform reduction during both early and late phases. Insulin and C-peptide responses were both significantly higher (by 105% and 43%, respectively) with whey pre-load. Notably, the early insulin response was 96% higher after whey. Similarly, both total GLP-1 (tGLP-1) and intact GLP-1 (iGLP-1) levels were significantly higher (by 141% and 298%, respectively) with whey pre-load. Dipeptidyl peptidase 4 plasma activity did not display any significant difference after breakfast between the groups. CONCLUSIONS/INTERPRETATION: In summary, consumption of whey protein shortly before a high-glycaemic-index breakfast increased the early prandial and late insulin secretion, augmented tGLP-1 and iGLP-1 responses and reduced postprandial glycaemia in type 2 diabetic patients. Whey protein may therefore represent a novel approach for enhancing glucose-lowering strategies in type 2 diabetes. Trial registration ClinicalTrials.gov NCT01571622 Funding The Israeli Ministry of Health and Milk Council funded the research.

Metformin affects the circadian clock and metabolic rhythms in a tissue-specific manner.

Metformin is a commonly-used treatment for type 2 diabetes, whose mechanism of action has been linked, in part, to activation of AMP-activated protein kinase (AMPK). However, little is known regarding its effect on circadian rhythms. Our aim was to evaluate the effect of metformin administration on metabolism, locomotor activity and circadian rhythms. We tested the effect of metformin treatment in the liver and muscle of young lean, healthy mice, as obesity and diabetes disrupt circadian rhythms. Metformin led to increased leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was similar leading to AMPK activation either by liver kinase B1 (LKB1) and/or other kinases in the muscle. AMPK activation resulted in the inhibition of acetyl CoA carboxylase (ACC), the rate limiting enzyme in fatty acid synthesis. Metformin also led to the activation of liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock. This effect was mainly of phase advances in the liver and phase delays in the muscle in clock and metabolic genes and/or protein expression. In conclusion, our results demonstrate the differential effects of metformin in the liver and muscle and the critical role the circadian clock has in orchestrating metabolic processes.

Effects of caloric intake timing on insulin resistance and hyperandrogenism in lean women with polycystic ovary syndrome.

In women with PCOS (polycystic ovary syndrome), hyperinsulinaemia stimulates ovarian cytochrome P450c17α activity that, in turn, stimulates ovarian androgen production. Our objective was to compare whether timed caloric intake differentially influences insulin resistance and hyperandrogenism in lean PCOS women. A total of 60 lean PCOS women [BMI (body mass index), 23.7±0.2 kg/m²] were randomized into two isocaloric (~1800 kcal; where 1 kcal≈4.184 J) maintenance diets with different meal timing distribution: a BF (breakfast diet) (980 kcal breakfast, 640 kcal lunch and 190 kcal dinner) or a D (dinner diet) group (190 kcal breakfast, 640 kcal lunch and 980 kcal dinner) for 90 days. In the BF group, a significant decrease was observed in both AUC(glucose) (glucose area under the curve) and AUC(insulin) (insulin area under the curve) by 7 and 54% respectively. In the BF group, free testosterone decreased by 50% and SHBG (sex hormone-binding globulin) increased by 105%. GnRH (gonadotropin-releasing hormone)-stimulated peak serum 17OHP (17α-hydroxyprogesterone) decreased by 39%. No change in these parameters was observed in the D group. In addition, women in the BF group had an increased ovulation rate. In lean PCOS women, a high caloric intake at breakfast with reduced intake at dinner results in improved insulin sensitivity indices and reduced cytochrome P450c17α activity, which ameliorates hyperandrogenism and improves ovulation rate. Meal timing and distribution should be considered as a therapeutic option for women with PCOS.

High-fat diet delays and fasting advances the circadian expression of adiponectin signaling components in mouse liver.

The circadian clock controls energy homeostasis by regulating circadian expression and/or activity of enzymes involved in metabolism. Disruption of circadian rhythms may lead to obesity and metabolic disorders. We tested whether the biological clock controls adiponectin signaling pathway in the liver and whether fasting and/or high-fat (HF) diet affects this control. Mice were fed low-fat or HF diet and fasted on the last day. The circadian expression of clock genes and components of adiponectin metabolic pathway in the liver was tested at the RNA, protein, or enzyme activity level. In addition, serum levels of glucose, adiponectin, and insulin were measured. Under low-fat diet, adiponectin signaling pathway components exhibited circadian rhythmicity. However, fasting and HF diet altered this circadian expression; fasting resulted in a phase advance, and HF diet caused a phase delay. In addition, adenosine monophosphate-activated protein kinase levels were high during fasting and low during HF diet. Changes in the phase and daily rhythm of clock genes and components of adiponectin signaling pathway as a result of HF diet may lead to obesity and may explain the disruption of other clock-controlled output systems, such as blood pressure and sleep/wake cycle, usually associated with metabolic disorders.

Reduction in Glycated Hemoglobin and Daily Insulin Dose Alongside Circadian Clock Upregulation in Patients With Type 2 Diabetes Consuming a Three-Meal Diet: A Randomized Clinical Trial.

OBJECTIVE: In type 2 diabetes, insulin resistance and progressive ß-cell failure require treatment with high insulin doses, leading to weight gain. Our aim was to study whether a three-meal diet (3Mdiet) with a carbohydrate-rich breakfast may upregulate clock gene expression and, as a result, allow dose reduction of insulin, leading to weight loss and better glycemic control compared with an isocaloric six-meal diet (6Mdiet). RESEARCH DESIGN AND METHODS: Twenty-eight volunteers with diabetes (BMI 32.4 ± 5.2 kg/m2 and HbA1c 8.1 ± 1.1% [64.5 ± 11.9 mmol/mol]) were randomly assigned to 3Mdiet or 6Mdiet. Body weight, glycemic control, continuous glucose monitoring (CGM), appetite, and clock gene expression were assessed at baseline, after 2 weeks, and after 12 weeks. RESULTS: 3Mdiet, but not 6Mdiet, led to a significant weight loss (-5.4 ± 0.9 kg) (P < 0.01) and decreased HbA1c (-12 mmol/mol [-1.2%]) (P < 0.0001) after 12 weeks. Fasting glucose and daily and nocturnal glucose levels were significantly lower on the 3Mdiet. CGM showed a significant decrease in the time spent in hyperglycemia only on the 3Mdiet. Total daily insulin dose was significantly reduced by 26 ± 7 units only on the 3Mdiet. There was a significant decrease in the hunger and cravings only in the 3Mdiet group. Clock genes exhibited oscillation, increased expression, and higher amplitude on the 3Mdiet compared with the 6Mdiet. CONCLUSIONS: A 3Mdiet, in contrast to an isocaloric 6Mdiet, leads to weight loss and significant reduction in HbA1c, appetite, and overall glycemia, with a decrease in daily insulin. Upregulation of clock genes seen in this diet intervention could contribute to the improved glucose metabolism.

Glucose and insulin are needed for optimal defensin expression in human cell lines.

Many infections are associated with diabetes, as the ability of the body to fight pathogens is impaired. Recently, low levels of defensins have been found in diabetic rodents. However, whether hyperglycemia and/or insulin deficiency/insensitivity is the reason for the reduced defensin levels is still unknown. To study the functionality of the innate immune system during hyperglycemia, the expression levels of human beta-defensin-1 (hBD-1) was measured in human embryonic kidney (HEK-293) and colon adenocarcinoma (HCT-116) cells treated with different concentrations of glucose and insulin. Increasing concentrations of glucose enhanced hBD-1 expression and these levels were further elevated after insulin treatment. Insulin treatment also led to the up-regulation of human sodium/glucose transporter 1 (hSGLT1), which further increases intracellular glucose levels. Thus, our findings suggest for the first time that insulin signaling is important for hBD-1 optimal expression by elevating intracellular glucose levels and by mediating gene expression.

High-energy breakfast based on whey protein reduces body weight, postprandial glycemia and HbA1C in Type 2 diabetes.

Acute studies show that addition of whey protein at breakfast has a glucose-lowering effect through increased incretin and insulin secretion. However, whether this is a long-term effect in Type 2 diabetes is unknown. Fifty-six Type 2 diabetes participants aged 58.9±4.5 years, BMI 32.1±0.9 kg/m2 and HbA1C 7.8±0.1% (61.6±0.79 mmol/mol) were randomized to one of 3 isocaloric diets with similar lunch and dinner, but different breakfast: 1) 42 g total protein, 28 g whey (WBdiet, n=19); 2) 42 g various protein sources (PBdiet, n=19); or 3) high-carbohydrate breakfast, 17 g protein from various sources (CBdiet, n=18). Body weight and HbA1C were examined after 12 weeks. All participants underwent three all-day meal challenges for postprandial glycemia, insulin, C-peptide, intact glucagon-like peptide 1 (iGLP-1), ghrelin and hunger and satiety scores. Overall postprandial AUCglucose was reduced by 12% in PBdiet and by 19% in WBdiet, compared with CBdiet (P<.0001). Compared with PBdiet and CBdiet, WBdiet led to a greater postprandial overall AUC for insulin, C-peptide, iGLP-1 and satiety scores, while postprandial overall AUC for ghrelin and hunger scores were reduced (P<.0001). After 12 weeks, HbA1C was reduced after WBdiet by 0.89±0.05% (11.5±0.6 mmol/mol), after PBdiet by 0.6±0.04% (7.1±0.31 mmol/mol) and after CBdiet by 0.36±0.04% (2.9±0.31 mmol/mol) (P<.0001). Furthermore, the participants on WBdiet lost 7.6±0.3 kg, PBdiet 6.1±0.3 kg and CBdiet 3.5±0.3 kg (P<.0001). Whey protein-based breakfast is an important adjuvant in the management of Type 2 diabetes.

Influences of Breakfast on Clock Gene Expression and Postprandial Glycemia in Healthy Individuals and Individuals With Diabetes: A Randomized Clinical Trial.

OBJECTIVE: The circadian clock regulates glucose metabolism by mediating the activity of metabolic enzymes, hormones, and transport systems. Breakfast skipping and night eating have been associated with high HbA1c and postprandial hyperglycemia after lunch and dinner. Our aim was to explore the acute effect of breakfast consumption or omission on glucose homeostasis and clock gene expression in healthy individuals and individuals with type 2 diabetes. RESEARCH DESIGN AND METHODS: In a crossover design, 18 healthy volunteers and 18 volunteers with 14.5 ± 1.5 years diabetes, BMI 30.7 ± 1.1 kg/m2, and HbA1c 7.6 ± 0.1% (59.6 ± 0.8 mmol/mol) were randomly assigned to a test day with breakfast and lunch (YesB) and a test day with only lunch (NoB). Postprandial clock and clock-controlled gene expression, plasma glucose, insulin, intact glucagon-like peptide 1 (iGLP-1), and dipeptidyl peptidase IV (DPP-IV) plasma activity were assessed after breakfast and lunch. RESULTS: In healthy individuals, the expression level of Per1, Cry1, Rorα, and Sirt1 was lower (P < 0.05) but Clock was higher (P < 0.05) after breakfast. In contrast, in individuals with type 2 diabetes, Per1, Per2, and Sirt1 only slightly, but significantly, decreased and Rorα increased (P < 0.05) after breakfast. In healthy individuals, the expression level of Bmal1, Rorα, and Sirt1 was higher (P < 0.05) after lunch on YesB day, whereas the other clock genes remained unchanged. In individuals with type 2 diabetes, Bmal1, Per1, Per2, Rev-erbα, and Ampk increased (P < 0.05) after lunch on the YesB day. Omission of breakfast altered clock and metabolic gene expression in both healthy and individuals with type 2 diabetes. CONCLUSIONS: Breakfast consumption acutely affects clock and clock-controlled gene expression leading to normal oscillation. Breakfast skipping adversely affects clock and clock-controlled gene expression and is correlated with increased postprandial glycemic response in both healthy individuals and individuals with diabetes.

The circadian clock machinery controls adiponectin expression.

Adiponectin, an adipokine involved in glucose and lipid metabolism, exhibits a circadian manner of expression. Adiponectin expression is mediated by the helix-loop-helix transcription factor sterol regulatory element binding protein (SREBP)-1c. In this study, we tested whether the circadian clock helix-loop-helix transcription factors CLOCK and BMAL1 regulate adiponectin expression. We found that adiponectin expression is regulated by the clock through the circadian expression of its transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and its co-activator PPARγ co-activator 1α (PGC1α) in mouse white adipose tissue and differentiated adipocytes. In addition, reconstitution of the core clock mechanism and siRNA experiments in cell culture suggest that the clock directly activates the adiponectin promoter and mediates its expression. In summary, adiponectin expression is regulated by the circadian clock and through the circadian expression of its transcription factor PPARγ and its co-activator PGC1α.

Effect of metformin and lipid emulsion on the circadian gene expression in muscle cells.

The circadian clock influences nearly all aspects of metabolism. However, little is known regarding the effect of the energy status on circadian rhythms. Our aim was to test the effect of two opposing energy situations, metformin and lipid emulsion (LE), on clock and metabolic circadian expression in differentiated C2C12 myotubes. Metformin treatment led to depleted ATP levels accompanied by elevated NADH levels, whereas LE treatment led to increased ATP and NAD(+) levels. Nevertheless, both LE and metformin treatments activated the AMP-activated protein kinase (AMPK) pathway. In contrast, the effect on circadian rhythms was completely different. LE led to disrupted clock and metabolic gene expression, whereas metformin led to mainly high-amplitude shifted rhythms. Combination of metformin and LE led to an antagonistic effect on circadian gene expression. Although metformin and LE have an opposing effect on circadian gene expression and on the cellular energy status, they both lead to AMPK activation.

Long-term commercial cow's milk consumption and its effects on metabolic parameters associated with obesity in young mice.

SCOPE: Research has demonstrated that consumption of milk promotes weight loss and satiety, however conflicting evidence also exists. Therefore, we tested the effect of long-term milk consumption on body weight and metabolic parameters. METHODS AND RESULTS: Newly weaned mice received whole milk, low-fat milk, or water as control for 17 weeks and serum, liver, and white adipose tissue (WAT) were tested for parameters associated with obesity and diabetes. Our results show that low-fat milk leads to the same overall caloric intake and body weight as the control group. However, the whole-milk group consumed more calories and reached a higher body weight. In addition, in the low-fat milk group, cholesterol, HDL-cholesterol, triglycerides, leptin, ghrelin, insulin, corticosterone, and glucagon were not significantly different than the control group. In contrast, in the whole-milk group, cholesterol, HDL-cholesterol, triglycerides, and glucagon were high compared with the control group. Metabolism in both liver and WAT showed only slight differences between the milk groups. Whereas the whole-milk group showed reduced insulin signaling in WAT, the low-fat milk group exhibited increased insulin signaling. CONCLUSION: Whole-milk consumption leads to increased body weight and caloric intake and reduced insulin signaling in WAT, as opposed to low-fat milk consumption.

Dexamethasone induces high-amplitude rhythms in preadipocytes, but hinders circadian expression in differentiated adipocytes.

Glucocorticoids induce circadian gene expression in cultured cells and change the phase of circadian gene expression in vivo. In addition, glucocorticoids induce differentiation of preadipocyte to adipocytes. We set out to test the effect of dexamethasone, a glucocorticoid receptor agonist, on circadian rhythms in 3T3-L1 differentiated adipocytes. Our results show that differentiated adipocytes exhibit robust circadian rhythms without dexamethasone. Dexamethasone induces phase changes and increases the amplitude of circadian gene expression in nondifferentiated 3T3-L1 preadipocytes. However, dexamethasone had an opposite effect on differentiated adipocytes, leading to low-amplitude circadian expression. In conclusion, although glucocorticoids reset circadian rhythms, once rhythms are reset, glucocorticoid administration hinders circadian expression.

High caloric intake at breakfast vs. dinner differentially influences weight loss of overweight and obese women.

OBJECTIVE: Few studies examined the association between time-of-day of nutrient intake and the metabolic syndrome. Our goal was to compare a weight loss diet with high caloric intake during breakfast to an isocaloric diet with high caloric intake at dinner. DESIGN AND METHODS: Overweight and obese women (BMI 32.4 ± 1.8 kg/m(2) ) with metabolic syndrome were randomized into two isocaloric (~1400 kcal) weight loss groups, a breakfast (BF) (700 kcal breakfast, 500 kcal lunch, 200 kcal dinner) or a dinner (D) group (200 kcal breakfast, 500 kcal lunch, 700 kcal dinner) for 12 weeks. RESULTS: The BF group showed greater weight loss and waist circumference reduction. Although fasting glucose, insulin, and ghrelin were reduced in both groups, fasting glucose, insulin, and HOMA-IR decreased significantly to a greater extent in the BF group. Mean triglyceride levels decreased by 33.6% in the BF group, but increased by 14.6% in the D group. Oral glucose tolerance test led to a greater decrease of glucose and insulin in the BF group. In response to meal challenges, the overall daily glucose, insulin, ghrelin, and mean hunger scores were significantly lower, whereas mean satiety scores were significantly higher in the BF group. CONCLUSIONS: High-calorie breakfast with reduced intake at dinner is beneficial and might be a useful alternative for the management of obesity and metabolic syndrome.

Association between phase shifts, expression levels, and amplitudes in peripheral circadian clocks.

Data analyses examining the relationship between circadian phase shifts and amplitudes are scarce. The aim of this data analysis was to explore the association between the phase shifts of gene expression, their amplitudes, and daily levels as a result of a given treatment under ad libitum or restricted feeding (RF) conditions. Two hundred forty data sets of gene expression (clock and metabolic genes) from various tissues and treatments were statistically analyzed. The data revealed a significant association between phase delays and increased amplitudes. Moreover, upon subgroup analyses, separating the RF from the non-RF groups, phase delays were significantly correlated with increased amplitudes and phase advances with decreased amplitudes. This picture was also achieved when clock genes, but not metabolic genes, were analyzed. In contrast, under RF, increased amplitude of metabolic genes correlated with phase advances. Moreover, phase advances under RF led to increased average daily levels in clock genes, but not in metabolic genes. In summary, these data demonstrated statistically significant association between phase shifts, daily levels, and amplitudes in circadian gene expression in peripheral tissues under timed versus ad libitum feeding conditions.

High-fat diet followed by fasting disrupts circadian expression of adiponectin signaling pathway in muscle and adipose tissue.

The circadian clock controls energy homeostasis by regulating circadian expression of proteins involved in metabolism. Disruption of circadian rhythms leads to obesity and metabolic disorders. Little is known regarding the control of the biological clock over adiponectin signaling pathway in adipose tissue, the adiponectin producer, and muscle, an adiponectin target tissue under fasting, low-fat (LF), or high-fat (HF) diet. Mice were fed LF or HF diet for 7 weeks and fasted on the last day. The circadian mRNA expression of clock genes and components of adiponectin metabolic pathway (mAdipoR1, mAdipoR2, mPparalpha, mPpargamma, mAmpk, and mAcc) in the muscle and adipose tissue were tested. Using average daily levels of multiple time points around the circadian cycle, we assessed mRNA levels of the different adiponectin signaling components. In addition, serum glucose, adiponectin, and insulin were measured. Under LF diet, adiponectin signaling pathway components exhibited circadian rhythmicity at the mRNA levels. Fasting and HF diet followed by fasting disrupted this circadian expression causing a phase advance or delay, respectively. Changes were also found in the expression levels of adiponectin receptor, mAmpk, mAcc, mPparalpha, and mPpargamma reflecting a defect in adiponectin signaling. As both peroxisome proliferator-activated receptor alpha (PPARalpha) and mAMPK are linked to the core clock mechanism, they could mediate the disruptions seen in clock gene expression under HF diet. In turn, the circadian clock affects the daily rhythm of these adiponectin signaling components.

A high-fat diet has a tissue-specific effect on adiponectin and related enzyme expression.

OBJECTIVE: This study was designed to test whether adiponectin plays a role in diet-induced obesity and insulin resistance and acts as a mediator to induce or inhibit specific metabolic pathways involved in lipid metabolism RESEARCH METHODS AND PROCEDURES: Forty C57BL/6J male mice were fed either a high-fat (HF) or control diet for 4 months, and adiponectin, its receptors, and enzyme expression in liver and muscle tissue were measured. RESULTS: Mice fed the HF diet exhibited significantly greater weight gain, abnormal oral glucose tolerance test curves, and elevated homeostasis model assessment of insulin resistance (5.3 +/- 0.89 vs. 2.8 +/- 0.39). A significant reduction of adiponectin RNA expression (51%) and protein levels (15%) was observed in the adipose tissue of HF animals; however, serum adiponectin levels did not differ between groups (7.12 +/- 0.34 mug/mL vs. 6.44 +/- 0.38 microg/mL). Expression of hepatic mRNA of AdipoR1 and AdipoR2 was reduced by 15% and 25%, respectively, in animals fed the HF diet. In contrast, receptor mRNA expression of AdipoR1 and AdipoR2 increased by 25% and 30%, respectively, in muscle tissue. No effect was found on hepatic adenosine monophosphate-activated protein kinase expression; however, a significant reduction of phosphoadenosine monophosphate kinase levels in muscles was observed. Hepatic acetyl-coenzyme A carboxylase was similar between groups, but in muscles, the inactive form phosphoacetyl-coenzyme A carboxylase was significantly reduced (p < 0.05). DISCUSSION: The HF diet led to decreased insulin sensitivity accompanied by impaired activity of adiponectin-related enzymes in skeletal muscles but not in the liver. These results suggest that the HF diet has a tissue-specific effect on adiponectin and associated enzyme expression.