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By Jennifer A Felsted, Alice Meng, Dominique Ameroso, and Maribel Rios

Excerpt from the article published in Endocrinology, Volume 161, Issue 7, July 2020, bqaa068, DOI: https://doi.org/10.1210/endocr/bqaa068

Editor’s Highlights

• The ventromedial hypothalamus (VMH) plays a well-established role in appetite and body weight regulation as well as in glucose homeostasis and lipid metabolism.
• Diverse leptin-induced, leptin-inhibited, and insulin-inhibited steroidogenic factor-1 (SF1)   neuronal subpopulations have been identified in the VMH, indicating functional heterogeneity. 
• The thrombospondin receptor alpha2delta-1 (α2δ-1) suppresses appetite and facilitates glycemic control, acting downstream of brain-derived neurotrophic factor (BDNF) in the VMH.
• Central BDNF depletion exhibit dramatic obesity, hyperglycemia, dyslipidemia, and decreased excitatory tone in the VMH as well as a significant reduction in cell-surface expression of α2δ-1.
• Inhibiting α2δ-1 via administration blunted the effects of thrombospondins inducing excitatory synaptogenesis, reducing the excitatory tone and activity of SF1 neurons without affecting calcium currents, implicating actions of α2δ-1 facilitating excitatory synapse homeostasis.
• Sex-specific effects of α2δ-1 in the VMH are evident in mechanisms controlling glucose homeostasis.
• A higher density of estrogen receptors in female versus male VMH and that estrogen signaling via ERα in the VMH is protective against obesity and glucose intolerance in females but not in males, revealing a female-specific and bimodal effects of VMH α2δ-1 impacting glucose and lipid balance control and sympathetic tone. 

Abstract

The thrombospondin receptor alpha2delta-1 (α2δ-1) plays essential roles promoting the activity of SF1 neurons in the ventromedial hypothalamus (VMH) and mediating glucose and lipid metabolism in male mice. Its role in the VMH of female mice remains to be defined, especially considering that this hypothalamic region is sexually dimorphic. We found that α2δ-1 depletion in SF1 neurons differentially affects glucose and lipid balance control and sympathetic tone in females compared to males. Mutant females show a modest increase in relative body weight gain when fed a high-fat diet (HFD) and normal energy expenditure, indicating that α2δ-1 is not a critical regulator of energy balance in females, similar to males. However, diminished α2δ-1 function in the VMH leads to enhanced glycemic control in females fed a chow diet, in contrast to the glucose intolerance reported previously in mutant males. Interestingly, the effects of α2δ-1 on glucose balance in females are influenced by diet. Accordingly, females but not males lacking α2δ-1 exhibit diminished glycemic control as well as susceptibility to hepatic steatosis when fed a HFD. Increased hepatic sympathetic tone and CD36 mRNA expression and reduced adiponectin levels underlie these diet-induced metabolic alterations in mutant females. The results indicate that α2δ-1 in VMH SF1 neurons critically regulates metabolic function through sexually dimorphic mechanisms. These findings are clinically relevant since metabolic alterations have been reported as a side effect in human patients prescribed gabapentinoid drugs, known to inhibit α2δ-1 function, for the treatment of seizure disorders, neuropathic pain, and anxiety disorders.

Introduction

The ventromedial hypothalamus (VMH) plays a well-established role in appetite and body weight regulation (1-3) as well as in glucose homeostasis (4-8) and lipid metabolism (9, 10). These effects are facilitated, in part, via regulation of sympathetic output to metabolic organs in the periphery (11, 12). The underlying mechanisms remain poorly understood. We reported a previously unrecognized and essential role of alpha2delta-1 (α2δ-1) in suppressing appetite and facilitating glycemic control, acting downstream of brain-derived neurotrophic factor (BDNF) in the VMH (13). α2δ-1 is an auxiliary subunit of voltage-gated calcium channels, facilitating calcium currents and neurotransmitter release (14). Additionally, it serves as a receptor for thrombospondins to induce excitatory synapse assembly through calcium-channel independent mechanisms (15). α2δ-1 is expressed in brain regions involved in metabolic control, with notably high expression in the VMH (16, 17).

Mice with central BDNF depletion exhibit dramatic obesity, hyperglycemia, dyslipidemia, and decreased excitatory tone in the VMH as well as a significant reduction in cell-surface expression of α2δ-1 in the VMH (13). Rescuing this deficit broadly in the VMH via viral gene delivery, significantly mitigated hyperphagia and body weight gain and normalized glycemic control in BDNF mutant mice. Moreover, a more discrete and selective deletion of α2δ-1 in steroidogenic factor-1 (SF1) neurons, which in the brain are exclusive to the VMH, elicited significant reductions in neuronal activity and sympathetic output without affecting calcium currents, ultimately resulting in glucose intolerance and alterations in lipid metabolism (18). In total, these studies support a chief role of α2δ-1 as increasing VMH neuronal excitability and activity in a calcium channel-independent manner to facilitate energy and glucose balance control. However, a caveat of these previous investigations is that they were performed exclusively in males. Considering that the VMH is a sexually dimorphic region of the brain, it is important to assess whether α2δ-1 plays similar roles in females.

Here, we examined the effects of depleting α2δ-1 from SF1 neurons in female mice. We found that in contrast to males with similar deficits in α2δ-1, mutant females exhibit normal sympathetic output and lipolysis in white adipose tissue and enhanced glycemic control under chow conditions when compared to female controls. However, mutant females display glucose intolerance and increased susceptibility to hepatic steatosis when challenged with a high-fat diet (HFD) compared with controls. The collective findings indicate that the effects of α2δ-1 in the VMH influencing metabolic function are sexually dimorphic. These findings are clinically relevant since metabolic alterations have been reported as a side effect in human patients prescribed gabapentinoid drugs, known to inhibit α2δ-1 function (19), for the treatment of seizure disorders, neuropathic pain, and anxiety disorders (20, 21).

…

Results

Α2 δ -1 in the VMH of female mice is not a critical regulator of energy balance

We showed previously that α2 δ -1 depletion in VMH SF1 neurons of male mice does not have an effect on feeding, energy expenditure or body weight when fed a chow (CD) or high-fat diet (HFD), negating a required role in the regulation of energy balance (18). Because the VMH is sexually dimorphic (32), we investigated whether α2 δ -1 is also dispensable in mutant females. For this, we crossed floxed α2 δ -1 mice with SF1-cre mice to generate α2 δ -1^{2L/2L:SF1-cre} mutant and α2 δ -1^2L/2L control females. Figs. 1A and 1B show that α2 δ -1 is depleted in the VMH of α2 δ -1^{2L/2L:SF1-cre}as previously reported for mutant males (18). Because SF1 is also expressed in the adrenal glands, cre-mediated recombination is expected to occur in this tissue. However, as we reported previously, α2δ-1 is not expressed in wild-type adrenal glands (18) and thus, its depletion in this region is not a confounding factor.

ANOVA analysis did not show a significant effect of genotype on body weight in mutant females fed CD (Fig. 1C). Moreover, no significant changes in CD intake were observed (Fig. 1D). Total body weights in α2 δ -1^{2L/2L:SF1-cre} females compared with α2 δ -1^2L/2L controls were not significantly different when challenged with a HFD starting at 8 weeks of age (Fig. 1E). When we calculated percentage body weight gain following transition from a CD to a HFD, we found that there was a significant effect of genotype (P = 0.003) and a significant interaction of genotype and time (P < 0.0001); however, this effect was modest, with mutants exhibiting a 38% weight gain compared with 28% in controls (data not shown). Measurements of HFD intake revealed a significant interaction of genotype and time (P = 0.02) (Fig. 1F), with mutants exhibiting a mild increase in food intake. Finally, measurements of lean and fat mass in females fed a CD (Fig. 1G) or a HFD (Fig. 1H) did not reveal any significant differences in mutants compared with controls.

Locomotor activity under CD conditions was normal in mutant females (Fig. 1I). Furthermore, energy expenditure was not affected in α2δ-1^{2L/2L:SF1-Cre} mutants fed a CD or HFD (Fig. 1J). In aggregate, the findings suggest that α2δ-1 expression in SF1 neurons is not required for the regulation of energy expenditure in female mice. These findings indicate that VMH α2δ-1 does not play a critical role in energy balance regulation in females.

α2δ-1^{2L/2L:SF1-cre} mutant females exhibit enhanced and diminished glycemic control under chow and HFD conditions, respectively

The VMH plays requisite roles in the regulation of glucose balance (4-8). We asked whether α2δ-1 in SF1 neurons is essential for those effects in female mice. α2δ-1^{2L/2L:SF1-Cre} females fed a CD had a significant increase in glucose tolerance compared with α2δ-1^2L/2L controls fed a similar diet (Fig. 2A and 2B). Accordingly, there was a significant effect of genotype (P = 0.0006) in the GTT (2A) and a trend (P= 0.09) towards a significant reduction in AUC in mutants compared with controls (Fig. 2B). Consistent with their enhanced glycemic control, α2δ-1^{2L/2L:SF1-Cre} females fed a CD showed a significant (P = 0.04) reduction in fasted levels of circulating insulin (Fig. 2C), suggesting increased insulin sensitivity. These findings in females are opposite to our previous observations in α2δ-1^{2L/2L:SF1-Cre} mutant males fed a similar diet, which display glucose intolerance (18) (Table 1).

Table 1.Effects of Depleting α2δ-1 in the VMH of Female and Male Mice: Metabolic Parameters in Female and Male α2δ-1^{2L/2L:SF1-Cre} Mice Compared With Their Sex- and Diet-Matched Controls

Metabolic Parameter	α2δ-1-12L/2L:SF1-cre Females	α2δ-1-12L/2L:SF1-cre Males
Food Intake (CD)	Normal	Normal*
Food Intake (HFD)	Mild increase	Normal*
Body Weight (CD)	Normal	Normal*
Body Weight (HFD)	10% increase in relative weight gain	Normal*
Energy Expenditure	Normal	Normal*
Fat and Lean Mass	Normal	Normal*
Glucose Tolerance (CD)	Improved	Reduced*
Glucose Tolerance (HFD)	Reduced	Normal
Fasted Insulin Levels (CD)	Reduced (44% of control levels)	Normal*
Fasted Insulin Levels (HFD)	Normal	Normal*
Liver TG (HFD)	Increased (120% of control levels)	Normal
Lipolysis in WAT	Normal	Elevated*
Serum Glycerol levels	Normal	Elevated (315% of control levels) *
Activated HSL levels in WAT	Normal	Elevated *
Serum NE Content (CD)	Normal	Reduced (27% of control levels)*
WAT NE Content (CD)	Normal	Reduced (55% of control levels)*
Liver NE Content (HFD)	Increased (450% of control levels)	Normal

We tested the effects of dietary challenge on glucose homeostasis in α2δ-1^{2L/2L:SF1-Cre}females. In contrast to our findings in females fed a CD, female mutants subjected to a HFD displayed mild glucose intolerance compared with α2δ-1^2L/2L females fed a similar diet (Fig. 2D and 2E). Accordingly, there was a main effect of genotype (P = 0.02) in the GTT (Fig. 2D). Furthermore, female mutants fed a HFD had similar circulating levels of insulin to those of diet-matched controls (Fig. 2F). Examination of α2δ-1^{2L/2L:SF1-Cre} and control males fed a HFD did not reveal significant differences in the GTT or AUC (Fig. 2G and 2H) (Table 1). In total, the data indicate that depletion of α2δ-1 in SF1 neurons produces sex-specific effects on glycemic control. Moreover, they suggest that perturbing VMH α2δ-1 function has a bidirectional effect on glucose balance influenced by diet in females but not in males.

Depletion of α2δ-1 in SF1 neurons in females increases susceptibility to diet-induced hepatic steatosis

Lipid metabolism is greatly influenced by neuronal activity in the VMH (9, 10). We asked whether α2δ-1 depletion in this hypothalamic region affected lipid balance control in female mice. For this, we measured levels of triglycerides (TG) and cholesterol in serum, WAT, and liver of fed α2δ-1^{2L/2L:SF1-Cre} mutants and α2δ-1^2L/2Lcontrols. There was a significant effect of diet on TG levels in serum (P = 0.003), WAT (P = 0.007), and liver (P = 0.03) (Fig. 3A, 3B and 3C). Notably, there was a significant effect of genotype (P = 0.05) and a trend (P = 0.07) towards a significant interaction of diet and genotype on TG content in the liver. Indeed, whereas hepatic content of TG significantly increased as a consequence of HFD administration in mutant females, it did not in controls. Accordingly, α2δ-1^{2L/2L:SF1-Cre} females fed a HFD exhibited a significant increase in TG levels in the liver compared with their α2δ-1^2L/2L counterparts (Fig. 3C). We reported previously that TG content in serum, WAT, and liver of α2δ-1^{2L/2L:SF1-Cre} males fed a CD was normal (18). Here, we investigated whether the effects of dietary challenge observed in mutant females were also present in mutant males. α2δ-1^{2L/2L:SF1-Cre} males fed a HFD contained normal and decreased levels of TG in serum and WAT, respectively (Fig. 3D and E). However, TG content in liver was not significantly different in α2δ-1^{2L/2L:SF1-Cre} males compared with α2δ-1^2L/2L controls (Fig. 3F) (Table 1). The results indicate that α2δ-1 depletion in SF1 neurons results in susceptibility to diet-induced hepatic steatosis in females.

Levels of cholesterol were also differentially affected by diet in α2δ-1^{2L/2L:SF1-Cre}females compared with α2δ-1^2L/2L controls. There was a significant effect of genotype (P = 0.02) on circulating levels of cholesterol (Fig. 3G). In WAT, there was a significant effect of genotype (P = 0.04) and a significant interaction of genotype and diet (P = 0.05) on cholesterol levels. Accordingly, WAT cholesterol levels were significantly higher in control HFD females compared with HFD mutants (Fig. 3H). There were no significant differences in cholesterol content in liver under either diet (Fig. 3I). Mutant males fed a CD exhibited normal and decreased levels of cholesterol in serum and WAT, respectively, as reported previously (18). However, cholesterol content in WAT, serum and liver were normal following administration of a HFD (data not shown).

Next, we interrogated mechanisms driving lipid accumulation in the livers of α2δ-1^{2L/2L:SF1-Cre} females fed a HFD. For this, we measured hepatic transcript content of factors involved in lipogenesis and TG synthesis, including Fas, SREBP-1c, DGAT1, DGAT2, CD36, SCD1, PPARγ and TNFα in control and mutant females fed CD or HFD. Quantitative RT-PCR analysis revealed that expression of Fas, SREBP-1c, DGAT1, DGAT2, SCD1, PPARγ, and TNFα was comparable in α2δ-1^{2L/2L:SF1-Cre} and α2δ-1^2L/2Lfemales fed similar diets. In contrast, whereas levels of the fatty acid translocase CD36 were similar in controls and mutants fed a CD, they were significantly elevated in mutants fed a HFD compared with controls fed a similar diet (Fig. 4). This finding suggests that CD36-mediated increases in hepatic fatty acid uptake contribute to susceptibility to diet-induced liver steatosis in α2δ-1^{2L/2L:SF1-Cre} females.

Normal lipolysis but diminished adiponectin secretion by WAT in α2δ-1^{2L/2L:SF1-Cre} females fed a HFD

During lipolysis, triglycerides are hydrolyzed and free fatty acids and glycerol are released from WAT into the circulation. This process is augmented during conditions of negative energy balance to provide the animal with substrates for oxidative metabolism and ATP production in peripheral tissues. Disinhibited lipolysis can result in increased hepatic lipid accumulation and reduced insulin sensitivity (33, 34). We reported previously that α2δ-1^{2L/2L:SF1-Cre} mutant males fed a CD exhibited an elevation in lipolysis as indicated by increased levels of the activated form of the lipolytic enzyme, hormone sensitive lipase (pHSL_S660) in WAT and a 315% elevation in circulating levels of glycerol (18). We investigated whether this was also the case in female mutants, contributing to the metabolic alterations that they exhibit. Measurements of total protein levels of HSL and its activated (pHSL₆₆₀) and inhibited forms (pHSL₅₆₅) in WAT were performed in the fed state, similar to measurements of TG content in liver and WAT. We found that levels of HSL, pHSL₆₆₀, and pHSL₅₆₅ were similar in α2δ-1^{2L/2L:SF1-Cre} and α2δ-1^2L/2L females administered similar diets (Fig. 5A, 5B and 5C). Consistent with these findings, whereas there was a significant effect of diet on circulating levels of glycerol and free fatty acids (FFA), there was no significant effect of genotype, indicating that lipolysis in WAT was not affected in mutant females (Fig. 5D and 5E) (Table 1).

Next, we asked whether adiponectin levels were altered in α2δ-1^{2L/2L:SF1-Cre} females, as this adipokine counters excess lipid storage in the liver (35, 36). Indeed, we found that whereas levels of adiponectin were similar in α2δ-1^{2L/2L:SF1-Cre} and α2δ-1^2L/2Lfemales fed a CD, they were significantly reduced in mutant females fed HFD compared with diet-matched controls (Fig. 5F). Serum levels of leptin were also measured and found to be comparable in α2δ-1^{2L/2L:SF1-Cre} and α2δ-1^2L/2L females fed a CD or HFD and only influenced by diet (data not shown).

In total, the results indicate that, unlike male α2δ-1^{2L/2L:SF1-Cre} mice, mutant females exhibit normal levels of lipolysis. Furthermore, they suggest that diminished circulating levels of adiponectin in α2δ-1^{2L/2L:SF1-Cre} females could underlie their susceptibility to diet-induced hepatic steatosis.

Hepatic sympathetic tone in α2δ-1^{2L/2L:SF1-Cre} females is significantly elevated by HFD administration.

The VMH regulates peripheral glucose and lipid metabolism through the control of sympathetic output to metabolic organs (5, 10, 37-39). We found previously that sympathetic tone was significantly reduced in α2δ-1^{2L/2L:SF1-Cre} males fed a CD as indicated by 45% and 63% reductions in norepinephrine (NE) content in WAT and serum, respectively (18). Because SF1 neurons critically regulate sympathetic tone in the periphery, we asked whether alterations in this branch of the autonomic system in α2δ-1^{2L/2L:SF1-Cre} females might contribute to the metabolic alterations that they exhibit. There was a significant effect of diet on NE content in WAT. However, levels of NE in serum, WAT, BAT, and smooth muscle were normal in mutant females compared with diet-matched controls (Fig. 6A-6D). In contrast, levels of NE in liver were significantly and dramatically elevated in α2δ-1^{2L/2L:SF1-Cre} females compared with α2δ-1^2L/2L controls fed a HFD (Fig. 6E). This effect of HFD on hepatic sympathetic outflow was not observed in mutant males (Fig. 6F) (Table 1), which also contained normal levels of NE in serum, WAT, BAT, and smooth muscle (data not shown). The data indicate that α2δ-1 depletion in the VMH exerts sexually dimorphic effects on sympathetic tone, diminishing sympathetic function in males but not in females fed a CD and increasing hepatic sympathetic outflow in females but not in males fed a HFD. Furthermore, considering previous findings showing that increased sympathetic tone onto the liver elicits hepatic steatosis (40), the increase in hepatic sympathetic outflow in α2δ-1^{2L/2L:SF1-Cre} females fed a HFD likely contributes to their susceptibility to TG accumulation.

Discussion

Here, we show that effects of α2δ-1 in the VMH influencing glucose and lipid balance and sympathetic outflow are sexually dimorphic. In females, α2δ-1 depletion results in improved glycemic control under CD conditions and in mild body weight gain, glucose intolerance, elevated sympathetic tone, and TG accumulation in the liver when fed a HFD. These bidirectional effects influenced by diet, suggest an important role of α2δ-1 coordinating adaptive mechanisms during challenging dietary conditions. They are also clinically relevant considering that alterations in metabolic function have been reported in individuals treated with gabapentinoid drugs, which inhibit α2δ-1 (19-21).

Depleting α2δ-1 in SF1 neurons had very mild effects on body weight of females and only when challenged with a HFD, indicating that α2δ-1 in those cells does not play a critical role in energy balance regulation, similar to males. However, sex-specific effects of α2δ-1 in the VMH were evident in mechanisms controlling glucose homeostasis. We reported previously that α2δ-1^{2L/2L:SF1-Cre} males fed a CD are glucose intolerant relative to littermate controls, indicating an essential and body weight-independent role of α2δ-1 facilitating glycemic control. In contrast, α2δ-1^{2L/2L:SF1-Cre}females fed a CD exhibited a significant increase in glucose tolerance and reduced fasted levels of serum insulin, suggesting enhanced insulin sensitivity. Diet influenced the consequences of depleting α2δ-1 in the female VMH. Accordingly, mutant females fed a HFD exhibited a mild reduction in glycemic control compared with α2δ-1^2L/2L females fed a similar diet. This effect was absent in mutant males fed a HFD, which performed similarly to control males in the GTT. Because there were no significant effects of genotype on total body weights of α2δ-1^2L/2L females fed a CD or a HFD, it is unlikely that observed alterations in glucose balance are driven by changes in body weight. Because SF1 is also expressed in the pituitary and we used the SF1-cre line of mice, it is also important to consider that altered α2δ-1 function in this region might contribute to the observed metabolic alterations. In total, the data suggest that α2δ-1 facilitates adaptive cellular mechanisms mediating metabolic flexibility in female mice.

Lipid balance control is also differentially influenced by VMH α2δ-1 in females versus males. Accordingly, α2δ-1^{2L/2L:SF1-Cre} females fed a CD did not exhibit the elevation in lipolysis in WAT observed previously in α2δ-1^{2L/2L:SF1-Cre} males administered the same diet (18). However, mutant females but not males had increased susceptibility to hepatic steatosis when fed a HFD. Decreased cholesterol content in WAT also suggests that lipid balance during dietary challenge is influenced by VMH α2δ-1 in female mice. This is a significant finding considering that dyslipidemia often precedes alterations in glycemic control, which are also exhibited by α2δ-1^{2L/2L:SF1-Cre} females fed a HFD. For example, previous studies showed that abnormal cholesterol levels greatly impact adipocyte metabolic activity, which can ultimately have deleterious effects on insulin sensitivity (41). Furthermore, we found that circulating levels of adiponectin were significantly reduced in α2δ-1^{2L/2L:SF1-Cre} females fed a HFD, suggesting impaired WAT function. Because adiponectin was reported to facilitate metabolic flexibility in mice, this deficit likely contributes to metabolic dysfunction in mutant females fed a HFD. Specifically, HFD-induced hepatic fat accumulation and reductions in insulin sensitivity were prevented in transgenic mice with elevated levels of circulating adiponectin (35, 36).

Identified elevations in hepatic sympathetic outflow are expected to contribute to the susceptibility to diet-induced liver steatosis observed in α2δ-1^{2L/2L:SF1-Cre} females. In support, Hurr and Morgan reported recently that diet-induced liver steatosis was associated with an increase in the firing rate of hepatic sympathetic fibers in mice and increased transcript content of the fatty acid translocase CD36 in the liver (40). Importantly, hepatic sympathetic denervation mitigated free fatty acid uptake and TG accumulation in livers of mice fed a HFD and this was mediated, in part, by blunting diet-induced elevations in CD36 mRNA expression in the liver. This is particularly relevant as a great proportion of hepatic TG is derived from FFA uptake from the circulation by cell membrane transporters (42) and as α2δ-1^{2L/2L:SF1-Cre}females fed a HFD also exhibited elevated expression of hepatic CD36. The collective evidence suggests that α2δ-1 in the VMH of female mice is a critical component of central mechanisms regulating hepatic sympathetic outflow and that in its absence, hepatic sympathetic overactivity leads to increased CD36 expression and TG accumulation in the liver. It is also important to note that unlike mutant females, sympathetic tone to the livers of α2δ-1^{2L/2L:SF1-Cre} males fed a HFD was similar to that of male controls. Sex-specific effects on sympathetic tone following depletion of VMH α2δ-1 were also evident in animals fed a CD. Whereas NE content in serum and WAT was dramatically reduced in α2δ-1^{2L/2L:SF1-Cre} males (18), it was normal in females as demonstrated here.

Altered SF1 neuronal activity is a plausible central mechanism driving metabolic alterations in female mutant mice. In support, activation of SF1 neurons via DREADD technology increases insulin sensitivity and glucose uptake in the periphery (5). α2δ-1 serves both as a voltage-gated calcium channel subunit and as a receptor for thrombospondins (TSPs), facilitating excitatory synapse assembly (14, 15). Eroglu et al (15) showed that cortical neurons in transgenic mice overexpressing α2δ-1 contained a higher density of excitatory synapses and elevated frequency of miniature EPSCs compared with wild-type mice (15). In contrast, inhibiting α2δ-1 via administration of gabapentin blunted the effects of thrombospondins inducing excitatory synaptogenesis. Finally, we showed that depleting α2δ-1 reduces the excitatory tone and activity of SF1 neurons without affecting calcium currents in male mice (18), implicating actions of α2δ-1 facilitating excitatory synapse assembly.

It remains unclear why α2δ-1 depletion results in sex-specific effects on glucose and lipid balance and sympathetic output in the periphery. One possibility is that α2δ-1 couples to different and opposing signaling cascades in females versus males, differentially affecting neuronal activity. However, considering that there are no examples in the literature indicating that α2δ-1 inhibits excitatory transmission and that there is evidence that it does not affect inhibitory transmission (15), this scenario is unlikely. Alternatively, there might be sex differences in the pattern of α2δ-1 expression or in the requirement of this thrombospondin receptor within distinct SF1⁺ neuronal subpopulations. This is important considering that diverse leptin-induced, leptin-inhibited and insulin-inhibited SF1 neuronal subpopulations have been identified in the VMH, indicating functional heterogeneity within this cell population (43). Future investigations outside the scope of the current study should examine this possibility. Sex-specific effects might also arise from α2δ-1 influencing effects of estrogen on VMH neuronal activity and energy and glucose balance control in females but not in males. This possibility is worth considering as there is a higher density of estrogen receptors in female versus male VMH (44) and that estrogen signaling via ERα in the VMH is protective against obesity and glucose intolerance in females but not in males (45). Finally, potential interactions between sex and genetic background should also be considered. Indeed, mice studied in our investigations were in a mixed C57Bl6 and FVB/NJ background, and females in both of those backgrounds exhibit resistance to diet-induced obesity and diet-induced alterations in glycemic control compared with males (46, 47).

In summary, our studies revealed female-specific and bimodal effects of VMH α2δ-1 impacting glucose and lipid balance control and sympathetic tone. The collective findings illustrate the importance of assessing sex-specific mechanisms regulating metabolic function and inform pathological mechanisms underlying metabolic disturbances in individuals administered the anti-epileptic and antinociceptive drugs gabapentin and pregabalin, which bind and inhibit α2δ-1 (20, 21).