Short chain fatty acids : the effect on adipose tissue metabolism and function

Adipose tissue homeostasis is regulated by a combination of extracellular and intracellular signalling pathways. Activation of surface G protein-coupled receptors (GPCR) and insulin receptors, influence the rate of lipolysis within adipocytes, the pathway responsible for triacylglycerol (TAG) breakdown into non-esterified fatty acids (NEFA) and glycerol. A GPCR for short chain fatty acids (SCFA) has been identified on the surface of mature adipocytes and activation of this receptor by SCFA including acetate, butyrate and propionate has resulted in a decrease in lipolysis, measured as a reduction in NEFA and glycerol concentration in the media. However, evidence for a mechanism of action of SCFA within adipocytes has remained unclear. Therefore, this thesis has aimed to better understand the mechanism(s) by which the SCFA, acetate, regulates adipose tissue metabolism and function, and in particular the pathway of lipolysis. Through the development of experimental methods in vitro, it was determined that the dose of isoproterenol, a β-adrenergic receptor activator, required to stimulate lipolysis by 50 % in murine 3T3-L1 adipocytes, was 5 μM, and that no interference with metabolic assays was observed in the presence of any potential treatment condition, either in the basal or stimulated state. In 3T3-L1 cells, in the basal state, treatment of cells with the short chain fatty acid acetate (4 mM), significantly reduced lipolysis, as a measure of NEFA and glycerol (P = 0.004 and P = 0.020, respectively) after a 180 min incubation. Similarly, in the stimulated state, acetate also reduced NEFA significantly (P = 0.020), however, glycerol was not reduced (P = 0.529) compared with controls. To evaluate whether the metabolic changes in NEFA and glycerol concentration reflected an intracellular change to the pathway of lipolysis, phosphorylation of the key enzyme, hormone sensitive lipase (HSL), was also analysed. It was identified that in the stimulated state, phosphorylation of HSL, at serine residue 563, was reduced by 15 % in the presence of 4 mM acetate, compared with control, complimentary to metabolic data. However, treatment of isolated primary mouse mature adipocytes with acetate did not produce results comparable to those found in the literature. Instead, accumulation of NEFA and glycerol in the media were found to be negligible. Cell viability may have been a limiting factor regarding the outcome of these studies, and therefore development of a protocol to improve cell viability without compromising cell yield would prove useful. Furthermore, future work should consider the lack of reduction in glycerol in the media in the stimulated state from 3T3-L1 mature adipocytes. This may be accounted for by fatty acid re-esterification, as the NEFA:glycerol ratios were 1.5:1, compared with the expected 3:1 ratio. Similarly, to re-enforce the effects of acetate on stimulated lipolysis, other phosphorylation sites within HSL may be considered, including SER565. Overall, the results obtained in this thesis demonstrate that in the mature adipocyte cell line 3T3-L1, an increase in the availability of the SCFA, acetate, resulted in a change in the pathway of lipolysis. This was evidenced by a reduction in the phosphorylation of HSL(SER563) under sub-maximal stimulation with isoproterenol, similar to levels observed in the presence of insulin. Complimentary to these data, under the same treatment conditions, incubation with acetate also resulted in a small but significant reduction in NEFA concentration in the media

Role of PTP1B in POMC neurons during chronic high fat diet: Sex differences in regulation of liver lipids and glucose tolerance

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of leptin receptor signalling and may contribute to leptin resistance in diet-induced obesity. Although PTP1B inhibition has been suggested as a potential weight loss therapy, the role of specific neuronal PTP1B signalling in cardiovascular and metabolic regulation and the importance of sex differences in this regulation are still unclear. In this study, we investigated the impact of pro-opiomelanocortin (POMC) neuronal PTP1B deficiency in cardiometabolic regulation in male and female mice fed a high fat diet (HFD). Compared to control mice (PTP1Bflox/flox), male and female mice deficient in POMC neuronal PTP1B (PTP1Bflox/flox/POMC-Cre) had attenuated body weight gain (Male: -18%; Female: -16%) and fat mass (Male: -33%; Female: -29%) in response to HFD. Glucose tolerance was improved by 40% and liver lipid accumulation was reduced by 40% in PTP1Bflox/flox/POMC-Cre males but not in females. Compared to control mice, deficiency of POMC neuronal PTP1B did not alter mean arterial pressure (MAP) in male or female mice (Male: 112±1 vs. 112±1 mmHg in controls; Female: 106±3 vs. 109±3 mmHg in controls). Deficiency of POMC neuronal PTP1B also did not alter MAP response to acute stress in male or female compared to control mice (Male: Δ32±0 vs. Δ29±4 mmHg; Female: Δ22±2 vs. Δ27±4 mmHg). These data demonstrate that POMC-specific PTP1B deficiency improved glucose tolerance and attenuated diet-induced fatty liver only in male mice, attenuated weight gain in males and females, but did not enhance the MAP and HR responses to a HFD or to acute stress

Role of SOCS3 in POMC Neurons in metabolic and cardiovascular regulation.

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin signaling. We previously showed that the chronic effects of leptin on blood pressure (BP) and glucose regulation are mediated by stimulation of pro-opiomelanocortin (POMC) neurons. In this study, we examined the importance of endogenous SOCS3 in POMC neurons in control of metabolic and cardiovascular function and potential sex differences. Male and female SOCS3flox/flox/POMC-Cre mice in which SOCS3 was selectively deleted in POMC neurons and control SOCS3flox/flox mice were studied during a control diet (CD) or high fat diet (HFD) and during chronic leptin infusion. On CD, male and female SOCS3flox/flox/POMC-Cre mice were lighter in body weight despite similar food intake compared to control mice. Male SOCS3flox/flox/POMC-Cre mice exhibited increased energy expenditure. BP and heart rate were similar in male and female SOCS3flox/flox/POMC-Cre and control mice on CD. On a HFD, male and female SOCS3flox/flox/POMC-Cre mice showed attenuated weight gain. Female SOCS3flox/flox/POMC-Cre mice exhibited greater HFD-induced elevations in baseline BP and BP responses to air jet stress test compared to control mice. Chronic leptin infusion produced similar responses in all groups for food intake, body weight, oxygen consumption, blood glucose, BP and heart rate. Thus, SOCS3 deficiency in POMC neurons influences body weight regulation in CD and HFD and differentially affects BP and energy balance in a sex specific manner, but does not amplify the dietary, glycemic or cardiovascular effects of leptin

Sodium Acetate decreases phosphorylation of hormone sensitive lipase in isoproterenol stimulated 3T3-L1 mature adipocytes

Lipolysis, the process of hydrolysis of stored triacylglycerol into glycerol and non-esterified fatty acids (NEFA), is reported to be reduced by short chain fatty acids (SCFA) but the mechanism of this inhibition is poorly understood. The aim of this study was to measure the phosphorylation at serine residue 563 of hormone sensitive lipase with and without exposure to sodium acetate. Using the 3T3-L1 cell line, we identified that stimulating the cells with isoproterenol increased phosphorylated hormone sensitive lipase (pHSL) expression by 60% compared with the basal state. In the presence of the SCFA acetate in stimulated cells, pHSL decreased by 15% compared with stimulated cells alone. These results were mirrored by the NEFA release from stimulated cells that had significantly decreased in the presence of sodium acetate after 60 min (from 0.53 µmol mg(−1) protein to 0.41 µmol mg(−1) protein, respectively, P = 0.004); and 180 min (1.73 µmol mg(−1) protein to 1.13 µmol mg(−1) protein, P = 0.020); however, treatment had no effect on glycerol release (P = 0.109). In conclusion, exposure to 4 mM acetate reduced the level of phosphorylation of HSL((SER563)) in mature 3T3-L1 adipocytes and led to a significant reduction in NEFA release, although glycerol release was not affected

Protein Tyrosine Phosphatase 1B (PTP1B) Deficiency In Pro-Opiomelanocortin (POMC) Neurons Does Not Enhance Leptin's Anorexigenic Effect, But Improves Glucose Tolerance And Increases Energy Expenditure In Mice Fed A High Fat Diet (Abstract only)

Obesity is a major cause of Type 2 diabetes (T2D), accounting for 90–95% of all diabetes mellitus, and is closely associated with resistance to the metabolic effects of insulin as well as leptin. Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of leptin signalling, has been implicated in the development of obesity-induced leptin and insulin resistance. We previously showed that leptin receptor activation in POMC neurons had powerful antidiabetic effects in non-obese and Type 1 diabetic rodents; whether PTP1B contributes to leptin resistance in POMC neurons in obese subjects is unclear. The objective of this study was to determine whether mice with selective deficiency of PTP1B signalling in POMC neurons have improved glucose regulation and enhanced metabolic responses to hyperleptinemia compared to control mice when fed a normal or high fat diet. Using Cre-loxP technology, PTP1B was deleted specifically in POMC neurons (POMC/PTP1B(−/−)), with PTP1B flox/flox littermates used as controls. Food intake responses to acute leptin (5mg/kg, IP) or saline were measured at 22 weeks of age in mice fed a normal diet. A glucose tolerance test (GTT) was performed at 19 weeks of age. Mice on a normal or high fat diet (HFD – from 6 to 22 weeks of age) were placed in specialized metabolic cages for continuous measurement of oxygen consumption (VO2) and heat production. Mice were infused with leptin for 7 days via IP osmotic minipump (4μg/kg/min). Blood samples were also analyzed for fasting glucose concentration at baseline, on the last day of leptin infusion, and after 7 days of recovery. The anorexigenic effects of leptin over 24 hours were not enhanced in POMC/PTP1B(−/−) mice compared with PTP1Bflox/flox (−1.3±0.3g vs. −1.5±0.2g) fed a normal diet. However, POMC/PTP1B(−/−) mice had decreased fasting blood glucose after chronic leptin infusion when on a HFD (189±6 vs. 135±6 mg/dL) compared to PTP1Bflox/flox (156±25 vs. 151±11 mg/dL). In addition, compared with controls, POMC/PTP1B(−/−) mice had improved glucose tolerance (AUC 27,107±1572 vs. 43,183±5971 mg/dL × 120 min). Chronic leptin infusion increased VO2 in POMC/PTP1B(−/−) mice fed a HFD compared to baseline (96±6 vs. 66±6 ml/kg/min respectively) but this increase was not as pronounced in control mice (97±4 vs. 89±5 ml/kg/min, respectively). Heat production was also increased in POMC/PTP1B(−/−) mice on a HFD during chronic leptin infusion (783±96 vs. 586±58 Cal/hr) but no change was observed in control mice (743±39 vs. 764±13 Cal/hr). These results demonstrate that PTP1B deficiency in POMC neurons does not enhance the anorexigenic effects of leptin in mice fed a normal diet. However PTP1B deficiency in POMC neurons improves glucose regulation in obese mice fed a HFD. Furthermore, POMC PTP1B deficient mice also demonstrated increased VO2 and heat production compared with controls during chronic hyperleptinemia. PTP1B could be an important drug target for T2D treatment via its effects to attenuate obesity-induced leptin resistance

Protein Tyrosine Phosphatase 1B (PTP1B) Deficiency in Proopiomelanocortin (POMC) Neurons Attenuates Body Weight, Fat Mass and Liver Lipid Accumulation in Mice Fed a High Fat Diet

Leptin acts on the central nervous system (CNS) to reduce appetite and body weight, improve glucose regulation, and increase energy expenditure and sympathetic nervous system (SNS) activity. Central leptin has also been shown to exert antisteatotic effects on non-adipose tissues. However, in severe obesity there may be resistance to leptin’s anorexic, antidiabetic and antisteatotic effects but sustained effects to increase SNS activity, blood pressure (BP), and heart rate (HR). Our previous studies showed that POMC neurons are important for mediating the BP, HR and glucose effects of leptin but the role of POMC neurons in mediating leptin’s effects on liver lipid accumulation remain less well defined. In this study we examined if deleting PTP1B, a negative regulator of leptin signaling, specifically in POMC neurons or in the entire forebrain, would protect mice against the adverse metabolic effects of a high fat diet (HFD). Male and female mice with forebrain (PTP1Bflox/flox/CamK2R5-Cre) or POMC specific (PTP1Bflox/flox/POMC-Cre) PTP1B deficiency and littermate controls (PTP1Bfox/flox) were fed a HFD from 6–20 weeks of age. Compared to PTP1Bflox/flox mice, mice with PTP1B deficiency in POMC neurons or in the entire forebrain had reduced body weight gain between 6 and 20 weeks of age when fed a HFD (Δ19.1 ±1.7g vs. Δ13.7±1.1g and Δ15.3±1.4g, respectively), and attenuated increase in fat mass (Δ13.3±1.2g vs. Δ7.3±0.7g and Δ10.2±1.0g, respectively). PTP1B deficiency attenuated food intake in forebrain specific PTP1B deficient mice compared to PTP1Bflox/flox and POMC specific PTP1B deficient mice (2.0±0.1g, 2.7±0.1g and 2.5±0.1 g, respectively). Glucose tolerance, expressed as area under the curve (AUC), was improved by PTP1B deficiency in forebrain and POMC specific neurons compared to PTP1Bflox/flox mice fed a HFD (AUC 19016±1172, 23207±1534 and 27561±3088, respectively). Liver fat mass, assessed by EchoMRI at 29±0.5 weeks of age, was reduced and liver lean mass was increased in mice with PTP1B deficiency in POMC neurons or in the entire forebrain (Fat: 8.8±1.8 and 6.0±3.3 mg/g, Lean: 93.6±2.8 and 97.2±5.6 mg/g, respectively) compared to PTP1Bflox/flox mice (Fat: 14.2±2., Lean: 79.4±10.3 mg/g), indicating a healthier liver profile. These findings indicate that PTP1B signaling in POMC neurons plays an important role in regulating body weight, fat mass, glucose tolerance and liver lipid accumulation in response to a chronic HFD but effects of PTP1B on food intake are mediated mainly in forebrain neurons other than POMC

Role of the brain melanocortins in blood pressure regulation

© 2017 Elsevier B.V. Melanocortins play an important role in regulating blood pressure (BP) and sympathetic nervous system (SNS) activity as well as energy balance, glucose and other metabolic functions in humans and experimental animals. In experimental models of hypertension with high SNS activity, blockade of the melanocortin-4 receptor (MC4R) reduces BP despite causing marked hyperphagia and obesity. Activation of the central nervous system (CNS) pro-opiomelanocortin (POMC)–MC4R pathway appears to be an important link between obesity, SNS activation and hypertension. Despite having severe obesity, subjects with MC4R deficiency exhibit reductions in BP, heart rate, and urinary catecholamine excretion, as well as attenuated SNS responses to cold stimuli compared to obese subjects with normal MC4R function. In this review we discuss the importance of the brain POMC-MC4R system in regulating SNS activity and BP in obesity and other forms of hypertension. We also highlight potential mechanisms and brain circuitry by which the melanocortin system regulates cardiovascular function